The constant region affects antigen binding of antibodies to DNA by altering secondary structure.

PubMed

Xia, Yumin; Janda, Alena; Eryilmaz, Ertan; Casadevall, Arturo; Putterman, Chaim

2013-11-01

We previously demonstrated an important role of the constant region in the pathogenicity of anti-DNA antibodies. To determine the mechanisms by which the constant region affects autoantibody binding, a panel of isotype-switch variants (IgG1, IgG2a, IgG2b) was generated from the murine PL9-11 IgG3 autoantibody. The affinity of the PL9-11 antibody panel for histone was measured by surface plasmon resonance (SPR). Tryptophan fluorescence was used to determine wavelength shifts of the antibody panel upon binding to DNA and histone. Finally, circular dichroism spectroscopy was used to measure changes in secondary structure. SPR analysis revealed significant differences in histone binding affinity between members of the PL9-11 panel. The wavelength shifts of tryptophan fluorescence emission were found to be dependent on the antibody isotype, while circular dichroism analysis determined that changes in antibody secondary structure content differed between isotypes upon antigen binding. Thus, the antigen binding affinity is dependent on the particular constant region expressed. Moreover, the effects of antibody binding to antigen were also constant region dependent. Alteration of secondary structures influenced by constant regions may explain differences in fine specificity of anti-DNA antibodies between antibodies with similar variable regions, as well as cross-reactivity of anti-DNA antibodies with non-DNA antigens. Copyright © 2013 Elsevier Ltd. All rights reserved.

Alu elements in primates are preferentially lost from areas of high GC content

PubMed Central

Brookfield, John FY

2013-01-01

The currently-accepted dogma when analysing human Alu transposable elements is that ‘young’ Alu elements are found in low GC regions and ‘old’ Alus in high GC regions. The correlation between high GC regions and high gene frequency regions make this observation particularly difficult to explain. Although a number of studies have tackled the problem, no analysis has definitively explained the reason for this trend. These observations have been made by relying on the subfamily as a proxy for age of an element. In this study, we suggest that this is a misleading assumption and instead analyse the relationship between the taxonomic distribution of an individual element and its surrounding GC environment. An analysis of 103906 Alu elements across 6 human chromosomes was carried out, using the presence of orthologous Alu elements in other primate species as a proxy for age. We show that the previously-reported effect of GC content correlating with subfamily age is not reflected by the ages of the individual elements. Instead, elements are preferentially lost from areas of high GC content over time. The correlation between GC content and subfamily may be due to a change in insertion bias in the young subfamilies. The link between Alu subfamily age and GC region was made due to an over-simplification of the data and is incorrect. We suggest that use of subfamilies as a proxy for age is inappropriate and that the analysis of ortholog presence in other primate species provides a deeper insight into the data. PMID:23717800

Hantavirus Gc induces long-term immune protection via LAMP-targeting DNA vaccine strategy.

PubMed

Jiang, Dong-Bo; Zhang, Jin-Peng; Cheng, Lin-Feng; Zhang, Guan-Wen; Li, Yun; Li, Zi-Chao; Lu, Zhen-Hua; Zhang, Zi-Xin; Lu, Yu-Chen; Zheng, Lian-He; Zhang, Fang-Lin; Yang, Kun

2018-02-01

Hemorrhagic fever with renal syndrome (HFRS) occurs widely throughout Eurasia. Unfortunately, there is no effective treatment, and prophylaxis remains the best option against the major pathogenic agent, hantaan virus (HTNV), which is an Old World hantavirus. However, the absence of cellular immune responses and immunological memory hampers acceptance of the current inactivated HFRS vaccine. Previous studies revealed that a lysosome-associated membrane protein 1 (LAMP1)-targeting strategy involving a DNA vaccine based on the HTNV glycoprotein Gn successfully conferred long-term immunity, and indicated that further research on Gc, another HTNV antigen, was warranted. Plasmids encoding Gc and lysosome-targeted Gc, designated pVAX-Gc and pVAX-LAMP/Gc, respectively, were constructed. Proteins of interest were identified by fluorescence microscopy following cell line transfection. Five groups of 20 female BALB/c mice were subjected to the following inoculations: inactivated HTNV vaccine, pVAX-LAMP/Gc, pVAX-Gc, and, as the negative controls, pVAX-LAMP or the blank vector pVAX1. Humoral and cellular immunity were assessed by enzyme-linked immunosorbent assays (ELISAs) and 15-mer peptide enzyme-linked immunospot (ELISpot) epitope mapping assays. Repeated immunization with pVAX-LAMP/Gc enhanced adaptive immune responses, as demonstrated by the specific and neutralizing antibody titers and increased IFN-γ production. The inactivated vaccine induced a comparable humoral reaction, but the negative controls only elicited insignificant responses. Using a mouse model of HTNV challenge, the in vivo protection conferred by the inactivated vaccine and Gc-based constructs (with/without LAMP recombination) was confirmed. Evidence of pan-epitope reactions highlighted the long-term cellular response to the LAMP-targeting strategy, and histological observations indicated the safety of the LAMP-targeting vaccines. The long-term protective immune responses induced by pVAX-LAMP/Gc may be

Methylation effect on the ohmic resistance of a poly-GC DNA-like chain

NASA Astrophysics Data System (ADS)

de Moura, F. A. B. F.; Lyra, M. L.; de Almeida, M. L.; Ourique, G. S.; Fulco, U. L.; Albuquerque, E. L.

2016-10-01

We determine, by using a tight-binding model Hamiltonian, the characteristic current-voltage (IxV) curves of a 5-methylated cytosine single strand poly-GC DNA-like finite segment, considering the methyl groups attached laterally to a random fraction of the cytosine basis. Striking, we found that the methylation significantly impacts the ohmic resistance (R) of the DNA-like segments, indicating that measurements of R can be used as a biosensor tool to probe the presence of anomalous methylation.

Ligation-mediated PCR with a back-to-back adapter reduces amplification bias resulting from variations in GC content.

PubMed

Ishihara, Satoru; Kotomura, Naoe; Yamamoto, Naoki; Ochiai, Hiroshi

2017-08-15

Ligation-mediated polymerase chain reaction (LM-PCR) is a common technique for amplification of a pool of DNA fragments. Here, a double-stranded oligonucleotide consisting of two primer sequences in back-to-back orientation was designed as an adapter for LM-PCR. When DNA fragments were ligated with this adapter, the fragments were sandwiched between two adapters in random orientations. In the ensuing PCR, ligation products linked at each end to an opposite side of the adapter, i.e. to a distinct primer sequence, were preferentially amplified compared with products linked at each end to an identical primer sequence. The use of this adapter in LM-PCR reduced the impairment of PCR by substrate DNA with a high GC content, compared with the use of traditional LM-PCR adapters. This result suggested that our method has the potential to contribute to reduction of the amplification bias that is caused by an intrinsic property of the sequence context in substrate DNA. A DNA preparation obtained from a chromatin immunoprecipitation assay using pulldown of a specific form of histone H3 was successfully amplified using the modified LM-PCR, and the amplified products could be used as probes in a fluorescence in situ hybridization analysis. Copyright © 2017 Elsevier Inc. All rights reserved.

Comparative Genomics of Burkholderia singularis sp. nov., a Low G+C Content, Free-Living Bacterium That Defies Taxonomic Dissection of the Genus Burkholderia

PubMed Central

Vandamme, Peter; Peeters, Charlotte; De Smet, Birgit; Price, Erin P.; Sarovich, Derek S.; Henry, Deborah A.; Hird, Trevor J.; Zlosnik, James E. A.; Mayo, Mark; Warner, Jeffrey; Baker, Anthony; Currie, Bart J.; Carlier, Aurélien

2017-01-01

Four Burkholderia pseudomallei-like isolates of human clinical origin were examined by a polyphasic taxonomic approach that included comparative whole genome analyses. The results demonstrated that these isolates represent a rare and unusual, novel Burkholderia species for which we propose the name B. singularis. The type strain is LMG 28154T (=CCUG 65685T). Its genome sequence has an average mol% G+C content of 64.34%, which is considerably lower than that of other Burkholderia species. The reduced G+C content of strain LMG 28154T was characterized by a genome wide AT bias that was not due to reduced GC-biased gene conversion or reductive genome evolution, but might have been caused by an altered DNA base excision repair pathway. B. singularis can be differentiated from other Burkholderia species by multilocus sequence analysis, MALDI-TOF mass spectrometry and a distinctive biochemical profile that includes the absence of nitrate reduction, a mucoid appearance on Columbia sheep blood agar, and a slowly positive oxidase reaction. Comparisons with publicly available whole genome sequences demonstrated that strain TSV85, an Australian water isolate, also represents the same species and therefore, to date, B. singularis has been recovered from human or environmental samples on three continents. PMID:28932212

Contribution of AT-, GC-, and methylated cytidine-rich DNA to chromatin composition in Malpighian tubule cell nuclei of Panstrongylus megistus (Hemiptera, Reduviidae).

PubMed

Alvarenga, Elenice M; Mondin, Mateus; Rodrigues, Vera L C C; Andrade, Larissa M; Vidal, Benedicto de Campos; Mello, Maria Luiza S

2012-11-01

The Malpighian tubule cell nuclei of male Panstrongylus megistus, a vector of Chagas disease, contain one chromocenter, which is composed solely of the Y chromosome. Considering that different chromosomes contribute to the composition of chromocenters in different triatomini species, the aim of this study was to determine the contribution of AT-, GC-, and methylated cytidine-rich DNA in the chromocenter as well as in euchromatin of Malpighian tubule cell nuclei of P. megistus in comparison with published data for Triatoma infestans. Staining with 4',6-diamidino-2-phenylindole/actinomycin D and chromomycin A(3)/distamycin, immunodetection of 5-methylcytidine and AgNOR test were used. The results revealed AT-rich/GC-poor DNA in the male chromocenter, but equally distributed AT and GC DNA sequences in male and female euchromatin, like in T. infestans. Accumulation of argyrophilic proteins encircling the chromocenter did not always correlate with that of GC-rich DNA. Methylated DNA identified by immunodetection was found sparsely distributed in the euchromatin of both sexes and at some points around the chromocenter edge, but it could not be considered responsible for chromatin condensation in the chromocenter, like in T. infestans. However, unlike in T. infestans, no correlation between the chromocenter AT-rich DNA and nucleolus organizing region (NOR) DNA was found in P. megistus. Copyright © 2011 Elsevier GmbH. All rights reserved.

Karyotype diversity and 2C DNA content in species of the Caesalpinia group.

PubMed

Rodrigues, Polliana Silva; Souza, Margarete Magalhães; Melo, Cláusio Antônio Ferreira; Pereira, Telma Nair Santana; Corrêa, Ronan Xavier

2018-04-11

The Leguminosae family is the third-largest family of angiosperms, and Caesalpinioideae is its second-largest subfamily. A great number of species (approximately 205) are found in the Caesalpinia group within this subfamily; together with these species' phenotypic plasticity and the similarities in their morphological descriptors, make this a complex group for taxonomic and phylogenetic studies. The objective of the present work was to evaluate the karyotypic diversity and the 2C DNA content variation in 10 species of the Caesalpinia group, representing six genera: Paubrasilia, Caesalpinia, Cenostigma, Poincianella, Erythrostemon and Libidibia. The GC-rich heterochromatin and 45S rDNA sites (which are used as chromosome markers) were located to evaluate the karyotype diversity in the clade. The variation in the 2C DNA content was determined through flow cytometry. The fluorochrome banding indicated that the chromomycin A 3 + /4',6-diamidino-2-phenylindole - blocks were exclusively in the terminal regions of the chromosomes, coinciding with 45S rDNA sites in all analyzed species. Physical mapping of the species (through fluorescence in situ hybridization) revealed variation in the size of the hybridization signals and in the number and distribution of the 45S rDNA sites. All hybridization sites were in the terminal regions of the chromosomes. In addition, all species had a hybridization site in the fourth chromosome pair. The 2C DNA content ranged from 1.54 pg in Erythrostemon calycina to 2.82 pg in the Paubrasilia echinata large-leaf variant. The Pa. echinata small-leaf variant was isolated from the other leaf variants through Scoot-Knott clustering. The chromosome diversity and the variation in the 2C DNA content reinforce that the actual taxonomy and clustering of the analyzed taxa requires more genera that were previously proposed. This fact indicates that taxonomy, phylogeny and cytoevolutionary inference related to the complex Caesalpinia group have to be

Sequence-Dependent Persistence Length of Long DNA

NASA Astrophysics Data System (ADS)

Chuang, Hui-Min; Reifenberger, Jeffrey G.; Cao, Han; Dorfman, Kevin D.

2017-12-01

Using a high-throughput genome-mapping approach, we obtained circa 50 million measurements of the extension of internal human DNA segments in a 41 nm ×41 nm nanochannel. The underlying DNA sequences, obtained by mapping to the reference human genome, are 2.5-393 kilobase pairs long and contain percent GC contents between 32.5% and 60%. Using Odijk's theory for a channel-confined wormlike chain, these data reveal that the DNA persistence length increases by almost 20% as the percent GC content increases. The increased persistence length is rationalized by a model, containing no adjustable parameters, that treats the DNA as a statistical terpolymer with a sequence-dependent intrinsic persistence length and a sequence-independent electrostatic persistence length.

Binding to the DNA Minor Groove by Heterocyclic Dications: From AT Specific Monomers to GC Recognition with Dimers

PubMed Central

Nanjunda, Rupesh; Wilson, W. David

2012-01-01

Compounds that bind in the DNA minor groove have provided critical information on DNA molecular recognition, they have found extensive uses in biotechnology and they are providing clinically useful drugs against diseases as diverse as cancer and sleeping sickness. This review focuses on the development of clinically useful heterocyclic diamidine minor groove binders. These compounds have shown us that the classical model for minor groove binding in AT DNA sequences must be expanded in several ways: compounds with nonstandard shapes can bind strongly to the groove, water can be directly incorporated into the minor groove complex in an interfacial interaction, and the compounds can form cooperative stacked dimers to recognize GC and mixed AT/GC base pair sequences. PMID:23255206

Complete chloroplast genome sequences of Drimys, Liriodendron, andPiper: Implications for the phylogeny of magnoliids and the evolution ofGC content

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhengqiu, C.; Penaflor, C.; Kuehl, J.V.

2006-06-01

highest in the inverted repeat due to the presence of rRNA genes and lowest in the small single copy region where most NADH genes are located. Phylogenetic analyses using maximum parsimony and maximum likelihood methods were performed on DNA sequences of 61 protein-coding genes. Trees from both analyses provided strong support for the monophyly of magnoliids and two strongly supported groups were identified, the Canellales/Piperales and the Laurales/Magnoliales. The phylogenies also provided moderate to strong support for the basal position of Amborella, and a sister relationship of magnoliids to a clade that includes monocots and eudicots. The complete sequences of three magnoliid chloroplast genomes provide new data from the largest basal angiosperm clade. Evolutionary comparisons of these new genome sequences, combined with other published angiosperm genome, confirm that GC content is unevenly distributed across the genome by location, codon position, and functional group. Furthermore, phylogenetic analyses provide the strongest support so far for the hypothesis that the magnoliids are sister to a large clade that includes both monocots and eudicots.« less

sup 60 Co. gamma. -rays induce predominantly C/G to G/C transversions in double-stranded M13 DNA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hoebee, B.; Loman, H.; Brouwer, J.

Upon irradiation with gamma rays of an oxygenated aqueous solution of double-stranded M13 DNA, a very specific mutation spectrum was found with respect to both the type and the positions in the DNA sequence. Of the 23 mutations, which were sequenced, 16 represent a C/G to G/C transversion. A C/G to T/A transition was found once and a G/C to T/A transversion twice. The remaining 4 mutations are frameshifts, 2 are identical and formed by the insertion of a G/C basepair; the other 2 mutations are due to a duplication of 10 basepairs situated at different positions but with amore » remarkable homology in base sequence. Fourteen mutations, including the 2 duplications are found in the neighborhood of a TGCT/ACGA sequence.« less

Accounting for GC-content bias reduces systematic errors and batch effects in ChIP-seq data.

PubMed

Teng, Mingxiang; Irizarry, Rafael A

2017-11-01

The main application of ChIP-seq technology is the detection of genomic regions that bind to a protein of interest. A large part of functional genomics' public catalogs is based on ChIP-seq data. These catalogs rely on peak calling algorithms that infer protein-binding sites by detecting genomic regions associated with more mapped reads (coverage) than expected by chance, as a result of the experimental protocol's lack of perfect specificity. We find that GC-content bias accounts for substantial variability in the observed coverage for ChIP-seq experiments and that this variability leads to false-positive peak calls. More concerning is that the GC effect varies across experiments, with the effect strong enough to result in a substantial number of peaks called differently when different laboratories perform experiments on the same cell line. However, accounting for GC content bias in ChIP-seq is challenging because the binding sites of interest tend to be more common in high GC-content regions, which confounds real biological signals with unwanted variability. To account for this challenge, we introduce a statistical approach that accounts for GC effects on both nonspecific noise and signal induced by the binding site. The method can be used to account for this bias in binding quantification as well to improve existing peak calling algorithms. We use this approach to show a reduction in false-positive peaks as well as improved consistency across laboratories. © 2017 Teng and Irizarry; Published by Cold Spring Harbor Laboratory Press.

Myonuclear transcription is responsive to mechanical load and DNA content but uncoupled from cell size during hypertrophy

PubMed Central

Kirby, Tyler J.; Patel, Rooshil M.; McClintock, Timothy S.; Dupont-Versteegden, Esther E.; Peterson, Charlotte A.; McCarthy, John J.

2016-01-01

Myofibers increase size and DNA content in response to a hypertrophic stimulus, thus providing a physiological model with which to study how these factors affect global transcription. Using 5-ethynyl uridine (EU) to metabolically label nascent RNA, we measured a sevenfold increase in myofiber transcription during early hypertrophy before a change in cell size and DNA content. The typical increase in myofiber DNA content observed at the later stage of hypertrophy was associated with a significant decrease in the percentage of EU-positive myonuclei; however, when DNA content was held constant by preventing myonuclear accretion via satellite cell depletion, both the number of transcriptionally active myonuclei and the amount of RNA generated by each myonucleus increased. During late hypertrophy, transcription did not scale with cell size, as smaller myofibers (<1000 μm2) demonstrated the highest transcriptional activity. Finally, transcription was primarily responsible for changes in the expression of genes known to regulate myofiber size. These findings show that resident myonuclei possess a significant reserve capacity to up-regulate transcription during hypertrophy and that myofiber transcription is responsive to DNA content but uncoupled from cell size during hypertrophy. PMID:26764089

Sequencing on the SOLiD 5500xl System - in-depth characterization of the GC bias.

PubMed

Roeh, Simone; Weber, Peter; Rex-Haffner, Monika; Deussing, Jan M; Binder, Elisabeth B; Jakovcevski, Mira

2017-07-04

Different types of sequencing biases have been described and subsequently improved for a variety of sequencing systems, mostly focusing on the widely used Illumina systems. Similar studies are missing for the SOLiD 5500xl system, a sequencer which produced many data sets available to researchers today. Describing and understanding the bias is important to accurately interpret and integrate these published data in various ongoing research projects. We report a particularly strong GC bias for this sequencing system when analyzing a defined gDNA mix of 5 microbes with a wide range of different GC contents (20-72%) when comparing to the expected distribution and Illumina MiSeq data from the same DNA pool. Since we observed this bias already under PCR-free conditions, changing the PCR conditions during library preparation - a common strategy to handle bias in the Illumina system - was not relevant. Source of the bias appeared to be an uneven heat distribution during the SOLiD emulsion PCR (ePCR) - for enrichment of libraries prior loading - since ePCR in either small pouches or in 96-well plates improved the GC bias. Sequencing of chromatin immunoprecipitated DNA (ChIP-seq) is a common approach in epigenetics. ChIP-seq of the mixed source histone mark H3K9ac (acetyl Histone H3 lysine 9), typically found on promoter regions and on gene bodies, including CpG islands, performed on a SOLiD 5500xl machine, resulted in major loss of reads at GC rich loci (GC content ≥ 62%), not explained by low sequencing depth. This was improved with adaptations of the ePCR.

Analysis of Cellular DNA Content by Flow Cytometry.

PubMed

Darzynkiewicz, Zbigniew; Huang, Xuan; Zhao, Hong

2017-10-02

Cellular DNA content can be measured by flow cytometry with the aim of : (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing DNA ploidy of the measured cell population. In this unit, simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and/or proteolytic treatment to permeabilize cells and make DNA accessible to fluorochrome. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells based on DNA-content differences for their subsequent culturing, is described. Also presented are methods for staining cell nuclei isolated from paraffin-embedded tissues. Available algorithms are listed for deconvolution of DNA-content-frequency histograms to estimate percentage of cells in major phases of the cell cycle and frequency of apoptotic cells with fractional DNA content. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley and Sons, Inc.

Analysis of Cellular DNA Content by Flow Cytometry.

PubMed

Darzynkiewicz, Zbigniew; Huang, Xuan; Zhao, Hong

2017-11-01

Cellular DNA content can be measured by flow cytometry with the aim of : (1) revealing cell distribution within the major phases of the cell cycle, (2) estimating frequency of apoptotic cells with fractional DNA content, and/or (3) disclosing DNA ploidy of the measured cell population. In this unit, simple and universally applicable methods for staining fixed cells are presented, as are methods that utilize detergents and/or proteolytic treatment to permeabilize cells and make DNA accessible to fluorochrome. Additionally, supravital cell staining with Hoechst 33342, which is primarily used for sorting live cells based on DNA-content differences for their subsequent culturing, is described. Also presented are methods for staining cell nuclei isolated from paraffin-embedded tissues. Available algorithms are listed for deconvolution of DNA-content-frequency histograms to estimate percentage of cells in major phases of the cell cycle and frequency of apoptotic cells with fractional DNA content. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley and Sons, Inc.

Mechanistic Investigation of the Bypass of a Bulky Aromatic DNA Adduct Catalyzed by a Y-family DNA Polymerase

PubMed Central

Gadkari, Varun V.; Tokarsky, E. John; Malik, Chanchal K.; Basu, Ashis K.; Suo, Zucai

2014-01-01

3-Nitrobenzanthrone (3-NBA), a nitropolyaromatic hydrocarbon (NitroPAH) pollutant in diesel exhaust, is a potent mutagen and carcinogen. After metabolic activation, the primary metabolites of 3-NBA react with DNA to form dG and dA adducts. One of the three major adducts identified is N-(2’-deoxyguanosin-8-yl)-3-aminobenzanthrone (dGC8-N-ABA). This bulky adduct likely stalls replicative DNA polymerases but can be traversed by lesion bypass polymerases in vivo. Here, we employed running start assays to show that a site-specifically placed dGC8-N-ABA is bypassed in vitro by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. However, the nucleotide incorporation rate of Dpo4 was significantly reduced opposite both the lesion and the template position immediately downstream from the lesion site, leading to two strong pause sites. To investigate the kinetic effect of dGC8-N-ABA on polymerization, we utilized pre-steady-state kinetic methods to determine the kinetic parameters for individual nucleotide incorporations upstream, opposite, and downstream from the dGC8-N-ABA lesion. Relative to the replication of the corresponding undamaged DNA template, both nucleotide incorporation efficiency and fidelity of Dpo4 were considerably decreased during dGC8-N-ABA lesion bypass and the subsequent extension step. The lower nucleotide incorporation efficiency caused by the lesion is a result of a significantly reduced dNTP incorporation rate constant and modestly weaker dNTP binding affinity. At both pause sites, nucleotide incorporation followed biphasic kinetics with a fast and a slow phase and their rates varied with nucleotide concentration. In contrast, only the fast phase was observed with undamaged DNA. A kinetic mechanism was proposed for the bypass of dGC8-N-ABA bypass catalyzed by Dpo4. PMID:25048879

Analysis of phylogeny and codon usage bias and relationship of GC content, amino acid composition with expression of the structural nif genes.

PubMed

Mondal, Sunil Kanti; Kundu, Sudip; Das, Rabindranath; Roy, Sujit

2016-08-01

Bacteria and archaea have evolved with the ability to fix atmospheric dinitrogen in the form of ammonia, catalyzed by the nitrogenase enzyme complex which comprises three structural genes nifK, nifD and nifH. The nifK and nifD encodes for the beta and alpha subunits, respectively, of component 1, while nifH encodes for component 2 of nitrogenase. Phylogeny based on nifDHK have indicated that Cyanobacteria is closer to Proteobacteria alpha and gamma but not supported by the tree based on 16SrRNA. The evolutionary ancestor for the different trees was also different. The GC1 and GC2% analysis showed more consistency than GC3% which appeared to below for Firmicutes, Cyanobacteria and Euarchaeota while highest in Proteobacteria beta and clearly showed the proportional effect on the codon usage with a few exceptions. Few genes from Firmicutes, Euryarchaeota, Proteobacteria alpha and delta were found under mutational pressure. These nif genes with low and high GC3% from different classes of organisms showed similar expected number of codons. Distribution of the genes and codons, based on codon usage demonstrated opposite pattern for different orientation of mirror plane when compared with each other. Overall our results provide a comprehensive analysis on the evolutionary relationship of the three structural nif genes, nifK, nifD and nifH, respectively, in the context of codon usage bias, GC content relationship and amino acid composition of the encoded proteins and exploration of crucial statistical method for the analysis of positive data with non-constant variance to identify the shape factors of codon adaptation index.

Preliminary analysis of length and GC content variation in the ribosomal first internal transcribed spacer (ITS1) of marine animals.

PubMed

Chow, S; Ueno, Y; Toyokawa, M; Oohara, I; Takeyama, H

2009-01-01

Length and guanine-cytosine (GC) content of the ribosomal first internal transcribed spacer (ITS1) were compared across a wide variety of marine animal species, and its phylogenetic utility was investigated. From a total of 773 individuals representing 599 species, we only failed to amplify the ITS1 sequence from 87 individuals by polymerase chain reaction with universal ITS1 primers. No species was found to have an ITS1 region shorter than 100 bp. In general, the ITS1 sequences of vertebrates were longer (318 to 2,318 bp) and richer in GC content (56.8% to 78%) than those of invertebrates (117 to 1,613 bp and 35.8% to 71.3%, respectively). Specifically, gelatinous animals (Cnidaria and Ctenophora) were observed to have short ITS1 sequences (118 to 422 bp) with lower GC content (35.8% to 61.7%) than the other animal taxa. Mollusca and Crustacea were diverse groups with respect to ITS1 length, ranging from 108 to 1,118 and 182 to 1,613 bp, respectively. No universal relationship between length and GC content was observed. Our data indicated that ITS1 has a limited utility for phylogenetic analysis as obtaining confident sequence alignment was often impossible between different genera of the same family and even between congeneric species.

Nuclear DNA content and base composition in 28 taxa of Musa.

PubMed

Kamaté, K; Brown, S; Durand, P; Bureau, J M; De Nay, D; Trinh, T H

2001-08-01

The nuclear DNA content of 28 taxa of Musa was assessed by flow cytometry, using line PxPC6 of Petunia hybrida as an internal standard. The 2C DNA value of Musa balbisiana (BB genome) was 1.16 pg, whereas Musa acuminata (AA genome) had an average 2C DNA value of 1.27 pg, with a difference of 11% between its subspecies. The two haploid (IC) genomes, A and B, comprising most of the edible bananas, are therefore of similar size, 0.63 pg (610 million bp) and 0.58 pg (560 million bp), respectively. The genome of diploid Musa is thus threefold that of Arabidopsis thaliana. The genome sizes in a set of triploid Musa cultivars or clones were quite different, with 2C DNA values ranging from 1.61 to 2.23 pg. Likewise, the genome sizes of tetraploid cultivars ranged from 1.94 to 2.37 pg (2C). Apparently, tetraploids (for instance, accession I.C.2) can have a genome size that falls within the range of triploid genome sizes, and vice versa (as in the case of accession Simili Radjah). The 2C values estimated for organs such as leaf, leaf sheath, rhizome, and flower were consistent, whereas root material gave atypical results, owing to browning. The genomic base composition of these Musa taxa had a median value of 40.8% GC (SD = 0.43%).

The circular dichroism properties of phi W-14 DNA containing alpha-putrescinylthymine.

PubMed

Spetter, S; Chen, C; Warren, R A; Hanlon, S

1985-03-08

The circular dichroism properties of phi W-14 DNA containing alpha-putrescinylthymine and its acetylated derivative have been examined in a number of aqueous solvents. Native phi W-14 DNA exhibits a B-type CD spectrum whose characteristics do not entirely conform to what would be expected for its GC content (51%). The conformationally sensitive positive band above 260 nm has a rotational strength greater than that normally found in prokaryotic DNAs of comparable GC content, such as Escherichia coli DNA. The rotational strength of this band in the spectrum of the heat-denatured form of phi W-14 DNA, however, is similar to that of heat denatured E. coli DNA. Abolition of the positive charge on the putrescine residues of native phi W-14 DNA by reaction with CH2O or by acetylation reduces the rotational strength to a level appropriate for its GC content. Increases in the electrolyte content of the solvent have the same effect, although the rotational strength of this band in phi W-14 DNA does not become comparable to that of E. coli DNA until 6-7 M LiCl. Titration to pH 10.6 in solvents of modest electrolyte content, however, fails to appreciably affect the CD spectral properties of either native phi W-14 DNA or the derivative in which half of the secondary and all of the primary amino groups have been acetylated. On the basis of these results we have concluded that the enhanced rotational strength of the positive band above 260 nm in the CD spectrum of phi W-14 DNA is due to a conformational difference caused by an ion-pair interaction of the positively charged primary amino groups of putrescine with the phosphate backbone. The CD spectral properties, however, reveal that these differences, averaged over the entire basepair population, appear to be relatively small. The average conformation, at least in dilute aqueous solutions, seems to be an unexceptional B variant with conformational properties which would be more appropriate for a DNA of higher CG content.

Theoretical rate constants of super-exchange hole transfer and thermally induced hopping in DNA.

PubMed

Shimazaki, Tomomi; Asai, Yoshihiro; Yamashita, Koichi

2005-01-27

Recently, the electronic properties of DNA have been extensively studied, because its conductivity is important not only to the study of fundamental biological problems, but also in the development of molecular-sized electronics and biosensors. We have studied theoretically the reorganization energies, the activation energies, the electronic coupling matrix elements, and the rate constants of hole transfer in B-form double-helix DNA in water. To accommodate the effects of DNA nuclear motions, a subset of reaction coordinates for hole transfer was extracted from classical molecular dynamics (MD) trajectories of DNA in water and then used for ab initio quantum chemical calculations of electron coupling constants based on the generalized Mulliken-Hush model. A molecular mechanics (MM) method was used to determine the nuclear Franck-Condon factor. The rate constants for two types of mechanisms of hole transfer-the thermally induced hopping (TIH) and the super-exchange mechanisms-were determined based on Marcus theory. We found that the calculated matrix elements are strongly dependent on the conformations of the nucleobase pairs of hole-transferable DNA and extend over a wide range of values for the "rise" base-step parameter but cluster around a particular value for the "twist" parameter. The calculated activation energies are in good agreement with experimental results. Whereas the rate constant for the TIH mechanism is not dependent on the number of A-T nucleobase pairs that act as a bridge, the rate constant for the super-exchange process rapidly decreases when the length of the bridge increases. These characteristic trends in the calculated rate constants effectively reproduce those in the experimental data of Giese et al. [Nature 2001, 412, 318]. The calculated rate constants were also compared with the experimental results of Lewis et al. [Nature 2000, 406, 51].

Content and persistence of extracellular DNA in native soils

NASA Astrophysics Data System (ADS)

Blagodatskaya, Evgenia; Blagodatsky, Sergey; Anderson, Traute-Heidi; Kuzyakov, Yakov

2014-05-01

The long-term persistence of soil extracellular DNA is questionable because of high potential activity of nucleases produced by soil microorganisms. By the other hand, the relative persistence of DNA-like biopolymers could be due to their adsorption on clay minerals and humus substances in soil. High-specific and ultra sensitive reagent PicoGreenTM (Molecular Probes) permits the quantitative assessment of microbial dsDNA in diluted soil extracts giving a good tool for tracing the DNA fate in soil. Our goal was to determine intracellular and extracellular DNA content in cambisol (loamy sand) and in chernozem (silty loam) soils and to investigate the possible adsorption and degradation of extracellular DNA in soil. Optimized procedure of mechanical and enzymatic destruction of cell walls was used for direct extraction of microbial DNA with Tris-EDTA buffer (Blagodatskaya et al., 2003). Extracellular dsDNA was determined in distilled water and in Tris-EDTA extracts without enzymatic or mechanical treatments. DNA content was determined after addition of PicoGreen to diluted soil extracts. Degradation of extracellular DNA was traced during 24 h incubation of 2 µg lambda-phage DNA in soil. Possible DNA adsorption to soil matrix was determined by recovery of lambda -phage DNA added to autoclaved soil. Extracellular dsDNA was absent in water extracts of both soils. The content of extracellular dsDNA extracted by Tris-EDTA buffer was 0.46 µg/g in chernozem and 1.59 µg/g in cambisol amounting 0.43 and 2.8% of total dsDNA content in these soils, respectively. 100% and 64.8% of added extracellular lambda -phage dsDNA was found in cambisol and chernozem soils, respectively, in 5 h after application. 39% and 73.5% of added DNA disappeared in cambisol and in chernozem, respectively, during 24 h incubation. Degradation rate of extracellular DNA depended on microbial biomass content, which was 2.5 times higher in chernozem as compared to cambisol. Maximum adsorption of DNA by

Acid-induced exchange of the imino proton in G.C pairs.

PubMed Central

Nonin, S; Leroy, J L; Gueron, M

1996-01-01

Acid-induced catalysis of imino proton exchange in G.C pairs of DNA duplexes is surprisingly fast, being nearly as fast as for the isolated nucleoside, despite base-pair dissociation constants in the range of 10(-5) at neutral or basic pH. It is also observed in terminal G.C pairs of duplexes and in base pairs of drug-DNA complexes. We have measured imino proton exchange in deoxyguanosine and in the duplex (ATATAGATCTATAT) as a function of pH. We show that acid-induced exchange can be assigned to proton transfer from N7-protonated guanosine to cytidine in the open state of the pair. This is faster than transfer from neutral guanosine (the process of intrinsic catalysis previously characterized at neutral ph) due to the lower imino proton pK of the protonated form, 7.2 instead of 9.4. Other interpretations are excluded by a study of exchange catalysis by formiate and cytidine as exchange catalysts. The cross-over pH between the regimes of pH-independent and acid-induced exchange rates is more basic in the case of base pairs than in the mononucleoside, suggestive of an increase by one to two decades in the dissociation constant of the base pair upon N7 protonation of G. Acid-induced catalysis is much weaker in A.T base pairs, as expected in view of the low pK for protonation of thymidine. PMID:8604298

Acid-induced exchange of the imino proton in G.C pairs.

PubMed

Nonin, S; Leroy, J L; Gueron, M

1996-02-15

Acid-induced catalysis of imino proton exchange in G.C pairs of DNA duplexes is surprisingly fast, being nearly as fast as for the isolated nucleoside, despite base-pair dissociation constants in the range of 10(-5) at neutral or basic pH. It is also observed in terminal G.C pairs of duplexes and in base pairs of drug-DNA complexes. We have measured imino proton exchange in deoxyguanosine and in the duplex (ATATAGATCTATAT) as a function of pH. We show that acid-induced exchange can be assigned to proton transfer from N7-protonated guanosine to cytidine in the open state of the pair. This is faster than transfer from neutral guanosine (the process of intrinsic catalysis previously characterized at neutral ph) due to the lower imino proton pK of the protonated form, 7.2 instead of 9.4. Other interpretations are excluded by a study of exchange catalysis by formiate and cytidine as exchange catalysts. The cross-over pH between the regimes of pH-independent and acid-induced exchange rates is more basic in the case of base pairs than in the mononucleoside, suggestive of an increase by one to two decades in the dissociation constant of the base pair upon N7 protonation of G. Acid-induced catalysis is much weaker in A.T base pairs, as expected in view of the low pK for protonation of thymidine.

Myonuclear transcription is responsive to mechanical load and DNA content but uncoupled from cell size during hypertrophy.

PubMed

Kirby, Tyler J; Patel, Rooshil M; McClintock, Timothy S; Dupont-Versteegden, Esther E; Peterson, Charlotte A; McCarthy, John J

2016-03-01

Myofibers increase size and DNA content in response to a hypertrophic stimulus, thus providing a physiological model with which to study how these factors affect global transcription. Using 5-ethynyl uridine (EU) to metabolically label nascent RNA, we measured a sevenfold increase in myofiber transcription during early hypertrophy before a change in cell size and DNA content. The typical increase in myofiber DNA content observed at the later stage of hypertrophy was associated with a significant decrease in the percentage of EU-positive myonuclei; however, when DNA content was held constant by preventing myonuclear accretion via satellite cell depletion, both the number of transcriptionally active myonuclei and the amount of RNA generated by each myonucleus increased. During late hypertrophy, transcription did not scale with cell size, as smaller myofibers (<1000 μm(2)) demonstrated the highest transcriptional activity. Finally, transcription was primarily responsible for changes in the expression of genes known to regulate myofiber size. These findings show that resident myonuclei possess a significant reserve capacity to up-regulate transcription during hypertrophy and that myofiber transcription is responsive to DNA content but uncoupled from cell size during hypertrophy. © 2016 Kirby et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Schematic for efficient computation of GC, GC3, and AT3 bias spectra of genome

PubMed Central

Rizvi, Ahsan Z; Venu Gopal, T; Bhattacharya, C

2012-01-01

Selection of synonymous codons for an amino acid is biased in protein translation process. This biased selection causes repetition of synonymous codons in structural parts of genome that stands for high N/3 peaks in DNA spectrum. Period-3 spectral property is utilized here to produce a 3-phase network model based on polyphase filterbank concepts for derivation of codon bias spectra (CBS). Modification of parameters in this model can produce GC, GC3, and AT3 bias spectra. Complete schematic in LabVIEW platform is presented here for efficient and parallel computation of GC, GC3, and AT3 bias spectra of genomes alongwith results of CBS patterns. We have performed the correlation coefficient analysis of GC, GC3, and AT3 bias spectra with codon bias patterns of CBS for biological and statistical significance of this model. PMID:22368390

Schematic for efficient computation of GC, GC3, and AT3 bias spectra of genome.

PubMed

Rizvi, Ahsan Z; Venu Gopal, T; Bhattacharya, C

2012-01-01

Selection of synonymous codons for an amino acid is biased in protein translation process. This biased selection causes repetition of synonymous codons in structural parts of genome that stands for high N/3 peaks in DNA spectrum. Period-3 spectral property is utilized here to produce a 3-phase network model based on polyphase filterbank concepts for derivation of codon bias spectra (CBS). Modification of parameters in this model can produce GC, GC3, and AT3 bias spectra. Complete schematic in LabVIEW platform is presented here for efficient and parallel computation of GC, GC3, and AT3 bias spectra of genomes alongwith results of CBS patterns. We have performed the correlation coefficient analysis of GC, GC3, and AT3 bias spectra with codon bias patterns of CBS for biological and statistical significance of this model.

Interaction of zanamivir with DNA and RNA: Models for drug DNA and drug RNA bindings

NASA Astrophysics Data System (ADS)

Nafisi, Shohreh; Kahangi, Fatemeh Ghoreyshi; Azizi, Ebrahim; Zebarjad, Nader; Tajmir-Riahi, Heidar-Ali

2007-03-01

Zanamivir (ZAN) is the first of a new generation of influenza virus-specific drugs known as neuraminidase inhibitors, which acts by interfering with life cycles of influenza viruses A and B. It prevents the virus spreading infection to other cells by blocking the neuraminidase enzyme present on the surface of the virus. The aim of this study was to examine the stability and structural features of calf thymus DNA and yeast RNA complexes with zanamivir in aqueous solution, using constant DNA or RNA concentration (12.5 mM) and various zanamivir/polynucleotide ( P) ratios of 1/20, 1/10, 1/4, and 1/2. FTIR and UV-visible spectroscopy are used to determine the drug external binding modes, the binding constant and the stability of zanamivir-DNA and RNA complexes in aqueous solution. Structural analysis showed major interaction of zanamivir with G-C (major groove) and A-T (minor groove) base pairs and minor perturbations of the backbone PO 2 group with overall binding constants of Kzanamivir-DNA = 1.30 × 10 4 M -1 and Kzanamivir-RNA = 1.38 × 10 4 M -1. The drug interaction induces a partial B to A-DNA transition, while RNA remains in A-conformation.

Nasopharyngeal carcinoma heterogeneity of DNA content identified on cytologic preparations.

PubMed

Maohuai, C; Chang, A R; Lo, D

2001-06-01

To evaluate tumor heterogeneity of DNA content in nasopharyngeal carcinoma (NPC) performed on cytologic specimens. Image cytometric analysis of DNA ploidy status of 40 NPCs was performed on nasopharyngeal brushing smears stained with the Feulgen method after hematoxylin eosin staining. If the DNA distribution pattern from the same tumor exhibited diploid, aneuploid or/and tetraploid peaks or some combination of these patterns, the presence of tumor heterogeneity of DNA content was identified. Thirty-four cases (85%) had a nondiploid DNA pattern among the 40 NPCs. Twenty-eight cases exhibited tumor heterogeneity of DNA content (70%). Of the 28 tumors, 13 (46%) had a combination of diploid and tetraploid patterns, 10 (37%) had a combination of diploid and aneuploid patterns, 3 cases (11%) had a combination of tetraploid and aneuploid patterns, and 2 cases had two aneuploid stem lines. The relationship between DNA ploidy pattern and tumor histologic and cytologic morphology was also examined. There is a high incidence of DNA content heterogeneity in NPC. The relevance of tumor heterogeneity to the biologic behavior of NPC awaits further study. DNA quantification with image cytometry on destained cytologic preparations is feasible and reliable.

Promoter hypermethylation of the DNA repair gene O(6)-methylguanine-DNA methyltransferase is associated with the presence of G:C to A:T transition mutations in p53 in human colorectal tumorigenesis.

PubMed

Esteller, M; Risques, R A; Toyota, M; Capella, G; Moreno, V; Peinado, M A; Baylin, S B; Herman, J G

2001-06-15

Defects in DNA repair may be responsible for the genesis of mutations in key genes in cancer cells. The tumor suppressor gene p53 is commonly mutated in human cancer by missense point mutations, most of them G:C to A:T transitions. A recognized cause for this type of change is spontaneous deamination of the methylcytosine. However, the persistence of a premutagenic O(6)-methylguanine can also be invoked. This last lesion is removed in the normal cell by the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT). In many tumor types, epigenetic silencing of MGMT by promoter hypermethylation has been demonstrated and linked to the appearance of G to A mutations in the K-ras oncogene in colorectal tumors. To study the relevance of defective MGMT function by aberrant methylation in relation to the presence of p53 mutations, we studied 314 colorectal tumors for MGMT promoter hypermethylation and p53 mutational spectrum. Inactivation of MGMT by aberrant methylation was associated with the appearance of G:C to A:T transition mutations at p53 (Fischer's exact test, two-tailed; P = 0.01). Overall, MGMT methylated tumors displayed p53 transition mutations in 43 of 126 (34%) cases, whereas MGMT unmethylated tumors only showed G:C to A:T changes in 37 of 188 (19%) tumors. A more striking association was found in G:C to A:T transitions in non-CpG dinucleotides; 71% (12 of 17) of the total non-CpG transition mutations in p53 were observed in MGMT aberrantly methylated tumors (Fischer's exact test, two-tailed; P = 0.008). Our data suggest that epigenetic silencing of MGMT by promoter hypermethylation may lead to G:C to A:T transition mutations in p53.

Rapid DNA analysis for automated processing and interpretation of low DNA content samples.

PubMed

Turingan, Rosemary S; Vasantgadkar, Sameer; Palombo, Luke; Hogan, Catherine; Jiang, Hua; Tan, Eugene; Selden, Richard F

2016-01-01

Short tandem repeat (STR) analysis of casework samples with low DNA content include those resulting from the transfer of epithelial cells from the skin to an object (e.g., cells on a water bottle, or brim of a cap), blood spatter stains, and small bone and tissue fragments. Low DNA content (LDC) samples are important in a wide range of settings, including disaster response teams to assist in victim identification and family reunification, military operations to identify friend or foe, criminal forensics to identify suspects and exonerate the innocent, and medical examiner and coroner offices to identify missing persons. Processing LDC samples requires experienced laboratory personnel, isolated workstations, and sophisticated equipment, requires transport time, and involves complex procedures. We present a rapid DNA analysis system designed specifically to generate STR profiles from LDC samples in field-forward settings by non-technical operators. By performing STR in the field, close to the site of collection, rapid DNA analysis has the potential to increase throughput and to provide actionable information in real time. A Low DNA Content BioChipSet (LDC BCS) was developed and manufactured by injection molding. It was designed to function in the fully integrated Accelerated Nuclear DNA Equipment (ANDE) instrument previously designed for analysis of buccal swab and other high DNA content samples (Investigative Genet. 4(1):1-15, 2013). The LDC BCS performs efficient DNA purification followed by microfluidic ultrafiltration of the purified DNA, maximizing the quantity of DNA available for subsequent amplification and electrophoretic separation and detection of amplified fragments. The system demonstrates accuracy, precision, resolution, signal strength, and peak height ratios appropriate for casework analysis. The LDC rapid DNA analysis system is effective for the generation of STR profiles from a wide range of sample types. The technology broadens the range of sample

Ribosomal DNA analysis of tsetse and non-tsetse transmitted Ethiopian Trypanosoma vivax strains in view of improved molecular diagnosis.

PubMed

Fikru, Regassa; Matetovici, Irina; Rogé, Stijn; Merga, Bekana; Goddeeris, Bruno Maria; Büscher, Philippe; Van Reet, Nick

2016-04-15

Animal trypanosomosis caused by Trypanosoma vivax (T. vivax) is a devastating disease causing serious economic losses. Most molecular diagnostics for T. vivax infection target the ribosomal DNA locus (rDNA) but are challenged by the heterogeneity among T. vivax strains. In this study, we investigated the rDNA heterogeneity of Ethiopian T. vivax strains in relation to their presence in tsetse-infested and tsetse-free areas and its effect on molecular diagnosis. We sequenced the rDNA loci of six Ethiopian (three from tsetse-infested and three from tsetse-free areas) and one Nigerian T. vivax strain. We analysed the obtained sequences in silico for primer-mismatches of some commonly used diagnostic PCR assays and for GC content. With these data, we selected some rDNA diagnostic PCR assays for evaluation of their diagnostic accuracy. Furthermore we constructed two phylogenetic networks based on sequences within the smaller subunit (SSU) of 18S and within the 5.8S and internal transcribed spacer 2 (ITS2) to assess the relatedness of Ethiopian T. vivax strains to strains from other African countries and from South America. In silico analysis of the rDNA sequence showed important mismatches of some published diagnostic PCR primers and high GC content of T. vivax rDNA. The evaluation of selected diagnostic PCR assays with specimens from cattle under natural T. vivax challenge showed that this high GC content interferes with the diagnostic accuracy of PCR, especially in cases of mixed infections with T. congolense. Adding betain to the PCR reaction mixture can enhance the amplification of T. vivax rDNA but decreases the sensitivity for T. congolense and Trypanozoon. The networks illustrated that Ethiopian T. vivax strains are considerably heterogeneous and two strains (one from tsetse-infested and one from tsetse-free area) are more related to the West African and South American strains than to the East African strains. The rDNA locus sequence of six Ethiopian T. vivax

NullSeq: A Tool for Generating Random Coding Sequences with Desired Amino Acid and GC Contents.

PubMed

Liu, Sophia S; Hockenberry, Adam J; Lancichinetti, Andrea; Jewett, Michael C; Amaral, Luís A N

2016-11-01

The existence of over- and under-represented sequence motifs in genomes provides evidence of selective evolutionary pressures on biological mechanisms such as transcription, translation, ligand-substrate binding, and host immunity. In order to accurately identify motifs and other genome-scale patterns of interest, it is essential to be able to generate accurate null models that are appropriate for the sequences under study. While many tools have been developed to create random nucleotide sequences, protein coding sequences are subject to a unique set of constraints that complicates the process of generating appropriate null models. There are currently no tools available that allow users to create random coding sequences with specified amino acid composition and GC content for the purpose of hypothesis testing. Using the principle of maximum entropy, we developed a method that generates unbiased random sequences with pre-specified amino acid and GC content, which we have developed into a python package. Our method is the simplest way to obtain maximally unbiased random sequences that are subject to GC usage and primary amino acid sequence constraints. Furthermore, this approach can easily be expanded to create unbiased random sequences that incorporate more complicated constraints such as individual nucleotide usage or even di-nucleotide frequencies. The ability to generate correctly specified null models will allow researchers to accurately identify sequence motifs which will lead to a better understanding of biological processes as well as more effective engineering of biological systems.

Effect of water content on thermal oxidation of oleic acid investigated by combination of EPR spectroscopy and SPME-GC-MS/MS.

PubMed

Chen, Hongjian; Cao, Peirang; Li, Bo; Sun, Dewei; Wang, Yong; Li, Jinwei; Liu, Yuanfa

2017-04-15

Promotion of water to the thermal oxidation of oleic acid was detected by the combination of EPR, SPME-GC-MS/MS and GC. Spin-trapping technique was used to identify and quantify the radical species formed during thermal oxidation of oleic acid by using DMPO as electron spin trap. The most abundant radical species were identified as DMPO-alkyl radical adducts. EPR intensity plateau of the samples with 5% water content was 140% higher than the samples without water. It implies oleic acid samples with high water content had high level of oxidation rates. The proportion of aldehydes of the samples with 2% water content was the maximum about 59.97%. Among the formed products, (E,E)-2,4-decadienal has genotoxic and cytotoxic effects, whose percentage was nearly twice comparing with that of 5-0% water content. This study demonstrated that higher water content in frying systems would contribute to seriously oxidation and degradation of oleic acids. Copyright © 2016 Elsevier Ltd. All rights reserved.

Blood Cell Mitochondrial DNA Content and Premature Ovarian Aging

PubMed Central

Cacciatore, Chiara; Busnelli, Marta; Rossetti, Raffaella; Bonetti, Silvia; Paffoni, Alessio; Mari, Daniela; Ragni, Guido; Persani, Luca; Arosio, M.; Beck-Peccoz, P.; Biondi, M.; Bione, S.; Bruni, V.; Brigante, C.; Cannavo`, S.; Cavallo, L.; Cisternino, M.; Colombo, I.; Corbetta, S.; Crosignani, P.G.; D'Avanzo, M.G.; Dalpra, L.; Danesino, C.; Di Battista, E.; Di Prospero, F.; Donti, E.; Einaudi, S.; Falorni, A.; Foresta, C.; Fusi, F.; Garofalo, N.; Giotti, I.; Lanzi, R.; Larizza, D.; Locatelli, N.; Loli, P.; Madaschi, S.; Maghnie, M.; Maiore, S.; Mantero, F.; Marozzi, A.; Marzotti, S.; Migone, N.; Nappi, R.; Palli, D.; Patricelli, M.G.; Pisani, C.; Prontera, P.; Petraglia, F.; Radetti, G.; Renieri, A.; Ricca, I.; Ripamonti, A.; Rossetti, R.; Russo, G.; Russo, S.; Tonacchera, M.; Toniolo, D.; Torricelli, F.; Vegetti, W.; Villa, N.; Vineis, P.; Wasniewsk, M.; Zuffardi, O.

2012-01-01

Primary ovarian insufficiency (POI) is a critical fertility defect characterized by an anticipated and silent impairment of the follicular reserve, but its pathogenesis is largely unexplained. The frequent maternal inheritance of POI together with a remarkable dependence of ovarian folliculogenesis upon mitochondrial biogenesis and bioenergetics suggested the possible involvement of a generalized mitochondrial defect. Here, we verified the existence of a significant correlation between blood and ovarian mitochondrial DNA (mtDNA) content in a group of women undergoing ovarian hyperstimulation (OH), and then aimed to verify whether mtDNA content was significantly altered in the blood cells of POI women. We recruited 101 women with an impaired ovarian reserve: 59 women with premature ovarian failure (POF) and 42 poor responders (PR) to OH. A Taqman copy number assay revealed a significant mtDNA depletion (P<0.001) in both POF and PR women in comparison with 43 women of similar age and intact ovarian reserve, or 53 very old women with a previous physiological menopause. No pathogenic variations in the mitochondrial DNA polymerase γ (POLG) gene were detected in 57 POF or PR women with low blood mtDNA content. In conclusion, blood cell mtDNA depletion is a frequent finding among women with premature ovarian aging, suggesting that a still undetermined but generalized mitochondrial defect may frequently predispose to POI which could then be considered a form of anticipated aging in which the ovarian defect may represent the first manifestation. The determination of mtDNA content in blood may become an useful tool for the POI risk prediction. PMID:22879975

Blood cell mitochondrial DNA content and premature ovarian aging.

PubMed

Bonomi, Marco; Somigliana, Edgardo; Cacciatore, Chiara; Busnelli, Marta; Rossetti, Raffaella; Bonetti, Silvia; Paffoni, Alessio; Mari, Daniela; Ragni, Guido; Persani, Luca

2012-01-01

Primary ovarian insufficiency (POI) is a critical fertility defect characterized by an anticipated and silent impairment of the follicular reserve, but its pathogenesis is largely unexplained. The frequent maternal inheritance of POI together with a remarkable dependence of ovarian folliculogenesis upon mitochondrial biogenesis and bioenergetics suggested the possible involvement of a generalized mitochondrial defect. Here, we verified the existence of a significant correlation between blood and ovarian mitochondrial DNA (mtDNA) content in a group of women undergoing ovarian hyperstimulation (OH), and then aimed to verify whether mtDNA content was significantly altered in the blood cells of POI women. We recruited 101 women with an impaired ovarian reserve: 59 women with premature ovarian failure (POF) and 42 poor responders (PR) to OH. A Taqman copy number assay revealed a significant mtDNA depletion (P<0.001) in both POF and PR women in comparison with 43 women of similar age and intact ovarian reserve, or 53 very old women with a previous physiological menopause. No pathogenic variations in the mitochondrial DNA polymerase γ (POLG) gene were detected in 57 POF or PR women with low blood mtDNA content. In conclusion, blood cell mtDNA depletion is a frequent finding among women with premature ovarian aging, suggesting that a still undetermined but generalized mitochondrial defect may frequently predispose to POI which could then be considered a form of anticipated aging in which the ovarian defect may represent the first manifestation. The determination of mtDNA content in blood may become an useful tool for the POI risk prediction.

[Under what conditions does G.C Watson-Crick DNA base pair acquire all four configurations characteristic for A.T Watson-Crick DNA base pair?].

PubMed

Brovarets', O O

2013-01-01

At the MP2/6-311++G(2df,pd)//B3LYP/6-311++G(d,p) level of theory it was established for the first time, that the Löwdin's G*.C* DNA base pair formed by the mutagenic tautomers can acquire, as the A-T Watson-Crick DNA base pair, four biologically important configurations, namely: Watson-Crick, reverse Watson-Crick, Hoogsteen and reverse Hoogsteen. This fact demonstrates rather unexpected role of the tautomerisation of the one of the Watson-Crick DNA base pairs, in particular, via double proton transfer: exactly the G.C-->G*.C* tautomerisation allows to overcome steric hindrances for the implementation of the above mentioned configurations. Geometric, electron-topological and energetic properties of the H-bonds that stabilise the studied pairs, as well as the energetic characteristics of the latters are presented.

Variation in nuclear DNA content in Malus species and cultivated apples.

PubMed

Tatum, Tatiana C; Stepanovic, Svetlana; Biradar, D P; Rayburn, A Lane; Korban, Schuyler S

2005-10-01

The nuclear DNA content for a group of 40 Malus species and hybrids has been estimated using flow cytometry. Estimates of nuclear DNA content for this germplasm collection range from 1.45 pg for Malus fusca (diploid) to 2.57 pg for Malus ioensis (triploid). Among diploids, the nuclear (2C) DNA ranges from 1.45 pg for M. fusca to 1.68 pg for Malus transitoria. Among triploids, the nuclear (3C) DNA content ranges from 2.37 pg / 3C for Malus sikkimensis to 2.57 pg / 3C for M. ioensis. Given the complexity of the apple genome and its suggested allopolyploid origin, the results obtained in this study confirm earlier reports that polyploids can easily withstand the loss of a certain amount of DNA, and that there is a slight tendency towards diminished haploid nuclear DNA content with increased polyploidy.

Diatom centromeres suggest a mechanism for nuclear DNA acquisition

DOE PAGES

Diner, Rachel E.; Noddings, Chari M.; Lian, Nathan C.; ...

2017-07-18

Centromeres are essential for cell division and growth in all eukaryotes, and knowledge of their sequence and structure guides the development of artificial chromosomes for functional cellular biology studies. Centromeric proteins are conserved among eukaryotes; however, centromeric DNA sequences are highly variable. We combined forward and reverse genetic approaches with chromatin immunoprecipitation to identify centromeres of the model diatom Phaeodactylum tricornutum. We observed 25 unique centromere sequences typically occurring once per chromosome, a finding that helps to resolve nuclear genome organization and indicates monocentric regional centromeres. Diatom centromere sequences contain low-GC content regions but lack repeats or other conserved sequencemore » features. Native and foreign sequences with similar GC content to P. tricornutum centromeres can maintain episomes and recruit the diatom centromeric histone protein CENH3, suggesting nonnative sequences can also function as diatom centromeres. Thus, simple sequence requirements may enable DNA from foreign sources to persist in the nucleus as extrachromosomal episomes, revealing a potential mechanism for organellar and foreign DNA acquisition.« less

Diatom centromeres suggest a mechanism for nuclear DNA acquisition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Diner, Rachel E.; Noddings, Chari M.; Lian, Nathan C.

Centromeres are essential for cell division and growth in all eukaryotes, and knowledge of their sequence and structure guides the development of artificial chromosomes for functional cellular biology studies. Centromeric proteins are conserved among eukaryotes; however, centromeric DNA sequences are highly variable. We combined forward and reverse genetic approaches with chromatin immunoprecipitation to identify centromeres of the model diatom Phaeodactylum tricornutum. We observed 25 unique centromere sequences typically occurring once per chromosome, a finding that helps to resolve nuclear genome organization and indicates monocentric regional centromeres. Diatom centromere sequences contain low-GC content regions but lack repeats or other conserved sequencemore » features. Native and foreign sequences with similar GC content to P. tricornutum centromeres can maintain episomes and recruit the diatom centromeric histone protein CENH3, suggesting nonnative sequences can also function as diatom centromeres. Thus, simple sequence requirements may enable DNA from foreign sources to persist in the nucleus as extrachromosomal episomes, revealing a potential mechanism for organellar and foreign DNA acquisition.« less

GC-rich coding sequences reduce transposon-like, small RNA-mediated transgene silencing.

PubMed

Sidorenko, Lyudmila V; Lee, Tzuu-Fen; Woosley, Aaron; Moskal, William A; Bevan, Scott A; Merlo, P Ann Owens; Walsh, Terence A; Wang, Xiujuan; Weaver, Staci; Glancy, Todd P; Wang, PoHao; Yang, Xiaozeng; Sriram, Shreedharan; Meyers, Blake C

2017-11-01

The molecular basis of transgene susceptibility to silencing is poorly characterized in plants; thus, we evaluated several transgene design parameters as means to reduce heritable transgene silencing. Analyses of Arabidopsis plants with transgenes encoding a microalgal polyunsaturated fatty acid (PUFA) synthase revealed that small RNA (sRNA)-mediated silencing, combined with the use of repetitive regulatory elements, led to aggressive transposon-like silencing of canola-biased PUFA synthase transgenes. Diversifying regulatory sequences and using native microalgal coding sequences (CDSs) with higher GC content improved transgene expression and resulted in a remarkable trans-generational stability via reduced accumulation of sRNAs and DNA methylation. Further experiments in maize with transgenes individually expressing three crystal (Cry) proteins from Bacillus thuringiensis (Bt) tested the impact of CDS recoding using different codon bias tables. Transgenes with higher GC content exhibited increased transcript and protein accumulation. These results demonstrate that the sequence composition of transgene CDSs can directly impact silencing, providing design strategies for increasing transgene expression levels and reducing risks of heritable loss of transgene expression.

Developmental stage related patterns of codon usage and genomic GC content: searching for evolutionary fingerprints with models of stem cell differentiation

PubMed Central

2007-01-01

Background The usage of synonymous codons shows considerable variation among mammalian genes. How and why this usage is non-random are fundamental biological questions and remain controversial. It is also important to explore whether mammalian genes that are selectively expressed at different developmental stages bear different molecular features. Results In two models of mouse stem cell differentiation, we established correlations between codon usage and the patterns of gene expression. We found that the optimal codons exhibited variation (AT- or GC-ending codons) in different cell types within the developmental hierarchy. We also found that genes that were enriched (developmental-pivotal genes) or specifically expressed (developmental-specific genes) at different developmental stages had different patterns of codon usage and local genomic GC (GCg) content. Moreover, at the same developmental stage, developmental-specific genes generally used more GC-ending codons and had higher GCg content compared with developmental-pivotal genes. Further analyses suggest that the model of translational selection might be consistent with the developmental stage-related patterns of codon usage, especially for the AT-ending optimal codons. In addition, our data show that after human-mouse divergence, the influence of selective constraints is still detectable. Conclusion Our findings suggest that developmental stage-related patterns of gene expression are correlated with codon usage (GC3) and GCg content in stem cell hierarchies. Moreover, this paper provides evidence for the influence of natural selection at synonymous sites in the mouse genome and novel clues for linking the molecular features of genes to their patterns of expression during mammalian ontogenesis. PMID:17349061

Hippophae leaf extract concentration regulates antioxidant and prooxidant effects on DNA.

PubMed

Saini, Manu; Tiwari, Sandhya; Prasad, Jagdish; Singh, Surender; Kumar, M S Yogendra; Bala, Madhu

2010-03-01

Extracts from Hippophae leaves constitute some commonly consumed beverages such as tea and wine. We had developed an extract of Hippophae leaves (SBL-1), which was rich in quercetin, had antimutagenic effects, radioprotective effects, and countered radiation-induced gene conversion in Saccharomyces cerevisiae. This study was designed to investigate the action of SBL-1 on guanine cytosine (GC)-rich nascent and mouse genomic DNA in vitro. The human and mouse liver DNA have about 43% GC content. Our results showed that at small concentration SBL-1 protected nascent as well as genomic DNA, while at large concentration SBL-1 damaged both types of DNA. The concentration of SBL-1 that protected DNA also demonstrated higher free radical scavenging activity. The reducing power of SBL-1 was greater than its free radical scavenging activity. The greater reducing power may have reduced the trace metals present in the SBL-1, leading to generation of hydroxyl radicals via Fenton reaction. The increased proportion of unscavenged hydroxyl radicals with increase in SBL-1 concentration may have been responsible for DNA damage or prooxidant effect of SBL-1 in vitro. This study suggests that the dietary supplements prepared from Hippophae should have low metal content.

Nuclear DNA contents of Echinchloa crus-galli and its Gaussian relationships with environments

NASA Astrophysics Data System (ADS)

Li, Dan-Dan; Lu, Yong-Liang; Guo, Shui-Liang; Yin, Li-Ping; Zhou, Ping; Lou, Yu-Xia

2017-02-01

Previous studies on plant nuclear DNA content variation and its relationships with environmental gradients produced conflicting results. We speculated that the relationships between nuclear DNA content of a widely-distributed species and its environmental gradients might be non-linear if it was sampled in a large geographical gradient. Echinochloa crus-galli (L.) P. Beauv. is a worldwide species, but without documents on its intraspecific variation of nuclear DNA content. Our objectives are: 1) to detect intraspecific variation scope of E. crus-galli in its nuclear DNA content, and 2) to testify whether nuclear DNA content of the species changes with environmental gradients following Gaussian models if its populations were sampled in a large geographical gradient. We collected seeds of 36 Chinese populations of E. crus-galli across a wide geographical gradient, and sowed them in a homogeneous field to get their offspring to determine their nuclear DNA content. We analyzed the relationships of nuclear DNA content of these populations with latitude, longitude, and nineteen bioclimatic variables by using Gaussian and linear models. (1) Nuclear DNA content varied from 2.113 to 2.410 pg among 36 Chinese populations of E. crus-galli, with a mean value of 2.256 pg. (2) Gaussian correlations of nuclear DNA content (y) with geographical gradients were detected, with latitude (x) following y = 2.2923*e -(x - 24.9360)2/2*63.79452 (r = 0.546, P < 0.001), and with longitude (x) following y = 2.2933*e -(x - 116.1801)2/2*44.74502 (r = 0.672, P < 0.001). (3) Among the nineteen bioclimatic variables, except temperature isothermality, precipitations of the wettest month, the wettest quarter and the warmest quarter, the others could be better fit with nuclear DNA content by using Gaussian models than by linear models. There exists intra-specific variation among 36 Chinese populations of E. crus-galli, Gaussian models could be applied to fit the correlations of its Nuclear DNA content

A Laboratory Experiment To Measure Henry's Law Constants of Volatile Organic Compounds with a Bubble Column and a Gas Chromatography Flame Ionization Detector (GC-FID)

ERIC Educational Resources Information Center

Lee, Shan-Hu; Mukherjee, Souptik; Brewer, Brittany; Ryan, Raphael; Yu, Huan; Gangoda, Mahinda

2013-01-01

An undergraduate laboratory experiment is described to measure Henry's law constants of organic compounds using a bubble column and gas chromatography flame ionization detector (GC-FID). This experiment is designed for upper-division undergraduate laboratory courses and can be implemented in conjunction with physical chemistry, analytical…

DNA Asymmetric Strand Bias Affects the Amino Acid Composition of Mitochondrial Proteins

PubMed Central

Min, Xiang Jia; Hickey, Donal A.

2007-01-01

Abstract Variations in GC content between genomes have been extensively documented. Genomes with comparable GC contents can, however, still differ in the apportionment of the G and C nucleotides between the two DNA strands. This asymmetric strand bias is known as GC skew. Here, we have investigated the impact of differences in nucleotide skew on the amino acid composition of the encoded proteins. We compared orthologous genes between animal mitochondrial genomes that show large differences in GC and AT skews. Specifically, we compared the mitochondrial genomes of mammals, which are characterized by a negative GC skew and a positive AT skew, to those of flatworms, which show the opposite skews for both GC and AT base pairs. We found that the mammalian proteins are highly enriched in amino acids encoded by CA-rich codons (as predicted by their negative GC and positive AT skews), whereas their flatworm orthologs were enriched in amino acids encoded by GT-rich codons (also as predicted from their skews). We found that these differences in mitochondrial strand asymmetry (measured as GC and AT skews) can have very large, predictable effects on the composition of the encoded proteins. PMID:17974594

DNA methylation detection based on difference of base content

NASA Astrophysics Data System (ADS)

Sato, Shinobu; Ohtsuka, Keiichi; Honda, Satoshi; Sato, Yusuke; Takenaka, Shigeori

2016-04-01

Methylation frequently occurs in cytosines of CpG sites to regulate gene expression. The identification of aberrant methylation of certain genes is important for cancer marker analysis. The aim of this study was to determine the methylation frequency in DNA samples of unknown length and/or concentration. Unmethylated cytosine is known to be converted to thymine following bisulfite treatment and subsequent PCR. For this reason, the AT content in DNA increases with an increasing number of methylation sites. In this study, the fluorescein-carrying bis-acridinyl peptide (FKA) molecule was used for the detection of methylation frequency. FKA contains fluorescein and two acridine moieties, which together allow for the determination of the AT content of double-stranded DNA fragments. Methylated and unmethylated human genomes were subjected to bisulfide treatment and subsequent PCR using primers specific for the CFTR, CDH4, DBC1, and NPY genes. The AT content in the resulting PCR products was estimated by FKA, and AT content estimations were found to be in good agreement with those determined by DNA sequencing. This newly developed method may be useful for determining methylation frequencies of many PCR products by measuring the fluorescence in samples excited at two different wavelengths.

Direct Determination of the Base-Pair Force Constant of DNA from the Acoustic Phonon Dispersion of the Double Helix

NASA Astrophysics Data System (ADS)

van Eijck, L.; Merzel, F.; Rols, S.; Ollivier, J.; Forsyth, V. T.; Johnson, M. R.

2011-08-01

Quantifying the molecular elasticity of DNA is fundamental to our understanding of its biological functions. Recently different groups, through experiments on tailored DNA samples and numerical models, have reported a range of stretching force constants (0.3 to 3N/m). However, the most direct, microscopic measurement of DNA stiffness is obtained from the dispersion of its vibrations. A new neutron scattering spectrometer and aligned, wet spun samples have enabled such measurements, which provide the first data of collective excitations of DNA and yield a force constant of 83N/m. Structural and dynamic order persists unchanged to within 15 K of the melting point of the sample, precluding the formation of bubbles. These findings are supported by large scale phonon and molecular dynamics calculations, which reconcile hard and soft force constants.

Substantial Regional Variation in Substitution Rates in the Human Genome: Importance of GC Content, Gene Density, and Telomere-Specific Effects

NASA Astrophysics Data System (ADS)

Arndt, Peter F.; Hwa, Terence; Petrov, Dmitri A.

2005-06-01

This study presents the first global, 1 Mbp level analysis of patterns of nucleotide substitutions along the human lineage. The study is based on the analysis of a large amount of repetitive elements deposited into the human genome since the mammalian radiation, yielding a number of results that would have been difficult to obtain using the more conventional comparative method of analysis. This analysis revealed substantial and consistent variability of rates of substitution, with the variability ranging up to 2-fold among different regions. The rates of substitutions of C or G nucleotides with A or T nucleotides vary much more sharply than the reverse rates suggesting that much of that variation is due to differences in mutation rates rather than in the probabilities of fixation of C/G vs. A/T nucleotides across the genome. For all types of substitution we observe substantially more hotspots than coldspots, with hotspots showing substantial clustering over tens of Mbp's. Our analysis revealed that GC-content of surrounding sequences is the best predictor of the rates of substitution. The pattern of substitution appears very different near telomeres compared to the rest of the genome and cannot be explained by the genome-wide correlations of the substitution rates with GC content or exon density. The telomere pattern of substitution is consistent with natural selection or biased gene conversion acting to increase the GC-content of the sequences that are within 10-15 Mbp away from the telomere.

Conformational Heterogeneity in a Fully Complementary DNA Three-Way Junction with a GC-Rich Branchpoint.

PubMed

Toulmin, Anita; Baltierra-Jasso, Laura E; Morten, Michael J; Sabir, Tara; McGlynn, Peter; Schröder, Gunnar F; Smith, Brian O; Magennis, Steven W

2017-09-19

DNA three-way junctions (3WJs) are branched structures that serve as important biological intermediates and as components in DNA nanostructures. We recently derived the global structure of a fully complementary 3WJ and found that it contained unpaired bases at the branchpoint, which is consistent with previous observations of branch flexibility and branchpoint reactivity. By combining high-resolution single-molecule Förster resonance energy transfer, molecular modeling, time-resolved ensemble fluorescence spectroscopy, and the first 19 F nuclear magnetic resonance observations of fully complementary 3WJs, we now show that the 3WJ structure can adopt multiple distinct conformations depending upon the sequence at the branchpoint. A 3WJ with a GC-rich branchpoint adopts an open conformation with unpaired bases at the branch and at least one additional conformation with an increased number of base interactions at the branchpoint. This structural diversity has implications for branch interactions and processing in vivo and for technological applications.

Immunotherapy of HIV-infected patients with Gc protein-derived macrophage activating factor (GcMAF).

PubMed

Yamamoto, Nobuto; Ushijima, Naofumi; Koga, Yoshihiko

2009-01-01

Serum Gc protein (known as vitamin D3-binding protein) is the precursor for the principal macrophage activating factor (MAF). The MAF precursor activity of serum Gc protein of HIV-infected patients was lost or reduced because Gc protein is deglycosylated by alpha-N-acetylgalactosaminidase (Nagalase) secreted from HIV-infected cells. Therefore, macrophages of HIV-infected patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Since Nagalase is the intrinsic component of the envelope protein gp120, serum Nagalase activity is the sum of enzyme activities carried by both HIV virions and envelope proteins. These Nagalase carriers were already complexed with anti-HIV immunoglobulin G (IgG) but retained Nagalase activity that is required for infectivity. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent macrophage activating factor (termed GcMAF), which produces no side effects in humans. Macrophages activated by administration of 100 ng GcMAF develop a large amount of Fc-receptors as well as an enormous variation of receptors that recognize IgG-bound and unbound HIV virions. Since latently HIV-infected cells are unstable and constantly release HIV virions, the activated macrophages rapidly intercept the released HIV virions to prevent reinfection resulting in exhaustion of infected cells. After less than 18 weekly administrations of 100 ng GcMAF for nonanemic patients, they exhibited low serum Nagalase activities equivalent to healthy controls, indicating eradication of HIV-infection, which was also confirmed by no infectious center formation by provirus inducing agent-treated patient PBMCs. No recurrence occurred and their healthy CD + cell counts were maintained for 7 years.

Mesoscopic modeling of DNA denaturation rates: Sequence dependence and experimental comparison

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dahlen, Oda, E-mail: oda.dahlen@ntnu.no; Erp, Titus S. van, E-mail: titus.van.erp@ntnu.no

Using rare event simulation techniques, we calculated DNA denaturation rate constants for a range of sequences and temperatures for the Peyrard-Bishop-Dauxois (PBD) model with two different parameter sets. We studied a larger variety of sequences compared to previous studies that only consider DNA homopolymers and DNA sequences containing an equal amount of weak AT- and strong GC-base pairs. Our results show that, contrary to previous findings, an even distribution of the strong GC-base pairs does not always result in the fastest possible denaturation. In addition, we applied an adaptation of the PBD model to study hairpin denaturation for which experimentalmore » data are available. This is the first quantitative study in which dynamical results from the mesoscopic PBD model have been compared with experiments. Our results show that present parameterized models, although giving good results regarding thermodynamic properties, overestimate denaturation rates by orders of magnitude. We believe that our dynamical approach is, therefore, an important tool for verifying DNA models and for developing next generation models that have higher predictive power than present ones.« less

Packaging of Dinoroseobacter shibae DNA into Gene Transfer Agent Particles Is Not Random

PubMed Central

Wang, Hui; Hall, April T K; Patzelt, Diana; Preusse, Matthias; Petersen, Jörn; Brinkmann, Henner; Bunk, Boyke; Bhuju, Sabin; Jarek, Michael; Geffers, Robert; Lang, Andrew S; Wagner-Döbler, Irene

2018-01-01

Abstract Gene transfer agents (GTAs) are phage-like particles which contain a fragment of genomic DNA of the bacterial or archaeal producer and deliver this to a recipient cell. GTA gene clusters are present in the genomes of almost all marine Rhodobacteraceae (Roseobacters) and might be important contributors to horizontal gene transfer in the world’s oceans. For all organisms studied so far, no obvious evidence of sequence specificity or other nonrandom process responsible for packaging genomic DNA into GTAs has been found. Here, we show that knock-out of an autoinducer synthase gene of Dinoroseobacter shibae resulted in overproduction and release of functional GTA particles (DsGTA). Next-generation sequencing of the 4.2-kb DNA fragments isolated from DsGTAs revealed that packaging was not random. DNA from low-GC conjugative plasmids but not from high-GC chromids was excluded from packaging. Seven chromosomal regions were strongly overrepresented in DNA isolated from DsGTA. These packaging peaks lacked identifiable conserved sequence motifs that might represent recognition sites for the GTA terminase complex. Low-GC regions of the chromosome, including the origin and terminus of replication, were underrepresented in DNA isolated from DsGTAs. DNA methylation reduced packaging frequency while the level of gene expression had no influence. Chromosomal regions found to be over- and underrepresented in DsGTA-DNA were regularly spaced. We propose that a “headful” type of packaging is initiated at the sites of coverage peaks and, after linearization of the chromosomal DNA, proceeds in both directions from the initiation site. GC-content, DNA-modifications, and chromatin structure might influence at which sides GTA packaging can be initiated. PMID:29325123

Reduced mitochondrial DNA content associates with poor prognosis of prostate cancer in African American men.

PubMed

Koochekpour, Shahriar; Marlowe, Timothy; Singh, Keshav K; Attwood, Kristopher; Chandra, Dhyan

2013-01-01

Reduction or depletion of mitochondrial DNA (mtDNA) has been associated with cancer progression. Although imbalanced mtDNA content is known to occur in prostate cancer, differences in mtDNA content between African American (AA) and Caucasian American (CA) men are not defined. We provide the first evidence that tumors in AA men possess reduced level of mtDNA compared to CA men. The median tumor mtDNA content was reduced in AA men. mtDNA content was also reduced in normal prostate tissues of AA men compared to CA men, suggesting a possible predisposition to cancer in AA men. mtDNA content was also reduced in benign prostatic hyperplasia (BPH) tissue from AA men. Tumor and BPH tissues from patients ≥ 60 years of age possess reduced mtDNA content compared to patients <60 years of age. In addition, mtDNA content was higher in normal tissues from patients with malignant T3 stage disease compared to patients with T2 stage disease. mtDNA levels in matched normal prostate tissues were nearly doubled in Gleason grade of >7 compared to ≤ 7, whereas reduced mtDNA content was observed in tumors of Gleason grade >7 compared to ≤ 7. Together, our data suggest that AA men possess lower mtDNA levels in normal and tumor tissues compared to CA men, which could contribute to higher risk and more aggressive prostate cancer in AA men.

HS-SPME/GC analysis reveals the population variability of terpene contents in Juniperus communis needles.

PubMed

Filipowicz, Natalia; Madanecki, Piotr; Gołebiowski, Marek; Stepnowski, Piotr; Ochocka, J Renata

2009-12-01

Juniperus communis var. communis L. is an aromatic plant - typical boreal element of flora. In the extensive literature concerning J. communis, there is much data on the composition and the content of essential oil of needles and coneberries, but a detailed analysis of terpene distribution within and between populations is missing. A representative pool of 74 J. communis individuals originating from ten populations of Northern Poland was investigated in order to evaluate the intra- and interpopulational variability of the terpene pattern. Headspace solid-phase microextraction (HS-SPME) coupled with GC/MS and GC/FID was applied in achiral and enantioselective analysis. The majority of the samples (85%), despite different origin, were similar in the terpene pattern. High diversity of terpenes was observed within the populations and low diversity between them. High variation of enantiomeric composition was in accordance with large variation of individual compounds in general (achiral analysis). J. communis samples from Northern Poland could be distinguished by the alpha-pinene/sabinene ratio, and they were divided into three chemical races.

Moisture content impacts the stability of DNA adsorbed onto gold microparticles.

PubMed

Smyth, Tyson J; Betker, Jamie; Wang, Wei; Anchordoquy, Thomas J

2011-11-01

Particle-mediated epidermal delivery (PMED) of small quantities of DNA (0.5-4.0 μg) has been reported to both induce an immune response and protect against disease in human subjects. In order for the PMED of DNA to be a viable technique for vaccination, the adsorbed DNA must be stable during shipping and storage. Here, we report that the storage stability of plasmid DNA adsorbed to 2-μm gold particles is strongly dependent on sample water content. Gold/DNA samples stored at 60°C and 6% relative humidity (RH) maintained supercoil content after 4-month storage, whereas storage at higher RHs facilitated degradation. Storage with desiccants had stabilizing effects on DNA similar to storage at 6% RH. However, storage with "indicating" Drierite and phosphorus pentoxide resulted in enhanced rates of DNA degradation. Copyright © 2011 Wiley-Liss, Inc.

Base compositions of genes encoding alpha-actin and lactate dehydrogenase-A from differently adapted vertebrates show no temperature-adaptive variation in G + C content.

PubMed

Ream, Rachael A; Johns, Glenn C; Somero, George N

2003-01-01

There is a long-standing debate in molecular evolution concerning the putative importance of GC content in adapting the thermal stabilities of DNA and RNA. Most studies of this relationship have examined broad-scale compositional patterns, for example, total GC percentages in genomes and occurrence of GC-rich isochores. Few studies have systematically examined the GC contents of individual orthologous genes from differently thermally adapted species. When this has been done, the emphasis has been on comparing large numbers of genes in only a few species. We have approached the GC-adaptation temperature hypothesis in a different manner by examining patterns of base composition of genes encoding lactate dehydrogenase-A (ldh-a) and alpha-actin (alpha-actin) from 51 species of vertebrates whose adaptation temperatures ranged from -1.86 degrees C (Antarctic fishes) to approximately 45 degrees C (desert reptile). No significant positive correlation was found between any index of GC content (GC content of the entire sequence, GC content of the third codon position [GC(3)], and GC content at fourfold degenerate sites [GC(4)]) and any index of adaptation temperature (maximal, mean, or minimal body temperature). For alpha-actin, slopes of regression lines for all comparisons did not differ significantly from zero. For ldh-a, negative correlations between adaptation temperature and total GC content, GC(3), and GC(4) were observed but were shown to be due entirely to phylogenetic influences (as revealed by independent contrast analyses). This comparison of GC content across a wide range of ectothermic ("cold-blooded") and endothermic ("warm-blooded") vertebrates revealed that frogs of the genus Xenopus, which have commonly been used as a representative cold-blooded species, in fact are outliers among ectotherms for the alpha-actin analyses, raising concern about the appropriateness of choosing these amphibians as representative of ectothermic vertebrates in general. Our study

DNA content analysis allows discrimination between Trypanosoma cruzi and Trypanosoma rangeli.

PubMed

Naves, Lucila Langoni; da Silva, Marcos Vinícius; Fajardo, Emanuella Francisco; da Silva, Raíssa Bernardes; De Vito, Fernanda Bernadelli; Rodrigues, Virmondes; Lages-Silva, Eliane; Ramírez, Luis Eduardo; Pedrosa, André Luiz

2017-01-01

Trypanosoma cruzi, a human protozoan parasite, is the causative agent of Chagas disease. Currently the species is divided into six taxonomic groups. The genome of the CL Brener clone has been estimated to be 106.4-110.7 Mb, and DNA content analyses revealed that it is a diploid hybrid clone. Trypanosoma rangeli is a hemoflagellate that has the same reservoirs and vectors as T. cruzi; however, it is non-pathogenic to vertebrate hosts. The haploid genome of T. rangeli was previously estimated to be 24 Mb. The parasitic strains of T. rangeli are divided into KP1(+) and KP1(-). Thus, the objective of this study was to investigate the DNA content in different strains of T. cruzi and T. rangeli by flow cytometry. All T. cruzi and T. rangeli strains yielded cell cycle profiles with clearly identifiable G1-0 (2n) and G2-M (4n) peaks. T. cruzi and T. rangeli genome sizes were estimated using the clone CL Brener and the Leishmania major CC1 as reference cell lines because their genome sequences have been previously determined. The DNA content of T. cruzi strains ranged from 87,41 to 108,16 Mb, and the DNA content of T. rangeli strains ranged from 63,25 Mb to 68,66 Mb. No differences in DNA content were observed between KP1(+) and KP1(-) T. rangeli strains. Cultures containing mixtures of the epimastigote forms of T. cruzi and T. rangeli strains resulted in cell cycle profiles with distinct G1 peaks for strains of each species. These results demonstrate that DNA content analysis by flow cytometry is a reliable technique for discrimination between T. cruzi and T. rangeli isolated from different hosts.

Packaging of Dinoroseobacter shibae DNA into Gene Transfer Agent Particles Is Not Random.

PubMed

Tomasch, Jürgen; Wang, Hui; Hall, April T K; Patzelt, Diana; Preusse, Matthias; Petersen, Jörn; Brinkmann, Henner; Bunk, Boyke; Bhuju, Sabin; Jarek, Michael; Geffers, Robert; Lang, Andrew S; Wagner-Döbler, Irene

2018-01-01

Gene transfer agents (GTAs) are phage-like particles which contain a fragment of genomic DNA of the bacterial or archaeal producer and deliver this to a recipient cell. GTA gene clusters are present in the genomes of almost all marine Rhodobacteraceae (Roseobacters) and might be important contributors to horizontal gene transfer in the world's oceans. For all organisms studied so far, no obvious evidence of sequence specificity or other nonrandom process responsible for packaging genomic DNA into GTAs has been found. Here, we show that knock-out of an autoinducer synthase gene of Dinoroseobacter shibae resulted in overproduction and release of functional GTA particles (DsGTA). Next-generation sequencing of the 4.2-kb DNA fragments isolated from DsGTAs revealed that packaging was not random. DNA from low-GC conjugative plasmids but not from high-GC chromids was excluded from packaging. Seven chromosomal regions were strongly overrepresented in DNA isolated from DsGTA. These packaging peaks lacked identifiable conserved sequence motifs that might represent recognition sites for the GTA terminase complex. Low-GC regions of the chromosome, including the origin and terminus of replication, were underrepresented in DNA isolated from DsGTAs. DNA methylation reduced packaging frequency while the level of gene expression had no influence. Chromosomal regions found to be over- and underrepresented in DsGTA-DNA were regularly spaced. We propose that a "headful" type of packaging is initiated at the sites of coverage peaks and, after linearization of the chromosomal DNA, proceeds in both directions from the initiation site. GC-content, DNA-modifications, and chromatin structure might influence at which sides GTA packaging can be initiated. © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

[Features of binding of proflavine to DNA at different DNA-ligand concentration ratios].

PubMed

Berezniak, E G; gladkovskaia, N A; Khrebtova, A S; Dukhopel'nikov, E V; Zinchenko, A V

2009-01-01

The binding of proflavine to calf thymus DNA has been studied using the methods of differential scanning calorimetry and spectrophotometry. It was shown that proflavine can interact with DNA by at least 3 binding modes. At high DNA-ligand concentration ratios (P/D), proflavine intercalates into both GC- and AT-sites, with a preference to GC-rich sequences. At low P/D ratios proflavine interacts with DNA by the external binding mode. From spectrophotometric concentration dependences, the parameters of complexing of proflavine with DNA were calculated. Thermodynamic parameters of DNA melting were calculated from differential scanning calorimetry data.

Gc protein (vitamin D-binding protein): Gc genotyping and GcMAF precursor activity.

PubMed

Nagasawa, Hideko; Uto, Yoshihiro; Sasaki, Hideyuki; Okamura, Natsuko; Murakami, Aya; Kubo, Shinichi; Kirk, Kenneth L; Hori, Hitoshi

2005-01-01

The Gc protein (human group-specific component (Gc), a vitamin D-binding protein or Gc globulin), has important physiological functions that include involvement in vitamin D transport and storage, scavenging of extracellular G-actin, enhancement of the chemotactic activity of C5a for neutrophils in inflammation and macrophage activation (mediated by a GalNAc-modified Gc protein (GcMAF)). In this review, the structure and function of the Gc protein is focused on especially with regard to Gc genotyping and GcMAF precursor activity. A discussion of the research strategy "GcMAF as a target for drug discovery" is included, based on our own research.

Transfer of the high-GC cyclohexane carboxylate degradation pathway from Rhodopseudomonas palustris to Escherichia coli for production of biotin.

PubMed

Bernstein, Jeffrey R; Bulter, Thomas; Liao, James C

2008-01-01

This work demonstrates the transfer of the five-gene cyclohexane carboxylate (CHC) degradation pathway from the high-GC alphaproteobacterium Rhodopseudomonas palustris to Escherichia coli, a gammaproteobacterium. The degradation product of this pathway is pimeloyl-CoA, a key metabolite in E. coli's biotin biosynthetic pathway. This pathway is useful for biotin overproduction in E. coli; however, the expression of GC-rich genes is troublesome in this host. When the native R. palustris CHC degradation pathway is transferred to a DeltabioH pimeloyl-CoA auxotroph of E. coli, it is unable to complement growth in the presence of CHC. To overcome this expression problem we redesigned the operon with decreased GC content and removed stretches of high-GC intergenic DNA which comprise the 5' untranslated region of each gene, replacing these features with shorter low-GC sequences. We show this synthetic construct enables growth of the DeltabioH strain in the presence of CHC. When the synthetic degradation pathway is overexpressed in conjunction with the downstream genes for biotin biosynthesis, we measured significant accumulation of biotin in the growth medium, showing that the pathway transfer is successfully integrated with the host metabolism.

High leukocyte mitochondrial DNA content contributes to poor prognosis in glioma patients through its immunosuppressive effect

PubMed Central

Chen, Y; Zhang, J; Huang, X; Zhang, J; Zhou, X; Hu, J; Li, G; He, S; Xing, J

2015-01-01

Background: Epidemiological studies have indicated significant associations of leukocyte mitochondrial DNA (mtDNA) copy number with risk of several malignancies, including glioma. However, whether mtDNA content can predict the clinical outcome of glioma patients has not been investigated. Methods: The mtDNA content of peripheral blood leukocytes from 336 glioma patients was examined using a real-time PCR-based method. Kaplan–Meier curves and Cox proportional hazards regression model were used to examine the association of mtDNA content with overall survival (OS) and progression-free survival (PFS) of patients. To explore the potential mechanism, the immune phenotypes of peripheral blood mononuclear cells (PBMCs) and plasma concentrations of several cytokines from another 20 glioma patients were detected by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. Results: Patients with high mtDNA content showed both poorer OS and PFS than those with low mtDNA content. Multivariate Cox regression analysis demonstrated that mtDNA content was an independent prognostic factor for both OS and PFS. Stratified analyses showed that high mtDNA content was significantly associated with poor prognosis of patients with younger age, high-grade glioma or adjuvant radiochemotherapy. Immunological analysis indicated that patients with high mtDNA content had significantly lower frequency of natural killer cells in PBMCs and higher plasma concentrations of interleukin-2 and tumour necrosis factor-α, suggesting an immunosuppression-related mechanism involved in mtDNA-mediated prognosis. Conclusions: Our study for the first time demonstrated that leukocyte mtDNA content could serve as an independent prognostic marker and an indicator of immune functions in glioma patients. PMID:26022928

Reduction of wobble-position GC bases in Corynebacteria genes and enhancement of PCR and heterologous expression.

PubMed

Sanli, G; Blaber, S I; Blaber, M

2001-01-01

Corynebacteria codon usage exhibits an overall GC content of 67%, and a wobble-position GC content of 88%. Escherichia coli, on the other hand has an overall GC content of 51%, and a wobble-position GC content of 55%. The high GC content of Corynebacteria genes results in an unfavorable codon preference for heterologous expression, and can present difficulties for polymerase-based manipulations due to secondary-structure effects. Since these characteristics are due primarily to base composition at the wobble-position, synthetic genes can, in principle, be designed to eliminate these problems and retain the wild-type amino acid sequence. Such genes would obviate the need for special additives or bases during in vitro polymerase-based manipulation and mutant host strains containing uncommon tRNA's for heterologous expression. We have evaluated synthetic genes with reduced wobble-position G/C content using two variants of the enzyme 2,5-diketo-D-gluconic acid reductase (2,5-DKGR A and B) from Corynebacterium. The wild-type genes are refractory to polymerase-based manipulations and exhibit poor heterologous expression in enteric bacteria. The results indicate that a subset of codons for five amino acids (alanine, arginine, glutamate, glycine and valine) contribute the greatest contribution to reduction in G/C content at the wobble-position. Furthermore, changes in codons for two amino acids (leucine and proline) enhance bias for expression in enteric bacteria without affecting the overall G/C content. The synthetic genes are readily amplified using polymerase-based methodologies, and exhibit high levels of heterologous expression in E. coli.

Nuclear DNA Content Estimates in Multicellular Green, Red and Brown Algae: Phylogenetic Considerations

PubMed Central

KAPRAUN, DONALD F.

2005-01-01

• Background and Aims Multicellular eukaryotic algae are phylogenetically disparate. Nuclear DNA content estimates have been published for fewer than 1 % of the described species of Chlorophyta, Phaeophyta and Rhodophyta. The present investigation aims to summarize the state of our knowledge and to add substantially to our database of C-values for theses algae. • Methods The DNA-localizing fluorochrome DAPI (4′, 6-diamidino-2-phenylindole) and RBC (chicken erythrocyte) standard were used to estimate 2C values with static microspectrophotometry. • Key Results 2C DNA contents for 85 species of Chlorophyta range from 0·2–6·1 pg, excluding the highly polyploidy Charales and Desmidiales with DNA contents of up to 39·2 and 20·7 pg, respectively. 2C DNA contents for 111 species of Rhodophyta range from 0·1–2·8 pg, and for 44 species of Phaeophyta range from 0·2–1·8 pg. • Conclusions New availability of consensus higher-level molecular phylogenies provides a framework for viewing C-value data in a phylogenetic context. Both DNA content ranges and mean values are greater in taxa considered to be basal. It is proposed that the basal, ancestral genome in each algal group was quite small. Both mechanistic and ecological processes are discussed that could have produced the observed C-value ranges. PMID:15596456

[A DNA study of rat liver oligonucleosomes enriched by transcriptionally active genes during induction due to the administration of an amino acid mixture].

PubMed

Vardevanian, P O; Davtian, A M; Tiratsuian, S G; Vardevanian, A O

1990-01-01

A highly active fraction of rat liver oligonucleosome DNA has been isolated and studied by means of thermal denaturation after induction by amino acid mixture or hydrocortisone. A considerable redistribution of DNA content has been shown in sucrose gradient fractions during these forms of induction. The changes are revealed in melting temperature, differential melting profile of DNA, isolated from actively transcribed chromatine fractions. Analysis of melting profiles shows changes of GC content of oligonucleosome DNA, suggesting that there are differences in activation during two studied forms of induction.

Nuclear DNA Content Variation in Life History Phases of the Bonnemasoniaceae (Rhodophyta)

PubMed Central

Salvador Soler, Noemi; Gómez Garreta, Amelia; Ribera Siguan, Mª Antonia; Kapraun, Donald F.

2014-01-01

Nuclear DNA content in gametophytes and sporophytes or the prostrate phases of the following species of Bonnemaisoniaceae (Asparagopsis armata, Asparagopsis taxiformis, Bonnemaisonia asparagoides, Bonnemaisonia clavata and Bonnemaisonia hamifera) were estimated by image analysis and static microspectrophotometry using the DNA-localizing fluorochrome DAPI (4′, 6-diamidino-2-phenylindole, dilactate) and the chicken erythrocytes standard. These estimates expand on the Kew database of DNA nuclear content. DNA content values for 1C nuclei in the gametophytes (spermatia and vegetative cells) range from 0.5 pg to 0.8 pg, and for 2C nuclei in the sporophytes or the prostrate phases range from 1.15–1.7 pg. Although only the 2C and 4C values were observed in the sporophyte or the prostrate phase, in the vegetative cells of the gametophyte the values oscillated from 1C to 4C, showing the possible start of endopolyploidy. The results confirm the alternation of nuclear phases in these Bonnemaisoniaceae species, in those that have tetrasporogenesis, as well as those that have somatic meiosis. The availability of a consensus phylogenetic tree for Bonnemaisoniaceae has opened the way to determine evolutionary trends in DNA contents. Both the estimated genome sizes and the published chromosome numbers for Bonnemaisoniaceae suggest a narrow range of values consistent with the conservation of an ancestral genome. PMID:24465835

Nuclear DNA content variation in life history phases of the Bonnemasoniaceae (Rhodophyta).

PubMed

Salvador Soler, Noemi; Gómez Garreta, Amelia; Ribera Siguan, Ma Antonia; Kapraun, Donald F

2014-01-01

Nuclear DNA content in gametophytes and sporophytes or the prostrate phases of the following species of Bonnemaisoniaceae (Asparagopsis armata, Asparagopsis taxiformis, Bonnemaisonia asparagoides, Bonnemaisonia clavata and Bonnemaisonia hamifera) were estimated by image analysis and static microspectrophotometry using the DNA-localizing fluorochrome DAPI (4', 6-diamidino-2-phenylindole, dilactate) and the chicken erythrocytes standard. These estimates expand on the Kew database of DNA nuclear content. DNA content values for 1C nuclei in the gametophytes (spermatia and vegetative cells) range from 0.5 pg to 0.8 pg, and for 2C nuclei in the sporophytes or the prostrate phases range from 1.15-1.7 pg. Although only the 2C and 4C values were observed in the sporophyte or the prostrate phase, in the vegetative cells of the gametophyte the values oscillated from 1C to 4C, showing the possible start of endopolyploidy. The results confirm the alternation of nuclear phases in these Bonnemaisoniaceae species, in those that have tetrasporogenesis, as well as those that have somatic meiosis. The availability of a consensus phylogenetic tree for Bonnemaisoniaceae has opened the way to determine evolutionary trends in DNA contents. Both the estimated genome sizes and the published chromosome numbers for Bonnemaisoniaceae suggest a narrow range of values consistent with the conservation of an ancestral genome.

ITS1: a DNA barcode better than ITS2 in eukaryotes?

PubMed

Wang, Xin-Cun; Liu, Chang; Huang, Liang; Bengtsson-Palme, Johan; Chen, Haimei; Zhang, Jian-Hui; Cai, Dayong; Li, Jian-Qin

2015-05-01

A DNA barcode is a short piece of DNA sequence used for species determination and discovery. The internal transcribed spacer (ITS/ITS2) region has been proposed as the standard DNA barcode for fungi and seed plants and has been widely used in DNA barcoding analyses for other biological groups, for example algae, protists and animals. The ITS region consists of both ITS1 and ITS2 regions. Here, a large-scale meta-analysis was carried out to compare ITS1 and ITS2 from three aspects: PCR amplification, DNA sequencing and species discrimination, in terms of the presence of DNA barcoding gaps, species discrimination efficiency, sequence length distribution, GC content distribution and primer universality. In total, 85 345 sequence pairs in 10 major groups of eukaryotes, including ascomycetes, basidiomycetes, liverworts, mosses, ferns, gymnosperms, monocotyledons, eudicotyledons, insects and fishes, covering 611 families, 3694 genera, and 19 060 species, were analysed. Using similarity-based methods, we calculated species discrimination efficiencies for ITS1 and ITS2 in all major groups, families and genera. Using Fisher's exact test, we found that ITS1 has significantly higher efficiencies than ITS2 in 17 of the 47 families and 20 of the 49 genera, which are sample-rich. By in silico PCR amplification evaluation, primer universality of the extensively applied ITS1 primers was found superior to that of ITS2 primers. Additionally, shorter length of amplification product and lower GC content was discovered to be two other advantages of ITS1 for sequencing. In summary, ITS1 represents a better DNA barcode than ITS2 for eukaryotic species. © 2014 John Wiley & Sons Ltd.

Mitochondrial DNA content in breast cancer: Impact on in vitro and in vivo phenotype and patient prognosis

PubMed Central

Weerts, Marjolein J.A.; Sieuwerts, Anieta M.; Smid, Marcel; Look, Maxime P.; Foekens, John A.; Sleijfer, Stefan; Martens, John W.M.

2016-01-01

Reduced mitochondrial DNA (mtDNA) content in breast cancer cell lines has been associated with transition towards a mesenchymal phenotype, but its clinical consequences concerning breast cancer dissemination remain unidentified. Here, we aimed to clarify the link between mtDNA content and a mesenchymal phenotype and its relation to prognosis of breast cancer patients. We analyzed mtDNA content in 42 breast cancer cell lines and 207 primary breast tumor specimens using a combination of quantitative PCR and array-based copy number analysis. By associating mtDNA content with expression levels of genes involved in epithelial-to-mesenchymal transition (EMT) and with the intrinsic breast cancer subtypes, we could not identify a relation between low mtDNA content and mesenchymal properties in the breast cancer cell lines or in the primary breast tumors. In addition, we explored the relation between mtDNA content and prognosis in our cohort of primary breast tumor specimens that originated from patients with lymph node-negative disease who did not receive any (neo)adjuvant systemic therapy. When patients were divided based on the tumor quartile levels of mtDNA content, those in the lowest quarter (≤ 350 mtDNA molecules per cell) showed a poorer 10-year distant metastasis-free survival than patients with > 350 mtDNA molecules per cell (HR 0.50 [95% CI 0.29–0.87], P = 0.015). The poor prognosis was independent of established clinicopathological markers (HR 0.54 [95% CI 0.30–0.97], P = 0.038). We conclude that, despite a lack of evidence between mtDNA content and EMT, low mtDNA content might provide meaningful prognostic value for distant metastasis in breast cancer. PMID:27081694

Comparison of GC-MS, GC-MRM-MS, and GC × GC to characterise higher plant biomarkers in Tertiary oils and rock extracts

NASA Astrophysics Data System (ADS)

Eiserbeck, Christiane; Nelson, Robert K.; Grice, Kliti; Curiale, Joseph; Reddy, Christopher M.

2012-06-01

Higher plant biomarkers occur in various compound classes with an array of isomers that are challenging to separate and identify. Traditional one-dimensional (1D) gas chromatographic (GC) techniques achieved impressive results in the past, but have reached limitations in many cases. Comprehensive two-dimensional gas chromatography (GC × GC) either coupled to a flame ionization detector (GC × GC-FID) or time-of-flight mass spectrometer (GC × GC-TOFMS) is a powerful tool to overcome the challenges of 1D GC, such as the resolution of unresolved complex mixture (UCM). We studied a number of Tertiary, terrigenous oils, and source rocks from the Arctic and Southeast Asia, with special focus on angiosperm biomarkers, such as oleanoids and lupanoids. Different chromatographic separation and detection techniques such as traditional 1D GC-MS, metastable reaction monitoring (GC-MRM-MS), GC × GC-FID, and GC × GC-TOFMS are compared and applied to evaluate the differences and advantages in their performance for biomarker identification. The measured 22S/(22S + 22R) homohopane ratios for all applied techniques were determined and compare exceptionally well (generally between 2% and 10%). Furthermore, we resolved a variety of angiosperm-derived compounds that co-eluted using 1D GC techniques, demonstrating the superior separation power of GC × GC for these biomarkers, which indicate terrigenous source input and Cretaceous or younger ages. Samples of varying thermal maturity and biodegradation contain higher plant biomarkers from various stages of diagenesis and catagenesis, which can be directly assessed in a GC × GC chromatogram. The analysis of whole crude oils and rock extracts without loss in resolution enables the separation of unstable compounds that are prone to rearrangement (e.g. unsaturated triterpenoids such as taraxer-14-ene) when exposed to fractionation techniques like molecular sieving. GC × GC-TOFMS is particularly valuable for the successful separation of

Decreased Integrity, Content, and Increased Transcript Level of Mitochondrial DNA Are Associated with Keratoconus

PubMed Central

Hao, Xiao-Dan; Chen, Zhao-Li; Qu, Ming-Li; Zhao, Xiao-Wen; Li, Su-Xia; Chen, Peng

2016-01-01

Oxidative stress may play an important role in the pathogenesis of keratoconus (KC). Mitochondrial DNA (mtDNA) is involved in mitochondrial function, and the mtDNA content, integrity, and transcript level may affect the generation of reactive oxygen species (ROS) and be involved in the pathogenesis of KC. We designed a case-control study to research the relationship between KC and mtDNA integrity, content and transcription. One-hundred ninety-eight KC corneas and 106 normal corneas from Chinese patients were studied. Quantitative real-time PCR was used to measure the relative mtDNA content, transcript levels of mtDNA and related genes. Long-extension PCR was used to detect mtDNA damage. ROS, mitochondrial membrane potential and ATP were measured by respective assay kit, and Mito-Tracker Green was used to label the mitochondria. The relative mtDNA content of KC corneas was significantly lower than that of normal corneas (P = 9.19×10−24), possibly due to decreased expression of the mitochondrial transcription factor A (TFAM) gene (P = 3.26×10−3). In contrast, the transcript levels of mtDNA genes were significantly increased in KC corneas compared with normal corneas (NADH dehydrogenase subunit 1 [ND1]: P = 1.79×10−3; cytochrome c oxidase subunit 1 [COX1]: P = 1.54×10−3; NADH dehydrogenase subunit 1, [ND6]: P = 4.62×10−3). The latter may be the result of increased expression levels of mtDNA transcription-related genes mitochondrial RNA polymerase (POLRMT) (P = 2.55×10−4) and transcription factor B2 mitochondrial (TFB2M) (P = 7.88×10−5). KC corneas also had increased mtDNA damage (P = 3.63×10−10), higher ROS levels, and lower mitochondrial membrane potential and ATP levels compared with normal corneas. Decreased integrity, content and increased transcript level of mtDNA are associated with KC. These changes may affect the generation of ROS and play a role in the pathogenesis of KC. PMID:27783701

Interaction between DNA and Drugs Having Protonable Basic Groups: Characterization through Affinity Constants, Drug Release Kinetics, and Conformational Changes

PubMed Central

Alarcón, Liliana P.; Baena, Yolima; Manzo, Rubén H.

2017-01-01

This paper reports the in vitro characterization of the interaction between the phosphate groups of DNA and the protonated species of drugs with basic groups through the determination of the affinity constants, the reversibility of the interaction, and the effect on the secondary structure of the macromolecule. Affinity constants of the counterionic condensation DNA–drug were in the order of 106. The negative electrokinetic potential of DNA decreased with the increase of the proportion of loading drugs. The drugs were slowly released from the DNA–drug complexes and had release kinetics consistent with the high degree of counterionic condensation. The circular dichroism profile of DNA was not modified by complexation with atenolol, lidocaine, or timolol, but was significantly altered by the more lipophilic drugs benzydamine and propranolol, revealing modifications in the secondary structure of the DNA. The in vitro characterization of such interactions provides a physicochemical basis that would contribute to identify the effects of this kind of drugs in cellular cultures, as well as side effects observed under their clinical use. Moreover, this methodology could also be projected to the fields of intracellular DNA transfection and the use of DNA as a carrier of active drugs. PMID:28054999

Use of a bacterial expression vector to map the varicella-zoster virus major glycoprotein gene, gC.

PubMed Central

Ellis, R W; Keller, P M; Lowe, R S; Zivin, R A

1985-01-01

The genome of varicella-zoster virus (VZV) encodes at least three major glycoprotein genes. Among viral gene products, the gC gene products are the most abundant glycoproteins and induce a substantial humoral immune response (Keller et al., J. Virol. 52:293-297, 1984). We utilized two independent approaches to map the gC gene. Small fragments of randomly digested VZV DNA were inserted into a bacterial expression vector. Bacterial colonies transformed by this vector library were screened serologically for antigen expression with monoclonal antibodies to gC. Hybridization of the plasmid DNA from a gC antigen-positive clone revealed homology to the 3' end of the VZV Us segment. In addition, mRNA from VZV-infected cells was hybrid selected by a set of VZV DNA recombinant plasmids and translated in vitro, and polypeptide products were immunoprecipitated by convalescent zoster serum or by monoclonal antibodies to gC. This analysis revealed that the mRNA encoding a 70,000-dalton polypeptide precipitable by anti-gC antibodies mapped to the HindIII C fragment, which circumscribes the entire Us region. We conclude that the VZV gC glycoprotein gene maps to the 3' end of the Us region and is expressed as a 70,000-dalton primary translational product. These results are consistent with the recently reported DNA sequence of Us (A.J. Davison, EMBO J. 2:2203-2209, 1983). Furthermore, glycosylation appears not to be required for a predominant portion of the antigenicity of gC glycoproteins. We also report the tentative map assignments for eight other VZV primary translational products. Images PMID:2981365

Mitochondrial DNA content and 4977 bp deletion in unfertilized oocytes.

PubMed

Chan, C C W; Liu, V W S; Lau, E Y L; Yeung, W S B; Ng, E H Y; Ho, P C

2005-12-01

Previous studies analysing the incidences of mitochondrial DNA (mtDNA) deletions and mtDNA content in unfertilized oocytes in relation to donors' age have been controversial. The objective of the study was to compare these two parameters in unfertilized oocytes and relate them to the donors' age. Fifty-two women donated 155 unfertilized metaphase II (MII) oocytes. The incidence of 4977 bp deletion was 34.6%, and the mtDNA copy number was 598 350 +/- 265 862. Women >or=35 years of age had a significantly higher incidence of 4977 bp deletion, lower mtDNA copy number, higher FSH level and poorer ovarian response when compared with younger women. The mtDNA copy number was negatively correlated with the donor's age. The higher incidence of mtDNA deletion and lower mtDNA copy number in older women suggested that these two parameters may reflect ovarian ageing.

Thermodynamic contributions for the incorporation of GTA triplets within canonical TAT/TAT and C+GC/C+GC base-triplet stacks of DNA triplexes.

PubMed

Soto, Ana Maria; Marky, Luis A

2002-10-15

Nucleic acid triple helices may be used in the control of gene expression. One limitation of using triplex-forming oligonucleotides as therapeutic agents is that their target sequences are limited to homopurine tracts. To increase the repertoire of sequences that can be targeted, it has been postulated that a guanine can target a thymidine forming a stable GTA mismatch triplet. In this work, we have used a combination of optical and calorimetric techniques to determine thermodynamic unfolding profiles of two triplexes containing a single GTA triplet, d(A(3)TA(3)C(5)T(3)AT(3)C(5)T(3)GT(3)) (ATA) and d(AGTGAC(5)TCACTC(5)TCGCT) (GTG), and their control triplexes, d(A(7)C(5)T(7)C(5)T(7)) (TAT7) and d(AGAGAC(5)TCTCTC(5)TCTCT) (AG5T). In general, the presence of a GTA mismatch in DNA triplexes is destabilizing; however, this destabilization is greater when placed in a C(+)GC/C(+)GC base-triplet stack than between a TAT/TAT stack. These destabilizations are accompanied by a reduced unfolding enthalpy of approximately 10 kcal/mol, suggesting a decrease in the base stacking contributions surrounding the mismatch. Relative to their corresponding control triplexes, the folding of ATA is accompanied by a lower counterion uptake and a similar proton uptake, while GTG folding is accompanied by an increase in the counterion and proton uptakes. These effects are consistent with the observed decrease in stacking interactions. The overall results indicate that the main difficulty of targeting pyrimidine interruptions is that the decrease in stacking contributions, due to the incorporation of a GTA mismatch, affects the stability of the neighboring base triplets. This suggests that nucleotide analogues that increase the strength of these base-triplet stacks will result in a more effective targeting of pyrimidine interruptions.

Energy barriers and rates of tautomeric transitions in DNA bases: ab initio quantum chemical study.

PubMed

Basu, Soumalee; Majumdar, Rabi; Das, Gourab K; Bhattacharyya, Dhananjay

2005-12-01

Tautomeric transitions of DNA bases are proton transfer reactions, which are important in biology. These reactions are involved in spontaneous point mutations of the genetic material. In the present study, intrinsic reaction coordinates (IRC) analyses through ab initio quantum chemical calculations have been carried out for the individual DNA bases A, T, G, C and also A:T and G:C base pairs to estimate the kinetic and thermodynamic barriers using MP2/6-31G** method for tautomeric transitions. Relatively higher values of kinetic barriers (about 50-60 kcal/mol) have been observed for the single bases, indicating that tautomeric alterations of isolated single bases are quite unlikely. On the other hand, relatively lower values of the kinetic barriers (about 20-25 kcal/mol) for the DNA base pairs A:T and G:C clearly suggest that the tautomeric shifts are much more favorable in DNA base pairs than in isolated single bases. The unusual base pairing A':C, T':G, C':A or G':T in the daughter DNA molecule, resulting from a parent DNA molecule with tautomeric shifts, is found to be stable enough to result in a mutation. The transition rate constants for the single DNA bases in addition to the base pairs are also calculated by computing the free energy differences between the transition states and the reactants.

Independent effects of leaf growth and light on the development of the plastid and its DNA content in Zea species.

PubMed

Zheng, Qi; Oldenburg, Delene J; Bendich, Arnold J

2011-05-01

In maize (Zea mays L.), chloroplast development progresses from the basal meristem to the mature leaf tip, and light is required for maturation to photosynthetic competence. During chloroplast greening, it was found that chloroplast DNA (cpDNA) is extensively degraded, falling to undetectable levels in many individual chloroplasts for three maize cultivars, as well as Zea mexicana (the ancestor of cultivated maize) and the perennial species Zea diploperennis. In dark-grown maize seedlings, the proplastid-to-etioplast transition is characterized by plastid enlargement, cpDNA replication, and the retention of high levels of cpDNA. When dark-grown seedlings are transferred to white light, the DNA content per plastid increases slightly during the first 4 h of illumination and then declines rapidly to a minimum at 24 h during the etioplast-to-chloroplast transition. Plastid autofluorescence (from chlorophyll) continues to increase as cpDNA declines, whereas plastid size remains constant. It is concluded that the increase in cpDNA that accompanies plastid enlargement is a consequence of cell and leaf growth, rather than illumination, whereas light stimulates photosynthetic capacity and cpDNA instability. When cpDNA from total tissue was monitored by blot hybridization and real-time quantitative PCR, no decline following transfer from dark to light was observed. The lack of agreement between DNA per plastid and cpDNA per cell may be attributed to nupts (nuclear sequences of plastid origin).

Charge transport through DNA based electronic barriers

NASA Astrophysics Data System (ADS)

Patil, Sunil R.; Chawda, Vivek; Qi, Jianqing; Anantram, M. P.; Sinha, Niraj

2018-05-01

We report charge transport in electronic 'barriers' constructed by sequence engineering in DNA. Considering the ionization potentials of Thymine-Adenine (AT) and Guanine-Cytosine (GC) base pairs, we treat AT as 'barriers'. The effect of DNA conformation (A and B form) on charge transport is also investigated. Particularly, the effect of width of 'barriers' on hole transport is investigated. Density functional theory (DFT) calculations are performed on energy minimized DNA structures to obtain the electronic Hamiltonian. The quantum transport calculations are performed using the Landauer-Buttiker framework. Our main findings are contrary to previous studies. We find that a longer A-DNA with more AT base pairs can conduct better than shorter A-DNA with a smaller number of AT base pairs. We also find that some sequences of A-DNA can conduct better than a corresponding B-DNA with the same sequence. The counterions mediated charge transport and long range interactions are speculated to be responsible for counter-intuitive length and AT content dependence of conductance of A-DNA.

Superstatistical model of bacterial DNA architecture

NASA Astrophysics Data System (ADS)

Bogachev, Mikhail I.; Markelov, Oleg A.; Kayumov, Airat R.; Bunde, Armin

2017-02-01

Understanding the physical principles that govern the complex DNA structural organization as well as its mechanical and thermodynamical properties is essential for the advancement in both life sciences and genetic engineering. Recently we have discovered that the complex DNA organization is explicitly reflected in the arrangement of nucleotides depicted by the universal power law tailed internucleotide interval distribution that is valid for complete genomes of various prokaryotic and eukaryotic organisms. Here we suggest a superstatistical model that represents a long DNA molecule by a series of consecutive ~150 bp DNA segments with the alternation of the local nucleotide composition between segments exhibiting long-range correlations. We show that the superstatistical model and the corresponding DNA generation algorithm explicitly reproduce the laws governing the empirical nucleotide arrangement properties of the DNA sequences for various global GC contents and optimal living temperatures. Finally, we discuss the relevance of our model in terms of the DNA mechanical properties. As an outlook, we focus on finding the DNA sequences that encode a given protein while simultaneously reproducing the nucleotide arrangement laws observed from empirical genomes, that may be of interest in the optimization of genetic engineering of long DNA molecules.

Chemical determination of free radical-induced damage to DNA.

PubMed

Dizdaroglu, M

1991-01-01

Free radical-induced damage to DNA in vivo can result in deleterious biological consequences such as the initiation and promotion of cancer. Chemical characterization and quantitation of such DNA damage is essential for an understanding of its biological consequences and cellular repair. Methodologies incorporating the technique of gas chromatography/mass spectrometry (GC/MS) have been developed in recent years for measurement of free radical-induced DNA damage. The use of GC/MS with selected-ion monitoring (SIM) facilitates unequivocal identification and quantitation of a large number of products of all four DNA bases produced in DNA by reactions with hydroxyl radical, hydrated electron, and H atom. Hydroxyl radical-induced DNA-protein cross-links in mammalian chromatin, and products of the sugar moiety in DNA are also unequivocally identified and quantitated. The sensitivity and selectivity of the GC/MS-SIM technique enables the measurement of DNA base products even in isolated mammalian chromatin without the necessity of first isolating DNA, and despite the presence of histones. Recent results reviewed in this article demonstrate the usefulness of the GC/MS technique for chemical determination of free radical-induced DNA damage in DNA as well as in mammalian chromatin under a vast variety of conditions of free radical production.

Estimation of terpene content in loblolly pine biomass using a hybrid fast-GC and pyrolysis-molecular beam mass spectrometry method

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harman-Ware, Anne E.; Davis, Mark F.; Peter, Gary F.

Terpenes can be used as renewable fuels and chemicals and quantifying their presence in biomass is becoming increasingly important. A novel method was developed to rapidly quantify total diterpenoid resin acids using pyrolysis-molecular beam mass spectrometry (py-MBMS). Pine sapling monoterpenes and diterpenoids were extracted from wood using a 1:1 (v/v) mixture of hexane and acetone and analyses were performed before and after extraction to determine the extraction efficiency of the solvent system. The resulting extract was analyzed for total diterpenoid content using py-MBMS and was combined with total monoterpene content that was determined using a low thermal mass modular acceleratedmore » column heater (LTM MACH) fast-GC/FID to measure the terpene content present in pine saplings. Oleoresin extruded from larger pine trees was used to validate mass balance closure of the terpene content in the extract solvent.« less

Estimation of terpene content in loblolly pine biomass using a hybrid fast-GC and pyrolysis-molecular beam mass spectrometry method

DOE PAGES

Harman-Ware, Anne E.; Davis, Mark F.; Peter, Gary F.; ...

2017-01-16

Terpenes can be used as renewable fuels and chemicals and quantifying their presence in biomass is becoming increasingly important. A novel method was developed to rapidly quantify total diterpenoid resin acids using pyrolysis-molecular beam mass spectrometry (py-MBMS). Pine sapling monoterpenes and diterpenoids were extracted from wood using a 1:1 (v/v) mixture of hexane and acetone and analyses were performed before and after extraction to determine the extraction efficiency of the solvent system. The resulting extract was analyzed for total diterpenoid content using py-MBMS and was combined with total monoterpene content that was determined using a low thermal mass modular acceleratedmore » column heater (LTM MACH) fast-GC/FID to measure the terpene content present in pine saplings. Oleoresin extruded from larger pine trees was used to validate mass balance closure of the terpene content in the extract solvent.« less

Bacteriophage GC1, a Novel Tectivirus Infecting Gluconobacter Cerinus, an Acetic Acid Bacterium Associated with Wine-Making.

PubMed

Philippe, Cécile; Krupovic, Mart; Jaomanjaka, Fety; Claisse, Olivier; Petrel, Melina; le Marrec, Claire

2018-01-16

The Gluconobacter phage GC1 is a novel member of the Tectiviridae family isolated from a juice sample collected during dry white wine making. The bacteriophage infects Gluconobacter cerinus , an acetic acid bacterium which represents a spoilage microorganism during wine making, mainly because it is able to produce ethyl alcohol and transform it into acetic acid. Transmission electron microscopy revealed tail-less icosahedral particles with a diameter of ~78 nm. The linear double-stranded DNA genome of GC1 (16,523 base pairs) contains terminal inverted repeats and carries 36 open reading frames, only a handful of which could be functionally annotated. These encode for the key proteins involved in DNA replication (protein-primed family B DNA polymerase) as well as in virion structure and assembly (major capsid protein, genome packaging ATPase (adenosine triphosphatase) and several minor capsid proteins). GC1 is the first tectivirus infecting an alphaproteobacterial host and is thus far the only temperate tectivirus of gram-negative bacteria. Based on distinctive sequence and life-style features, we propose that GC1 represents a new genus within the Tectiviridae , which we tentatively named " Gammatectivirus ". Furthermore, GC1 helps to bridge the gap in the sequence space between alphatectiviruses and betatectiviruses.

Charge transport and ac response under light illumination in gate-modulated DNA molecular junctions.

PubMed

Zhang, Yan; Zhu, Wen-Huan; Ding, Guo-Hui; Dong, Bing; Wang, Xue-Feng

2015-05-22

Using a two-strand tight-binding model and within nonequilibrium Green's function approach, we study charge transport through DNA sequences (GC)NGC and (GC)1(TA)NTA (GC)3 sandwiched between two Pt electrodes. We show that at low temperature DNA sequence (GC)NGC exhibits coherent charge carrier transport at very small bias, since the highest occupied molecular orbital in the GC base pair can be aligned with the Fermi energy of the metallic electrodes by a gate voltage. A weak distance dependent conductance is found in DNA sequence (GC)1(TA)NTA (GC)3 with large NTA. Different from the mechanism of thermally induced hopping of charges proposed by the previous experiments, we find that this phenomenon is dominated by quantum tunnelling through discrete quantum well states in the TA base pairs. In addition, ac response of this DNA junction under light illumination is also investigated. The suppression of ac conductances of the left and right lead of DNA sequences at some particular frequencies is attributed to the excitation of electrons in the DNA to the lead Fermi surface by ac potential, or the excitation of electrons in deep DNA energy levels to partially occupied energy levels in the transport window. Therefore, measuring ac response of DNA junctions can reveal a wealth of information about the intrinsic dynamics of DNA molecules.

Using Electrophoretic Mobility Shift Assays to Measure Equilibrium Dissociation Constants: GAL4-p53 Binding DNA as a Model System

ERIC Educational Resources Information Center

Heffler, Michael A.; Walters, Ryan D.; Kugel, Jennifer F.

2012-01-01

An undergraduate biochemistry laboratory experiment is described that will teach students the practical and theoretical considerations for measuring the equilibrium dissociation constant (K[subscript D]) for a protein/DNA interaction using electrophoretic mobility shift assays (EMSAs). An EMSA monitors the migration of DNA through a native gel;…

Detection of virgin coconut oil adulteration with animal fats using quantitative cholesterol by GC × GC-TOF/MS analysis.

PubMed

Xu, Baocheng; Li, Peiwu; Ma, Fei; Wang, Xiuping; Matthäus, Bertrand; Chen, Ran; Yang, Qingqing; Zhang, Wen; Zhang, Qi

2015-07-01

A new method based on the cholesterol level was developed to detect the presence of animal fats in virgin coconut oil (VCO). In this study, the sterols in VCO and animal fats was separated using conventional one-dimensional gas chromatography (1D GC) and comprehensive two-dimensional gas chromatography (GC×GC). Compared with 1D GC, the GC×GC system could obtain a complete baseline separation of the sterol trimethylsilyl ethers derived from cholesterol and cholestanol, so that the cholesterol content in pure VCO and false VCO adulterated with animal fats could be accurately determined. Cholesterol, a main sterol found in animal fats, represented less than 5mg/kg of VCO. The study demonstrated that the determination of the cholesterol level in VCO could be used for reliable detection of the presence of lard, chicken fat, mutton tallow, beef tallow, or their mixture in VCO at a level as little as 0.25%. Copyright © 2015 Elsevier Ltd. All rights reserved.

On-line Analysis of Catalytic Reaction Products Using a High-Pressure Tandem Micro-reactor GC/MS.

PubMed

Watanabe, Atsushi; Kim, Young-Min; Hosaka, Akihiko; Watanabe, Chuichi; Teramae, Norio; Ohtani, Hajime; Kim, Seungdo; Park, Young-Kwon; Wang, Kaige; Freeman, Robert R

2017-01-01

When a GC/MS system is coupled with a pressurized reactor, the separation efficiency and the retention time are directly affected by the reactor pressure. To keep the GC column flow rate constant irrespective of the reaction pressure, a restrictor capillary tube and an open split interface are attached between the GC injection port and the head of a GC separation column. The capability of the attached modules is demonstrated for the on-line GC/MS analysis of catalytic reaction products of a bio-oil model sample (guaiacol), produced under a pressure of 1 to 3 MPa.

DNA content alterations in Tetrahymena pyriformis macronucleus after exposure to food preservatives sodium nitrate and sodium benzoate.

PubMed

Loutsidou, Ariadni C; Hatzi, Vasiliki I; Chasapis, C T; Terzoudi, Georgia I; Spiliopoulou, Chara A; Stefanidou, Maria E

2012-12-01

The toxicity, in terms of changes in the DNA content, of two food preservatives, sodium nitrate and sodium benzoate was studied on the protozoan Tetrahymena pyriformis using DNA image analysis technology. For this purpose, selected doses of both food additives were administered for 2 h to protozoa cultures and DNA image analysis of T. pyriformis nuclei was performed. The analysis was based on the measurement of the Mean Optical Density which represents the cellular DNA content. The results have shown that after exposure of the protozoan cultures to doses equivalent to ADI, a statistically significant increase in the macronuclear DNA content compared to the unexposed control samples was observed. The observed increase in the macronuclear DNA content is indicative of the stimulation of the mitotic process and the observed increase in MOD, accompanied by a stimulation of the protozoan proliferation activity is in consistence with this assumption. Since alterations at the DNA level such as DNA content and uncontrolled mitogenic stimulation have been linked with chemical carcinogenesis, the results of the present study add information on the toxicogenomic profile of the selected chemicals and may potentially lead to reconsideration of the excessive use of nitrates aiming to protect public health.

Flow cytometry reliability analysis and variations in sugarcane DNA content.

PubMed

Oliveira, A C L; Pasqual, M; Bruzi, A T; Pio, L A S; Mendonça, P M S; Soares, J D R

2015-06-29

The aim of this study was to evaluate the reliability of flow cytometry analysis and the use of this technique to differentiate species and varieties of sugarcane (Saccharum spp) according to their relative DNA content. We analyzed 16 varieties and three species belonging to this genus. To determine a reliable protocol, we evaluated three extraction buffers (LB01, Marie, and Tris·MgCl2), the presence and absence of RNase, six doses of propidium iodide (10, 15, 20, 25, and 30 μg), four periods of exposure to propidium iodide (0, 5, 10, and 20 min), and seven external reference standards (peas, beans, corn, radish, rye, soybean, and tomato) with reference to the coefficient of variation and the DNA content. For statistical analyses, we used the programs Sisvar(®) and Xlstat(®). We recommend using the Marie extraction buffer and at least 15 μg propidium iodide. The samples should not be analyzed immediately after the addition of propidium iodide. The use of RNase is optional, and tomato should be used as an external reference standard. The results show that sugarcane has a variable genome size (8.42 to 12.12 pg/2C) and the individuals analyzed could be separated into four groups according to their DNA content with relative equality in the genome sizes of the commercial varieties.

Deppdb--DNA electrostatic potential properties database: electrostatic properties of genome DNA.

PubMed

Osypov, Alexander A; Krutinin, Gleb G; Kamzolova, Svetlana G

2010-06-01

The electrostatic properties of genome DNA influence its interactions with different proteins, in particular, the regulation of transcription by RNA-polymerases. DEPPDB--DNA Electrostatic Potential Properties Database--was developed to hold and provide all available information on the electrostatic properties of genome DNA combined with its sequence and annotation of biological and structural properties of genome elements and whole genomes. Genomes in DEPPDB are organized on a taxonomical basis. Currently, the database contains all the completely sequenced bacterial and viral genomes according to NCBI RefSeq. General properties of the genome DNA electrostatic potential profile and principles of its formation are revealed. This potential correlates with the GC content but does not correspond to it exactly and strongly depends on both the sequence arrangement and its context (flanking regions). Analysis of the promoter regions for bacterial and viral RNA polymerases revealed a correspondence between the scale of these proteins' physical properties and electrostatic profile patterns. We also discovered a direct correlation between the potential value and the binding frequency of RNA polymerase to DNA, supporting the idea of the role of electrostatics in these interactions. This matches a pronounced tendency of the promoter regions to possess higher values of the electrostatic potential.

Hybridization and genome size evolution: timing and magnitude of nuclear DNA content increases in Helianthus homoploid hybrid species

PubMed Central

Baack, Eric J.; Whitney, Kenneth D.; Rieseberg, Loren H.

2008-01-01

Summary Hybridization and polyploidy can induce rapid genomic changes, including the gain or loss of DNA, but the magnitude and timing of such changes are not well understood. The homoploid hybrid system in Helianthus (three hybrid-derived species and their two parents) provides an opportunity to examine the link between hybridization and genome size changes in a replicated fashion. Flow cytometry was used to estimate the nuclear DNA content in multiple populations of three homoploid hybrid Helianthus species (Helianthus anomalus, Helianthus deserticola, and Helianthus paradoxus), the parental species (Helianthus annuus and Helianthus petiolaris), synthetic hybrids, and natural hybrid-zone populations. Results confirm that hybrid-derived species have 50% more nuclear DNA than the parental species. Despite multiple origins, hybrid species were largely consistent in their DNA content across populations, although H. deserticola showed significant interpopulation differences. First- and sixth-generation synthetic hybrids and hybrid-zone plants did not show an increase from parental DNA content. First-generation hybrids differed in DNA content according to the maternal parent. In summary, hybridization by itself does not lead to increased nuclear DNA content in Helianthus, and the evolutionary forces responsible for the repeated increases in DNA content seen in the hybrid-derived species remain mysterious. PMID:15998412

Nonneutral GC3 and retroelement codon mimicry in Phytophthora.

PubMed

Jiang, Rays H Y; Govers, Francine

2006-10-01

Phytophthora is a genus entirely comprised of destructive plant pathogens. It belongs to the Stramenopila, a unique branch of eukaryotes, phylogenetically distinct from plants, animals, or fungi. Phytophthora genes show a strong preference for usage of codons ending with G or C (high GC3). The presence of high GC3 in genes can be utilized to differentiate coding regions from noncoding regions in the genome. We found that both selective pressure and mutation bias drive codon bias in Phytophthora. Indicative for selection pressure is the higher GC3 value of highly expressed genes in different Phytophthora species. Lineage specific GC increase of noncoding regions is reminiscent of whole-genome mutation bias, whereas the elevated Phytophthora GC3 is primarily a result of translation efficiency-driven selection. Heterogeneous retrotransposons exist in Phytophthora genomes and many of them vary in their GC content. Interestingly, the most widespread groups of retroelements in Phytophthora show high GC3 and a codon bias that is similar to host genes. Apparently, selection pressure has been exerted on the retroelement's codon usage, and such mimicry of host codon bias might be beneficial for the propagation of retrotransposons.

Genetic stock assessment of spawning arctic cisco (Coregonus autumnalis) populations by flow cytometric determination of DNA content.

PubMed

Lockwood, S F; Bickham, J W

1991-01-01

Intraspecific variation in cellular DNA content was measured in five Coregonus autumnalis spawning populations from the Mackenzie River drainage, Canada, using flow cytometry. The rivers assayed were the Peel, Arctic Red, Mountain, Carcajou, and Liard rivers. DNA content was determined from whole blood preparations of fish from all rivers except the Carcajou, for which kidney tissue was used. DNA content measurements of kidney and blood preparations of the same fish from the Mountain River revealed statistically indistinguishable results. Mosaicism was found in blood preparations from the Peel, Arctic Red, Mountain, and Liard rivers, but was not observed in kidney tissue preparations from the Mountain or Carcajou rivers. The Liard River sample had significantly elevated mean DNA content relative to the other four samples; all other samples were statistically indistinguishable. Significant differences in mean DNA content among spawning stocks of a single species reinforces the need for adequate sample sizes of both individuals and populations when reporting "C" values for a particular species.

Can mutational GC-pressure create new linear B-cell epitopes in herpes simplex virus type 1 glycoprotein B?

PubMed

Khrustalev, Vladislav Victorovich

2009-01-01

We showed that GC-content of nucleotide sequences coding for linear B-cell epitopes of herpes simplex virus type 1 (HSV1) glycoprotein B (gB) is higher than GC-content of sequences coding for epitope-free regions of this glycoprotein (G + C = 73 and 64%, respectively). Linear B-cell epitopes have been predicted in HSV1 gB by BepiPred algorithm ( www.cbs.dtu.dk/services/BepiPred ). Proline is an acrophilic amino acid residue (it is usually situated on the surface of protein globules, and so included in linear B-cell epitopes). Indeed, the level of proline is much higher in predicted epitopes of gB than in epitope-free regions (17.8% versus 1.8%). This amino acid is coded by GC-rich codons (CCX) that can be produced due to nucleotide substitutions caused by mutational GC-pressure. GC-pressure will also lead to disappearance of acrophobic phenylalanine, isoleucine, methionine and tyrosine coded by GC-poor codons. Results of our "in-silico directed mutagenesis" showed that single nonsynonymous substitutions in AT to GC direction in two long epitope-free regions of gB will cause formation of new linear epitopes or elongation of previously existing epitopes flanking these regions in 25% of 539 possible cases. The calculations of GC-content and amino acid content have been performed by CodonChanges algorithm ( www.barkovsky.hotmail.ru ).

Characterization of species-specific repeated DNA sequences from B. nigra.

PubMed

Gupta, V; Lakshmisita, G; Shaila, M S; Jagannathan, V; Lakshmikumaran, M S

1992-07-01

The construction and characterization of two genome-specific recombinant DNA clones from B. nigra are described. Southern analysis showed that the two clones belong to a dispersed repeat family. They differ from each other in their length, distribution and sequence, though the average GC content is nearly the same (45%). These B genome-specific repeats have been used to analyse the phylogenetic relationships between cultivated and wild species of the family Brassicaceae.

Analysis of Alkaloids from Physalis peruviana by Capillary GC, Capillary GC-MS, and GC-FTIR.

PubMed

Kubwabo, C; Rollmann, B; Tilquin, B

1993-04-01

The alkaloid composition of the aerial parts and roots of PHYSALIS PERUVIANA was analysed by capillary GC (GC (2)), GC (2)-MS and GC (2)-FTIR. Eight alkaloids were identified, three of those alkaloids are 3beta-acetoxytropane and two N-methylpyrrolidinylhygrine isomers, which were not previously found in the genus PHYSALIS. A reproduction of the identification of alkaloids detected in the plant by the use of retention indices has been proposed.

Soil Bacterial Community Shift Correlated with Change from Forest to Pasture Vegetation in a Tropical Soil

PubMed Central

Nüsslein, Klaus; Tiedje, James M.

1999-01-01

The change in vegetative cover of a Hawaiian soil from forest to pasture led to significant changes in the composition of the soil bacterial community. DNAs were extracted from both soil habitats and compared for the abundance of guanine-plus-cytosine (G+C) content, by analysis of abundance of phylotypes of small-subunit ribosomal DNA (SSU rDNA) amplified from fractions with 63 and 35% G+C contents, and by phylogenetic analysis of the dominant rDNA clones in the 63% G+C content fraction. All three methods showed differences between the forest and pasture habitats, providing evidence that vegetation had a strong influence on microbial community composition at three levels of taxon resolution. The forest soil DNA had a peak in G+C content of 61%, while the DNA of the pasture soil had a peak in G+C content of 67%. None of the dominant phylotypes found in the forest soil were detected in the pasture soil. For the 63% G+C fraction SSU rDNA sequence analysis of the three most dominant members revealed that their phyla changed from Fibrobacter and Syntrophomonas assemblages in the forest soil to Burkholderia and Rhizobium–Agrobacterium assemblages in the pasture soil. PMID:10427058

DNA barcoding Australia's fish species

PubMed Central

Ward, Robert D; Zemlak, Tyler S; Innes, Bronwyn H; Last, Peter R; Hebert, Paul D.N

2005-01-01

Two hundred and seven species of fish, mostly Australian marine fish, were sequenced (barcoded) for a 655 bp region of the mitochondrial cytochrome oxidase subunit I gene (cox1). Most species were represented by multiple specimens, and 754 sequences were generated. The GC content of the 143 species of teleosts was higher than the 61 species of sharks and rays (47.1% versus 42.2%), largely due to a higher GC content of codon position 3 in the former (41.1% versus 29.9%). Rays had higher GC than sharks (44.7% versus 41.0%), again largely due to higher GC in the 3rd codon position in the former (36.3% versus 26.8%). Average within-species, genus, family, order and class Kimura two parameter (K2P) distances were 0.39%, 9.93%, 15.46%, 22.18% and 23.27%, respectively. All species could be differentiated by their cox1 sequence, although single individuals of each of two species had haplotypes characteristic of a congener. Although DNA barcoding aims to develop species identification systems, some phylogenetic signal was apparent in the data. In the neighbour-joining tree for all 754 sequences, four major clusters were apparent: chimaerids, rays, sharks and teleosts. Species within genera invariably clustered, and generally so did genera within families. Three taxonomic groups—dogfishes of the genus Squalus, flatheads of the family Platycephalidae, and tunas of the genus Thunnus—were examined more closely. The clades revealed after bootstrapping generally corresponded well with expectations. Individuals from operational taxonomic units designated as Squalus species B through F formed individual clades, supporting morphological evidence for each of these being separate species. We conclude that cox1 sequencing, or ‘barcoding’, can be used to identify fish species. PMID:16214743

Strong spurious transcription likely contributes to DNA insert bias in typical metagenomic clone libraries.

PubMed

Lam, Kathy N; Charles, Trevor C

2015-01-01

Clone libraries provide researchers with a powerful resource to study nucleic acid from diverse sources. Metagenomic clone libraries in particular have aided in studies of microbial biodiversity and function, and allowed the mining of novel enzymes. Libraries are often constructed by cloning large inserts into cosmid or fosmid vectors. Recently, there have been reports of GC bias in fosmid metagenomic libraries, and it was speculated to be a result of fragmentation and loss of AT-rich sequences during cloning. However, evidence in the literature suggests that transcriptional activity or gene product toxicity may play a role. To explore possible mechanisms responsible for sequence bias in clone libraries, we constructed a cosmid library from a human microbiome sample and sequenced DNA from different steps during library construction: crude extract DNA, size-selected DNA, and cosmid library DNA. We confirmed a GC bias in the final cosmid library, and we provide evidence that the bias is not due to fragmentation and loss of AT-rich sequences but is likely occurring after DNA is introduced into Escherichia coli. To investigate the influence of strong constitutive transcription, we searched the sequence data for promoters and found that rpoD/σ(70) promoter sequences were underrepresented in the cosmid library. Furthermore, when we examined the genomes of taxa that were differentially abundant in the cosmid library relative to the original sample, we found the bias to be more correlated with the number of rpoD/σ(70) consensus sequences in the genome than with simple GC content. The GC bias of metagenomic libraries does not appear to be due to DNA fragmentation. Rather, analysis of promoter sequences provides support for the hypothesis that strong constitutive transcription from sequences recognized as rpoD/σ(70) consensus-like in E. coli may lead to instability, causing loss of the plasmid or loss of the insert DNA that gives rise to the transcription. Despite

Cytological study of DNA content and nuclear morphometric analysis for aid in the diagnosis of high-grade dysplasia within oral leukoplakia.

PubMed

Yang, Xi; Xiao, Xuan; Wu, Wenyan; Shen, Xuemin; Zhou, Zengtong; Liu, Wei; Shi, Linjun

2017-09-01

To quantitatively examine the DNA content and nuclear morphometric status of oral leukoplakia (OL) and investigate its association with the degree of dysplasia in a cytologic study. Oral cytobrush biopsy was carried out to obtain exfoliative epithelial cells from lesions before scalpel biopsy at the same location in a blinded series of 70 patients with OL. Analysis of nuclear morphometry and DNA content status using image cytometry was performed with oral smears stained with the Feulgen-thionin method. Nuclear morphometric analysis revealed significant differences in DNA content amount, DNA index, nuclear area, nuclear radius, nuclear intensity, sphericity, entropy, and fractal dimension (all P < .01) between low-grade and high-grade dysplasia. DNA content analysis identified 34 patients with OL (48.6%) with DNA content abnormality. Nonhomogeneous lesion (P = .018) and high-grade dysplasia (P = .008) were significantly associated with abnormal DNA content. Importantly, the positive correlation between the degree of oral dysplasia and DNA content status was significant (P = .004, correlation coefficient = 0.342). Cytology analysis of DNA content and nuclear morphometric status using image cytometry may support their use as a screening and monitoring tool for OL progression. Copyright © 2017 Elsevier Inc. All rights reserved.

Nuclear DNA Content Variation and Species Relationships in the Genus Lupinus (Fabaceae)

PubMed Central

NAGANOWSKA, BARBARA; WOLKO, BOGDAN; ŚLIWIŃSKA, ELWIRA; KACZMAREK, ZYGMUNT

2003-01-01

The 2C nuclear DNA content has been estimated by flow cytometry in 18 species and botanical forms of the genus Lupinus (family Fabaceae), using propidium iodide as a fluorescent dye. They represented distinct infrageneric taxonomic groups and differed in somatic chromosome numbers. Estimated 2C DNA values ranged from 0·97 pg in L. princei to 2·44 pg in L. luteus, which gives a more than 2·5-fold variation. Statistical analysis of the data obtained resulted in a grouping that supports the generally accepted taxonomic classification of the Old World lupins. The rough-seeded L. princei turned out to be an interesting exception, getting closer to smooth-seeded species. Results of DNA content analyses are discussed with regards to the phylogenetic relationships among the Old World lupins and some aspects of the evolution of the genus. PMID:12853281

DNA content of hepatocyte and erythrocyte nuclei of the spined loach (Cobitis taenia L.) and its polyploid forms.

PubMed

Juchno, Dorota; Lackowska, Bozena; Boron, Alicja; Kilarski, Wincenty

2010-09-01

We analyzed the DNA content of hepatocyte and erythrocyte nuclei of the spined loach Cobitis taenia (diploid) and its allopolyploid forms. Twenty triploid females and one tetraploid were used. At least 20,000 hepatocyte and erythrocyte nuclei were acquired and analyzed by flow cytometry. C. taenia erythrocyte nuclei contain 3.15 +/- 0.21 pg of DNA and the hepatocyte nuclei 4.45 +/- 0.46 pg of DNA. Triploid Cobitis have 5.08 +/- 0.41 pg of DNA in erythrocyte nuclei and 6.11 +/- 0.40 pg of DNA in hepatocyte nuclei, whereas the tetraploid erythrocyte and hepatocyte nuclei contained 6.60 and 7.40 pg of DNA, respectively. In general, the DNA contents correlate positively with the ploidy level of the fish investigated. The DNA content variation in the hepatocyte and erythrocyte nuclei may be due to differences in extent of chromatin condensation, which is more pronounced in the erythrocyte than hepatocyte nuclei, or to the several orders of ploidy that occur in the parenchymal liver cells.

Transcriptome Analysis of Core Dinoflagellates Reveals a Universal Bias towards "GC" Rich Codons.

PubMed

Williams, Ernest; Place, Allen; Bachvaroff, Tsvetan

2017-04-27

Although dinoflagellates are a potential source of pharmaceuticals and natural products, the mechanisms for regulating and producing these compounds are largely unknown because of extensive post-transcriptional control of gene expression. One well-documented mechanism for controlling gene expression during translation is codon bias, whereby specific codons slow or even terminate protein synthesis. Approximately 10,000 annotatable genes from fifteen "core" dinoflagellate transcriptomes along a range of overall guanine and cytosine (GC) content were used for codonW analysis to determine the relative synonymous codon usage (RSCU) and the GC content at each codon position. GC bias in the analyzed dataset and at the third codon position varied from 51% and 54% to 66% and 88%, respectively. Codons poor in GC were observed to be universally absent, but bias was most pronounced for codons ending in uracil followed by adenine (UA). GC bias at the third codon position was able to explain low abundance codons as well as the low effective number of codons. Thus, we propose that a bias towards codons rich in GC bases is a universal feature of core dinoflagellates, possibly relating to their unique chromosome structure, and not likely a major mechanism for controlling gene expression.

qPCR-based mitochondrial DNA quantification: Influence of template DNA fragmentation on accuracy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jackson, Christopher B., E-mail: Christopher.jackson@insel.ch; Gallati, Sabina, E-mail: sabina.gallati@insel.ch; Schaller, Andre, E-mail: andre.schaller@insel.ch

2012-07-06

Highlights: Black-Right-Pointing-Pointer Serial qPCR accurately determines fragmentation state of any given DNA sample. Black-Right-Pointing-Pointer Serial qPCR demonstrates different preservation of the nuclear and mitochondrial genome. Black-Right-Pointing-Pointer Serial qPCR provides a diagnostic tool to validate the integrity of bioptic material. Black-Right-Pointing-Pointer Serial qPCR excludes degradation-induced erroneous quantification. -- Abstract: Real-time PCR (qPCR) is the method of choice for quantification of mitochondrial DNA (mtDNA) by relative comparison of a nuclear to a mitochondrial locus. Quantitative abnormal mtDNA content is indicative of mitochondrial disorders and mostly confines in a tissue-specific manner. Thus handling of degradation-prone bioptic material is inevitable. We established a serialmore » qPCR assay based on increasing amplicon size to measure degradation status of any DNA sample. Using this approach we can exclude erroneous mtDNA quantification due to degraded samples (e.g. long post-exicision time, autolytic processus, freeze-thaw cycles) and ensure abnormal DNA content measurements (e.g. depletion) in non-degraded patient material. By preparation of degraded DNA under controlled conditions using sonification and DNaseI digestion we show that erroneous quantification is due to the different preservation qualities of the nuclear and the mitochondrial genome. This disparate degradation of the two genomes results in over- or underestimation of mtDNA copy number in degraded samples. Moreover, as analysis of defined archival tissue would allow to precise the molecular pathomechanism of mitochondrial disorders presenting with abnormal mtDNA content, we compared fresh frozen (FF) with formalin-fixed paraffin-embedded (FFPE) skeletal muscle tissue of the same sample. By extrapolation of measured decay constants for nuclear DNA ({lambda}{sub nDNA}) and mtDNA ({lambda}{sub mtDNA}) we present an approach to possibly correct

Structural evolution of nrDNA ITS in Pinaceae and its phylogenetic implications.

PubMed

Kan, Xian-Zhao; Wang, Shan-Shan; Ding, Xin; Wang, Xiao-Quan

2007-08-01

Nuclear ribosomal DNA (nrDNA) has been considered as an important tool for inferring phylogenetic relationships at many taxonomic levels. In comparison with its fast concerted evolution in angiosperms, nrDNA is symbolized by slow concerted evolution and substantial ITS region length variation in gymnosperms, particularly in Pinaceae. Here we studied structure characteristics, including subrepeat composition, size, GC content and secondary structure, of nrDNA ITS regions of all Pinaceae genera. The results showed that the ITS regions of all taxa studied contained subrepeat units, ranging from 2 to 9 in number, and these units could be divided into two types, longer subrepeat (LSR) without the motif (5'-GGCCACCCTAGTC) and shorter subrepeat (SSR) with the motif. Phylogenetic analyses indicate that the homology of some SSRs still can be recognized, providing important informations for the evolutionary history of nrDNA ITS and phylogeny of Pinaceae. In particular, the adjacent tandem SSRs are not more closely related to one another than they are to remote SSRs in some genera, which may imply that multiple structure variations such as recombination have occurred in the ITS1 region of these groups. This study also found that GC content in the ITS1 region is relevant to its sequence length and subrepeat number, and could provide some phylogenetic information, especially supporting the close relationships among Picea, Pinus, and Cathaya. Moreover, several characteristics of the secondary structure of Pinaceae ITS1 were found as follows: (1) the structure is dominated by several extended hairpins; (2) the configuration complexity is positively correlated with subrepeat number; (3) paired subrepeats often partially overlap at the conserved motif (5'-GGCCACCCTAGTC), and form a long stem, while other subrepeats fold onto itself, leaving part of the conserved motif exposed in hairpin loops.

Effect of fiber content on the thermal conductivity and dielectric constant of hair fiber reinforced epoxy composite

NASA Astrophysics Data System (ADS)

Prasad Nanda, Bishnu; Satapathy, Alok

2018-03-01

This paper reports on the dielectric and thermal properties of hair fibers reinforced epoxy composites. Hair is an important part of human body which also offers protection to the human body. It is also viewed as a biological waste which is responsible for creating environmental pollution due to its low decomposition rate. But at the same time it has unique microstructural, mechanical and thermal properties. In the present work, epoxy composites are made by solution casting method with different proportions of short hair fiber (SHF). Effects of fiber content on the thermal conductivity and dielectric constant of epoxy resin are studied. Thermal conductivities of the composites are obtained using a UnithermTM Model 2022 tester. An HIOKI-3532-50 Hi Tester Elsier Analyzer is used for measuring the capacitance of the epoxy-SHF composite, from which dielectric constant (Dk) of the composite are calculated. A reduction in thermal conductivity of the composite is noticed with the increase in wt. % of fiber. The dielectric constant value of the composites also found to be significantly affected by the fiber content.

Identifying sensitive windows for prenatal particulate air pollution exposure and mitochondrial DNA content in cord blood.

PubMed

Rosa, Maria José; Just, Allan C; Guerra, Marco Sánchez; Kloog, Itai; Hsu, Hsiao-Hsien Leon; Brennan, Kasey J; García, Adriana Mercado; Coull, Brent; Wright, Rosalind J; Téllez Rojo, Martha María; Baccarelli, Andrea A; Wright, Robert O

2017-01-01

Changes in mitochondrial DNA (mtDNA) can serve as a marker of cumulative oxidative stress (OS) due to the mitochondria's unique genome and relative lack of repair systems. In utero particulate matter ≤2.5μm (PM 2.5 ) exposure can enhance oxidative stress. Our objective was to identify sensitive windows to predict mtDNA damage experienced in the prenatal period due to PM 2.5 exposure using mtDNA content measured in cord blood. Women affiliated with the Mexican social security system were recruited during pregnancy in the Programming Research in Obesity, Growth, Environment and Social Stressors (PROGRESS) study. Mothers with cord blood collected at delivery and complete covariate data were included (n=456). Mothers' prenatal daily exposure to PM 2.5 was estimated using a satellite-based spatio-temporally resolved prediction model and place of residence during pregnancy. DNA was extracted from umbilical cord leukocytes. Quantitative real-time polymerase chain reaction (qPCR) was used to determine mtDNA content. A distributive lag regression model (DLM) incorporating weekly averages of daily PM 2.5 predictions was constructed to plot the association between exposure and OS over the length of pregnancy. In models that included child's sex, mother's age at delivery, prenatal environmental tobacco smoke exposure, birth year, maternal education, and assay batch, we found significant associations between higher PM 2.5 exposure during late pregnancy (35-40weeks) and lower mtDNA content in cord blood. Increased PM 2.5 during a specific prenatal window in the third trimester was associated with decreased mtDNA content suggesting heightened sensitivity to PM-induced OS during this life stage. Copyright © 2016 Elsevier Ltd. All rights reserved.

Gc protein-derived macrophage activating factor (GcMAF): isoelectric focusing pattern and tumoricidal activity.

PubMed

Mohamad, Saharuddin Bin; Nagasawa, Hideko; Sasaki, Hideyuki; Uto, Yoshihiro; Nakagawa, Yoshinori; Kawashima, Ken; Hori, Hitoshi

2003-01-01

Gc protein is the precursor for Gc protein-derived macrophage activating factor (GcMAF), with three phenotypes: Gc1f, Gc1s and Gc2, based on its electrophoretic mobility. The difference in electrophoretic mobility is because of the difference in its posttranslational sugar moiety composition. We compared the difference between Gc protein and GcMAF electrophoretic mobility using the isoelectric focusing (IEF) method. The tumoricidal activity of GcMAF-treated macrophage was evaluated after coculture with L-929 cell. The tumoricidal mechanism was investigated using TNF bioassay and nitric oxide (NO) release. The difference in Gc protein and GcMAF electrophoretic mobility was detected. The tumoricidal activity of GcMAF-treated macrophage was detected, but no release of TNF and NO was detected. The difference of isoelectric focusing mobility in Gc protein and GcMAF would be useful to develop a GcMAF detection method. GcMAF increased macrophage tumoricidal activity but TNF and NO release were not involved in the mechanism.

Higher success rate with transcranial electrical stimulation of motor-evoked potentials using constant-voltage stimulation compared with constant-current stimulation in patients undergoing spinal surgery.

PubMed

Shigematsu, Hideki; Kawaguchi, Masahiko; Hayashi, Hironobu; Takatani, Tsunenori; Iwata, Eiichiro; Tanaka, Masato; Okuda, Akinori; Morimoto, Yasuhiko; Masuda, Keisuke; Tanaka, Yuu; Tanaka, Yasuhito

2017-10-01

During spine surgery, the spinal cord is electrophysiologically monitored via transcranial electrical stimulation of motor-evoked potentials (TES-MEPs) to prevent injury. Transcranial electrical stimulation of motor-evoked potential involves the use of either constant-current or constant-voltage stimulation; however, there are few comparative data available regarding their ability to adequately elicit compound motor action potentials. We hypothesized that the success rates of TES-MEP recordings would be similar between constant-current and constant-voltage stimulations in patients undergoing spine surgery. The objective of this study was to compare the success rates of TES-MEP recordings between constant-current and constant-voltage stimulation. This is a prospective, within-subject study. Data from 100 patients undergoing spinal surgery at the cervical, thoracic, or lumbar level were analyzed. The success rates of the TES-MEP recordings from each muscle were examined. Transcranial electrical stimulation with constant-current and constant-voltage stimulations at the C3 and C4 electrode positions (international "10-20" system) was applied to each patient. Compound muscle action potentials were bilaterally recorded from the abductor pollicis brevis (APB), deltoid (Del), abductor hallucis (AH), tibialis anterior (TA), gastrocnemius (GC), and quadriceps (Quad) muscles. The success rates of the TES-MEP recordings from the right Del, right APB, bilateral Quad, right TA, right GC, and bilateral AH muscles were significantly higher using constant-voltage stimulation than those using constant-current stimulation. The overall success rates with constant-voltage and constant-current stimulations were 86.3% and 68.8%, respectively (risk ratio 1.25 [95% confidence interval: 1.20-1.31]). The success rates of TES-MEP recordings were higher using constant-voltage stimulation compared with constant-current stimulation in patients undergoing spinal surgery. Copyright © 2017

A Sequence-Dependent DNA Condensation Induced by Prion Protein

PubMed Central

2018-01-01

Different studies indicated that the prion protein induces hybridization of complementary DNA strands. Cell culture studies showed that the scrapie isoform of prion protein remained bound with the chromosome. In present work, we used an oxazole dye, YOYO, as a reporter to quantitative characterization of the DNA condensation by prion protein. We observe that the prion protein induces greater fluorescence quenching of YOYO intercalated in DNA containing only GC bases compared to the DNA containing four bases whereas the effect of dye bound to DNA containing only AT bases is marginal. DNA-condensing biological polyamines are less effective than prion protein in quenching of DNA-bound YOYO fluorescence. The prion protein induces marginal quenching of fluorescence of the dye bound to oligonucleotides, which are resistant to condensation. The ultrastructural studies with electron microscope also validate the biophysical data. The GC bases of the target DNA are probably responsible for increased condensation in the presence of prion protein. To our knowledge, this is the first report of a human cellular protein inducing a sequence-dependent DNA condensation. The increased condensation of GC-rich DNA by prion protein may suggest a biological function of the prion protein and a role in its pathogenesis. PMID:29657864

A Sequence-Dependent DNA Condensation Induced by Prion Protein.

PubMed

Bera, Alakesh; Biring, Sajal

2018-01-01

Different studies indicated that the prion protein induces hybridization of complementary DNA strands. Cell culture studies showed that the scrapie isoform of prion protein remained bound with the chromosome. In present work, we used an oxazole dye, YOYO, as a reporter to quantitative characterization of the DNA condensation by prion protein. We observe that the prion protein induces greater fluorescence quenching of YOYO intercalated in DNA containing only GC bases compared to the DNA containing four bases whereas the effect of dye bound to DNA containing only AT bases is marginal. DNA-condensing biological polyamines are less effective than prion protein in quenching of DNA-bound YOYO fluorescence. The prion protein induces marginal quenching of fluorescence of the dye bound to oligonucleotides, which are resistant to condensation. The ultrastructural studies with electron microscope also validate the biophysical data. The GC bases of the target DNA are probably responsible for increased condensation in the presence of prion protein. To our knowledge, this is the first report of a human cellular protein inducing a sequence-dependent DNA condensation. The increased condensation of GC-rich DNA by prion protein may suggest a biological function of the prion protein and a role in its pathogenesis.

Combined analysis by GC (RI), GC-MS and 13C NMR of the supercritical fluid extract of Abies alba twigs.

PubMed

Duquesnoy, Emilie; Marongiu, Bruno; Castola, Vincent; Piras, Alessandra; Porcedda, Silvia; Casanova, Joseph

2010-12-01

Two samples (leaves and twigs) of Abies alba Miller from Corsica were extracted using supercritical CO2 and their chemical compositions were compared with those of the essential oils obtained from the same batch of plant material. In total 45 components were identified using combined analysis by GC (RI), GC-MS and 13C NMR. It was observed that the contents of monoterpenes (mainly represented by limonene, alpha-pinene and camphene) were significantly lower in the supercritical fluid extract (SFE) than in the essential oil (EO). Conversely, the proportions of sesquiterpenes were much higher in CO2 extracts than in essential oils (around 30% vs 4%). Cis-abienol, a diterpene alcohol, was identified only in SFE, and the proportions of this constituent (7.5% and 17.3%) were determined using quantitative 13C NMR since it was under estimated using the standard conditions of GC.

Study on the interaction of triadimenol with calf thymus DNA by multispectroscopic methods and molecular modeling

NASA Astrophysics Data System (ADS)

Zhang, Yepeng; Zhang, Guowen; Fu, Peng; Ma, Yadi; Zhou, Jia

2012-10-01

The binding mechanism of triadimenol (NOL) to calf thymus DNA (ctDNA) in physiological buffer (pH 7.4) was investigated by multispectroscopic methods including UV-vis absorption, fluorescence, circular dichroism (CD), Fourier transform infrared (FT-IR), and nuclear magnetic resonance (1H NMR) spectroscopy, coupled with viscosity measurements and atomic force microscopy (AFM) technique. The results suggested that NOL interacted with ctDNA by intercalation mode. CD and AFM assays showed that NOL can damage the base stacking of ctDNA and result in regional cleavage of the two DNA strands. FT-IR and 1H NMR spectra coupled with molecular docking revealed that a specific binding mainly exists between NOL and G-C base pairs of the ctDNA where two hydrogen bonds form. Moreover, the association constants of NOL with DNA at three different temperatures were determined to be in the 103 L mol-1 range. The calculated thermodynamic parameters suggested that the binding of NOL to ctDNA was driven mainly by hydrogen bond and van der Waals.

DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation.

PubMed

Nacheva, Elizabeth; Mokretar, Katya; Soenmez, Aynur; Pittman, Alan M; Grace, Colin; Valli, Roberto; Ejaz, Ayesha; Vattathil, Selina; Maserati, Emanuela; Houlden, Henry; Taanman, Jan-Willem; Schapira, Anthony H; Proukakis, Christos

2017-01-01

Potential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol / chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues. This may contribute to array "waves", and could affect copy number determination, particularly if mosaicism is being sought, and sequencing coverage. Variations in isolation protocol may also affect apparent mtDNA abundance.

Overcoming PCR Inhibition During DNA-Based Gut Content Analysis of Ants.

PubMed

Penn, Hannah J; Chapman, Eric G; Harwood, James D

2016-10-01

Generalist predators play an important role in many terrestrial systems, especially within agricultural settings, and ants (Hymenoptera: Formicidae) often constitute important linkages of these food webs, as they are abundant and influential in these ecosystems. Molecular gut content analysis provides a means of delineating food web linkages of ants based on the presence of prey DNA within their guts. Although this method can provide insight, its use on ants has been limited, potentially due to inhibition when amplifying gut content DNA. We designed a series of experiments to determine those ant organs responsible for inhibition and identified variation in inhibition among three species (Tetramorium caespitum (L.), Solenopsis invicta Buren, and Camponotus floridanus (Buckley)). No body segment, other than the gaster, caused significant inhibition. Following dissection, we determined that within the gaster, the digestive tract and crop cause significant levels of inhibition. We found significant differences in the frequency of inhibition between the three species tested, with inhibition most evident in T. caespitum The most effective method to prevent inhibition before DNA extraction was to exude crop contents and crop structures onto UV-sterilized tissue. However, if extracted samples exhibit inhibition, addition of bovine serum albumin to PCR reagents will overcome this problem. These methods will circumvent gut content inhibition within selected species of ants, thereby allowing more detailed and reliable studies of ant food webs. As little is known about the prevalence of this inhibition in other species, it is recommended that the protocols in this study are used until otherwise shown to be unnecessary. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Single-Walled Carbon Nanotubes Modulate the B- to A-DNA Transition

PubMed Central

2015-01-01

We study the conformational equilibrium between B-to-A forms of ds-DNA adsorbed onto a single-walled carbon nanotube (SWNT) using free energy profile calculations based on all-atom molecular dynamics simulations. The potential of mean force (PMF) of the B-to-A transition of ds-DNA in the presence of an uncharged (10,0) carbon nanotube for two dodecamers with poly-AT or poly-GC sequences is calculated as a function of a root-mean-square-distance (ΔRMSD) difference metric for the B-to-A transition. The calculations reveal that in the presence of a SWNT DNA favors B-form DNA significantly in both poly-GC and poly-AT sequences. Furthermore, the poly-AT DNA:SWNT complex shows a higher energy penalty for adopting an A-like conformation than poly-GC DNA:SWNT by several kcal/mol. The presence of a SWNT on either poly-AT or poly-GC DNA affects the PMF of the transition such that the B form is favored by as much as 10 kcal/mol. In agreement with published data, we find a potential energy minimum between A and B-form DNA at ΔRMSD ≈ −1.5 Å and that the presence of the SWNT moves this minimum by as much as ΔRMSD = 3 Å. PMID:25553205

DNA interactions with a Methylene Blue redox indicator depend on the DNA length and are sequence specific.

PubMed

Farjami, Elaheh; Clima, Lilia; Gothelf, Kurt V; Ferapontova, Elena E

2010-06-01

A DNA molecular beacon approach was used for the analysis of interactions between DNA and Methylene Blue (MB) as a redox indicator of a hybridization event. DNA hairpin structures of different length and guanine (G) content were immobilized onto gold electrodes in their folded states through the alkanethiol linker at the 5'-end. Binding of MB to the folded hairpin DNA was electrochemically studied and compared with binding to the duplex structure formed by hybridization of the hairpin DNA to a complementary DNA strand. Variation of the electrochemical signal from the DNA-MB complex was shown to depend primarily on the DNA length and sequence used: the G-C base pairs were the preferential sites of MB binding in the duplex. For short 20 nts long DNA sequences, the increased electrochemical response from MB bound to the duplex structure was consistent with the increased amount of bound and electrochemically readable MB molecules (i.e. MB molecules that are available for the electron transfer (ET) reaction with the electrode). With longer DNA sequences, the balance between the amounts of the electrochemically readable MB molecules bound to the hairpin DNA and to the hybrid was opposite: a part of the MB molecules bound to the long-sequence DNA duplex seem to be electrochemically mute due to long ET distance. The increasing electrochemical response from MB bound to the short-length DNA hybrid contrasts with the decreasing signal from MB bound to the long-length DNA hybrid and allows an "off"-"on" genosensor development.

Alterations of DNA content in human endometrial stromal cells transfected with a temperature-sensitive SV40: tetraploidization and physiological consequences.

PubMed

Rinehart, C A; Mayben, J P; Butler, T D; Haskill, J S; Kaufman, D G

1992-01-01

The normal genomic stability of human cells is reversed during neoplastic transformation. The SV40 large T antigen alters the DNA content in human endometrial stromal cells in a manner that relates to neoplastic progression. Human endometrial stromal cells were transfected with a plasmid containing the A209 temperature-sensitive mutant of SV40 (tsSV40), which is also defective in the viral origin of replication. Ninety-seven clonal transfectants from seven different primary cell strains were isolated. Initial analysis revealed that 20% of the clonal populations (19/97) had an apparent diploid DNA content, 35% (34/97) had an apparent tetraploid DNA content, and the remainder were mixed populations of diploid and tetraploid cells. No aneuploid populations were observed. Diploid tsSV40 transformed cells always give rise to a population of cells with a tetraploid DNA content when continuously cultured at the permissive temperature. The doubling of DNA content can be vastly accelerated by the sudden reintroduction of large T antigen activity following a shift from non-permissive to permissive temperature. Tetraploid tsSV40 transfected cells have a lower capacity for anchorage-independent growth and earlier entry into 'crisis' than diploid cells. These results indicate that during the pre-crisis, extended lifespan phase of growth, the SV40 large T antigen causes a doubling of DNA content. This apparent doubling of DNA content does not confer growth advantage during the extended lifespan that precedes 'crisis'.

Disentangling oil weathering using GC x GC. 2. Mass transfer calculations.

PubMed

Arey, J Samuel; Nelson, Robert K; Plata, Desiree L; Reddy, Christopher M

2007-08-15

Hydrocarbon mass transfers to the atmosphere and water column drive the early weathering of oil spills and also control the chemical exposures of many coastal wildlife species. However, in the field, mass transfer rates of individual hydrocarbons to air and water are often uncertain. In the Part 1 companion to this paper, we used comprehensive two-dimensional gas chromatography (GC x GC) to identify distinct signatures of evaporation and dissolution encoded in the compositional evolution of weathered oils. In Part 2, we further investigate patterns of mass removal in GC x GC chromatograms using a mass transfer model. The model was tailored to conditions at a contaminated beach on Buzzards Bay, MA, after the 2003 Bouchard 120 oil spill. The model was applied to all resolved hydrocarbon compounds in the C11-C24 boiling range, based on their GC x GC-estimated vapor pressures and aqueous solubilities. With no fitted parameters, the model successfully predicted GC x GC chromatogram patterns of mass removal associated with evaporation, water-washing, and diffusion-limited transport. This enabled a critical field evaluation of the mass transfer model and also allowed mass apportionment estimates of hundreds of individual hydrocarbon compounds to air and water. Ultimately, this method should improve assessments of wildlife exposures to oil spill hydrocarbons.

Investigating the dynamics of surface-immobilized DNA nanomachines

NASA Astrophysics Data System (ADS)

Dunn, Katherine E.; Trefzer, Martin A.; Johnson, Steven; Tyrrell, Andy M.

2016-07-01

Surface-immobilization of molecules can have a profound influence on their structure, function and dynamics. Toehold-mediated strand displacement is often used in solution to drive synthetic nanomachines made from DNA, but the effects of surface-immobilization on the mechanism and kinetics of this reaction have not yet been fully elucidated. Here we show that the kinetics of strand displacement in surface-immobilized nanomachines are significantly different to those of the solution phase reaction, and we attribute this to the effects of intermolecular interactions within the DNA layer. We demonstrate that the dynamics of strand displacement can be manipulated by changing strand length, concentration and G/C content. By inserting mismatched bases it is also possible to tune the rates of the constituent displacement processes (toehold-binding and branch migration) independently, and information can be encoded in the time-dependence of the overall reaction. Our findings will facilitate the rational design of surface-immobilized dynamic DNA nanomachines, including computing devices and track-based motors.

Investigating the dynamics of surface-immobilized DNA nanomachines

PubMed Central

Dunn, Katherine E.; Trefzer, Martin A.; Johnson, Steven; Tyrrell, Andy M.

2016-01-01

Surface-immobilization of molecules can have a profound influence on their structure, function and dynamics. Toehold-mediated strand displacement is often used in solution to drive synthetic nanomachines made from DNA, but the effects of surface-immobilization on the mechanism and kinetics of this reaction have not yet been fully elucidated. Here we show that the kinetics of strand displacement in surface-immobilized nanomachines are significantly different to those of the solution phase reaction, and we attribute this to the effects of intermolecular interactions within the DNA layer. We demonstrate that the dynamics of strand displacement can be manipulated by changing strand length, concentration and G/C content. By inserting mismatched bases it is also possible to tune the rates of the constituent displacement processes (toehold-binding and branch migration) independently, and information can be encoded in the time-dependence of the overall reaction. Our findings will facilitate the rational design of surface-immobilized dynamic DNA nanomachines, including computing devices and track-based motors. PMID:27387252

Patterns and effects of GC3 heterogeneity and parsimony informative sites on the phylogenetic tree of genes.

PubMed

Ma, Shuai; Wu, Qi; Hu, Yibo; Wei, Fuwen

2018-05-20

The explosive growth in genomic data has provided novel insights into the conflicting signals hidden in phylogenetic trees. Although some studies have explored the effects of the GC content and parsimony informative sites (PIS) on the phylogenetic tree, the effect of the heterogeneity of the GC content at the first/second/third codon position on parsimony informative sites (GC1/2/3 PIS ) among different species and the effect of PIS on phylogenetic tree construction remain largely unexplored. Here, we used two different mammal genomic datasets to explore the patterns of GC1/2/3 PIS heterogeneity and the effect of PIS on the phylogenetic tree of genes: (i) all GC1/2/3 PIS have obvious heterogeneity between different mammals, and the levels of heterogeneity are GC3 PIS  > GC2 PIS  > GC1 PIS ; (ii) the number of PIS is positively correlated with the metrics of "good" gene tree topologies, and excluding the third codon position (C3) decreases the quality of gene trees by removing too many PIS. These results provide novel insights into the heterogeneity pattern of GC1/2/3 PIS in mammals and the relationship between GC3/PIS and gene trees. Additionally, it is necessary to carefully consider whether to exclude C3 to improve the quality of gene trees, especially in the super-tree method. Copyright © 2018 Elsevier B.V. All rights reserved.

Analysis of black carbon molecular markers by two chromatographic methods (GC-FID and HPLC-DAD)

NASA Astrophysics Data System (ADS)

Schneider, Maximilian P. W.; Smittenberg, Rienk H.; Dittmar, Thorsten; Schmidt, Michael W. I.

2010-05-01

The analysis of benzenepolycarboxylic acids (BPCA) as a quantitative measure for black carbon (BC) in soil and sediment samples is a well-established method [1, 2]. Briefly, the oxidation of polycondensated BC molecules forms seven molecular markers, which can be assigned to BC, and which subsequently can be quantified by GC-FID (gas chromatography with flame ionization detector). Recently this method has been refined for BC quantification in seawater samples measuring BPCA on HPLC-DAD (High performance liquid chromatography with diode array detector) [3]. However, a systematic comparison of BC as determined by both analytical techniques would be essential to the calculation of global BC budgets, but is lacking. Here we present data for the systematic comparison of the two BPCA methods, both for quantity and quality. We prepared chars under well-defined laboratory conditions. Chestnut hardwood chips and rice straw were pyrolysed at temperatures between 200 and 1000°C under constant N2 stream. The BC contents of the chars have been analysed using the BPCA extraction method followed by either GC-FID or HPLC-DAD quantification [4]. It appears that the GC-FID method yields systematically lower concentrations of BPCA in the chars compared to the HPLC-DAD method. Possible reasons for the observed difference are i) higher losses of sample material during preparation for GC-FID; ii) different quality of the linear regression used for quantification; iii) incomplete derivatisation of B5CA and B6CA, which is needed for GC-FID analysis. In a next step, we will test different derivatisation procedures (methylation with dimethyl sulfate or diazomethane, and silylation) for their influence on the GC-FID results. The aim of this study is to test if black carbon can be quantified in soil, sediment and water samples using one single method - a crucial step when attempting a global BC budget. References: [1] Brodowski, S., Rodionov, A., Haumeier L., Glaser, B., Amelung, W. (2005

DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation

PubMed Central

Nacheva, Elizabeth; Mokretar, Katya; Soenmez, Aynur; Pittman, Alan M.; Grace, Colin; Valli, Roberto; Ejaz, Ayesha; Vattathil, Selina; Maserati, Emanuela; Houlden, Henry; Taanman, Jan-Willem; Schapira, Anthony H.

2017-01-01

Potential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol / chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues. This may contribute to array “waves”, and could affect copy number determination, particularly if mosaicism is being sought, and sequencing coverage. Variations in isolation protocol may also affect apparent mtDNA abundance. PMID:28683077

Recombination Analysis of Herpes Simplex Virus 1 Reveals a Bias toward GC Content and the Inverted Repeat Regions

PubMed Central

Lee, Kyubin; Kolb, Aaron W.; Sverchkov, Yuriy; Cuellar, Jacqueline A.; Craven, Mark

2015-01-01

ABSTRACT Herpes simplex virus 1 (HSV-1) causes recurrent mucocutaneous ulcers and is the leading cause of infectious blindness and sporadic encephalitis in the United States. HSV-1 has been shown to be highly recombinogenic; however, to date, there has been no genome-wide analysis of recombination. To address this, we generated 40 HSV-1 recombinants derived from two parental strains, OD4 and CJ994. The 40 OD4-CJ994 HSV-1 recombinants were sequenced using the Illumina sequencing system, and recombination breakpoints were determined for each of the recombinants using the Bootscan program. Breakpoints occurring in the terminal inverted repeats were excluded from analysis to prevent double counting, resulting in a total of 272 breakpoints in the data set. By placing windows around the 272 breakpoints followed by Monte Carlo analysis comparing actual data to simulated data, we identified a recombination bias toward both high GC content and intergenic regions. A Monte Carlo analysis also suggested that recombination did not appear to be responsible for the generation of the spontaneous nucleotide mutations detected following sequencing. Additionally, kernel density estimation analysis across the genome found that the large, inverted repeats comprise a recombination hot spot. IMPORTANCE Herpes simplex virus 1 (HSV-1) virus is the leading cause of sporadic encephalitis and blinding keratitis in developed countries. HSV-1 has been shown to be highly recombinogenic, and recombination itself appears to be a significant component of genome replication. To date, there has been no genome-wide analysis of recombination. Here we present the findings of the first genome-wide study of recombination performed by generating and sequencing 40 HSV-1 recombinants derived from the OD4 and CJ994 parental strains, followed by bioinformatics analysis. Recombination breakpoints were determined, yielding 272 breakpoints in the full data set. Kernel density analysis determined that the large

E. coli mismatch repair enhances AT-to-GC mutagenesis caused by alkylating agents.

PubMed

Nakano, Kota; Yamada, Yoko; Takahashi, Eizo; Arimoto, Sakae; Okamoto, Keinosuke; Negishi, Kazuo; Negishi, Tomoe

2017-03-01

Alkylating agents are known to induce the formation of O 6 -alkylguanine (O 6 -alkG) and O 4 -alkylthymine (O 4 -alkT) in DNA. These lesions have been widely investigated as major sources of mutations. We previously showed that mismatch repair (MMR) facilitates the suppression of GC-to-AT mutations caused by O 6 -methylguanine more efficiently than the suppression of GC-to-AT mutations caused by O 6 -ethylguanine. However, the manner by which O 4 -alkyT lesions are repaired remains unclear. In the present study, we investigated the repair pathway involved in the repair of O 4 -alkT. The E. coli CC106 strain, which harbors Δprolac in its genomic DNA and carries the F'CC106 episome, can be used to detect AT-to-GC reverse-mutation of the gene encoding β-galactosidase. Such AT-to-GC mutations should be induced through the formation of O 4 -alkT at AT base pairs. As expected, an O 6 -alkylguanine-DNA alkyltransferase (AGT) -deficient CC106 strain, which is defective in both ada and agt genes, exhibited elevated mutant frequencies in the presence of methylating agents and ethylating agents. However, in the UvrA-deficient strain, the methylating agents were less mutagenic than in wild-type, while ethylating agents were more mutagenic than in wild-type, as observed with agents that induce O 6 -alkylguanine modifications. Unexpectedly, the mutant frequencies decreased in a MutS-deficient strain, and a similar tendency was observed in MutL- or MutH-deficient strains. Thus, MMR appears to promote mutation at AT base pairs. Similar results were obtained in experiments employing double-mutant strains harboring defects in both MMR and AGT, or MMR and NER. E. coli MMR enhances AT-to-GC mutagenesis, such as that caused by O 4 -alkylthymine. We hypothesize that the MutS protein recognizes the O 4 -alkT:A base pair more efficiently than O 4 -alkT:G. Such a distinction would result in misincorporation of G at the O 4 -alkT site, followed by higher mutation frequencies in wild

Decreased mitochondrial DNA content in subcutaneous fat from HIV-infected women taking antiretroviral therapy as measured at delivery.

PubMed

Nasi, Milena; Pinti, Marcello; Chiesa, Elisabetta; Fiore, Simona; Manzini, Serena; Del Giovane, Cinzia; D'Amico, Roberto; Palai, Nicoletta; Campatelli, Carlo; Sabbatini, Francesca; Roccio, Marianna; Tibaldi, Cecilia; Masuelli, Giulia; Mussini, Cristina; Ferrazzi, Enrico; d'Arminio Monforte, Antonella; Cossarizza, Andrea

2011-01-01

Increasing numbers of pregnant HIV-positive women are receiving combination antiretroviral regimens for preventing mother-to-child virus transmission or for treating the infection itself. Several studies have demonstrated that nucleoside reverse transcriptase inhibitors (NRTIs) induce mitochondrial toxicity by several mechanisms, including depletion of mitochondrial DNA (mtDNA). By the quantification of mtDNA levels, we studied mitochondrial toxicity in HIV-positive women at delivery and the possible correlations with antiretroviral regimens, viroimmunological and metabolic parameters. We analysed 68 HIV-positive women enrolled in the Italian Prospective Cohort Study on Efficacy and Toxicity of Antiretroviral in Pregnancy (TARGET Study); all were taking ≥1 NRTI. We quantified mtDNA copies per cell in subcutaneous fat samples collected during delivery. At the 3rd, 6th and 9th month of pregnancy, we collected data concerning CD4(+) T-cell count, plasma HIV RNA, total and high-density lipoprotein (HDL) cholesterol, fasting plasma glucose and triglycerides. As a control, we analysed mtDNA levels in abdominal subcutaneous fat samples from 23 HIV-seronegative women at delivery. mtDNA content was significantly lower in HIV-infected women when compared with HIV-negative controls. mtDNA content varied independently from viroimmunological, lipid and glucose parameters at the different months, with the exceptions of triglycerides at the 9th month and of HDL at the 6th month of pregnancy. In subcutaneous tissue from women taking NRTI-based antiretroviral regimens, we observed a significant decrease of mtDNA content, compared with uninfected women not on antiviral treatment. Moreover, a significant correlation was noted between mtDNA content and HDL cholesterol and triglycerides.

Mitochondria DNA replication and DNA methylation in the metabolic memory associated with continued progression of diabetic retinopathy.

PubMed

Tewari, Shikha; Zhong, Qing; Santos, Julia M; Kowluru, Renu A

2012-07-24

Diabetic retinopathy fails to halt after cessation of hyperglycemic insult, and a vicious cycle of mitochondria damage continues. The aim of our study was to investigate the effect of termination of hyperglycemia on retinal mtDNA replication, and elucidate the mechanism responsible for the continued mtDNA damage. Polymerase gamma 1 (POLG1), the catalytic subunit of the mitochondrial DNA replication enzyme, and the damage to the displacement loop region of mtDNA (D-loop) were analyzed in the retina from streptozotocin-diabetic rats maintained in poor glycemic control (PC, glycated hemoglobin ∼11%) or in good glycemic control (GC, glycated hemoglobin ∼6%) for 6 months, or in PC for three months followed by GC for three months (Rev). To understand the mechanism DNA methylation status of POLG1 promoter was investigated by methylation-specific PCR. The key parameters were confirmed in the isolated retinal endothelial cells exposed to high glucose, followed by normal glucose. POLG1 continued to be down-regulated, the D-loop region damaged, and the CpG islands at the regulatory region of POLG hyper-methylated even after three months of GC that had followed three months of PC (Rev group). Similar results were observed in the retinal endothelial cells exposed to normal glucose after being exposed to high glucose. Continued hypermethylation of the CpG sites at the regulatory region of POLG affects its binding to the mtDNA, compromising the transcriptional activity. Modulation of DNA methylation using pharmaceutic or molecular means could help maintain mitochondria homeostasis, and prevent further progression of diabetic retinopathy.

Spectroscopic studies of the binding of Cu(II) complexes of oxicam NSAIDs to alternating G-C and homopolymeric G-C sequences

NASA Astrophysics Data System (ADS)

Chakraborty, Sreeja; Bose, Madhuparna; Sarkar, Munna

2014-03-01

Drugs belonging to the Non-steroidal anti-inflammatory (NSAID) group are not only used as anti-inflammatory, analgesic and anti-pyretic agents, but also show anti-cancer effects. Complexing them with a bioactive metal like copper, show an enhancement in their anti-cancer effects compared to the bare drugs, whose exact mechanism of action is not yet fully understood. For the first time, it was shown by our group that Cu(II)-NSAIDs can directly bind to the DNA backbone. The ability of the copper complexes of NSAIDs namely meloxicam and piroxicam to bind to the DNA backbone could be a possible molecular mechanism behind their enhanced anticancer effects. Elucidating base sequence specific interaction of Cu(II)-NSAIDs to the DNA will provide information on their possible binding sites in the genome sequence. In this work, we present how these complexes respond to differences in structure and hydration pattern of GC rich sequences. For this, binding studies of Cu(II) complexes of piroxicam [Cu(II)-(Px)2 (L)2] and meloxicam [Cu(II)-(Mx)2 (L)] with alternating GC (polydG-dC) and homopolymeric GC (polydG-polydC) sequences were carried out using a combination of spectroscopic techniques that include UV-Vis absorption, fluorescence and circular dichroism (CD) spectroscopy. The Cu(II)-NSAIDs show strong binding affinity to both polydG-dC and polydG-polydC. The role reversal of Cu(II)-meloxicam from a strong binder of polydG-dC (Kb = 11.5 × 103 M-1) to a weak binder of polydG-polydC (Kb = 5.02 × 103 M-1), while Cu(II)-piroxicam changes from a strong binder of polydG-polydC (Kb = 8.18 × 103 M-1) to a weak one of polydG-dC (Kb = 2.18 × 103 M-1), point to the sensitivity of these complexes to changes in the backbone structures/hydration. Changes in the profiles of UV absorption band and CD difference spectra, upon complex binding to polynucleotides and the results of competitive binding assay using ethidium bromide (EtBr) fluorescence indicate different binding modes in each

Comparison of traditional gas chromatography (GC), headspace GC, and the microbial identification library GC system for the identification of Clostridium difficile.

PubMed Central

Cundy, K V; Willard, K E; Valeri, L J; Shanholtzer, C J; Singh, J; Peterson, L R

1991-01-01

Three gas chromatography (GC) methods were compared for the identification of 52 clinical Clostridium difficile isolates, as well as 17 non-C. difficile Clostridium isolates. Headspace GC and Microbial Identification System (MIS) GC, an automated system which utilizes a software library developed at the Virginia Polytechnic Institute to identify organisms based on the fatty acids extracted from the bacterial cell wall, were compared against the reference method of traditional GC. Headspace GC and MIS were of approximately equivalent accuracy in identifying the 52 C. difficile isolates (52 of 52 versus 51 of 52, respectively). However, 7 of 52 organisms required repeated sample preparation before an identification was achieved by the MIS method. Both systems effectively differentiated C. difficile from non-C. difficile clostridia, although the MIS method correctly identified only 9 of 17. We conclude that the headspace GC system is an accurate method of C. difficile identification, which requires only one-fifth of the sample preparation time of MIS GC and one-half of the sample preparation time of traditional GC. PMID:2007632

Immunotherapy for Prostate Cancer with Gc Protein-Derived Macrophage-Activating Factor, GcMAF.

PubMed

Yamamoto, Nobuto; Suyama, Hirofumi; Yamamoto, Nobuyuki

2008-07-01

Serum Gc protein (known as vitamin D(3)-binding protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of prostate cancer patients was lost or reduced because Gc protein was deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Therefore, macrophages of prostate cancer patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized beta-galactosidase and sialidase generated the most potent MAF (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages activated by GcMAF develop a considerable variation of receptors that recognize the abnormality in malignant cell surface and are highly tumoricidal. Sixteen nonanemic prostate cancer patients received weekly administration of 100 ng of GcMAF. As the MAF precursor activity increased, their serum Nagalase activity decreased. Because serum Nagalase activity is proportional to tumor burden, the entire time course analysis for GcMAF therapy was monitored by measuring the serum Nagalase activity. After 14 to 25 weekly administrations of GcMAF (100 ng/week), all 16 patients had very low serum Nagalase levels equivalent to those of healthy control values, indicating that these patients are tumor-free. No recurrence occurred for 7 years.

[DNA content in the organs of animals in space flight on the Kosmos-690 satellite].

PubMed

Guseĭnov, F T; Komolova, G S; Egorov, I A; Tigranian, R A; Serova, L V

1978-01-01

The DNA content in the liver, spleen and bone marrow of white rats exposed to a prolonged gamma-irradiation at a dose of 220 and 800 rad on the 10th day of the 20.5-day space flight and the ground-based synchronous experiment was measured. Space flight factors produced a modifying effect on the postradiation changes in the DNA content. This modifying influence was detected in all organs tested, although in a different degree, and involved an enhancement of the radiation effect which was associated with retardation of postradiation regenerative processes.

[The value of low-molecular-weight DNA of blood plasma in the diagnostic of the patological processes of different genesis].

PubMed

Vasil'eva, I N; Zinkin, V N

2013-01-01

The low-molecular-weight DNA appears in blood plasma of irradiated rats, and its content correlates directly with the irradiation dose. Cloning has shown, that enrichment of low-molecular-weight DNA with G+C content and features of its nucleotide sequences point to its ability to form rather stable nucleosomes. DNA obtained after irradiation of rats with principally different doses 8 and 100 Gy differed not only quantitatively, but also by content of the dinucleotides CpG and CpT; this suggests their origin from different sites of genome. For the first time it has been shown that exposure to low-frequency noise results in an increase of the contents of blood plasma low-molecular-weight DNA. In stroke patients blood concentrations of this DNA increased 3 days after the beginning of the acute period, and dynamics of its excretion differs in ischemic and hemorrhagic forms; in the case of ischemia low-molecular-weight DNA appears in cerebrospinal fluid. The chronic obstructive pulmonary disease in the state of remission is characterized by the decline of the level of low-molecular-weight DNA in the blood plasma unlike in the case of the chronic nonobstructive bronchitis. The clear dependence between formation and special features of the low-molecular-weight DNA fraction in blood plasma makes it possible to consider the low-molecular fraction as an universal index of apoptosis, which allows to distinguish basically different conditions of the body.

Direct comparisons of Illumina vs. Roche 454 sequencing technologies on the same microbial community DNA sample.

PubMed

Luo, Chengwei; Tsementzi, Despina; Kyrpides, Nikos; Read, Timothy; Konstantinidis, Konstantinos T

2012-01-01

Next-generation sequencing (NGS) is commonly used in metagenomic studies of complex microbial communities but whether or not different NGS platforms recover the same diversity from a sample and their assembled sequences are of comparable quality remain unclear. We compared the two most frequently used platforms, the Roche 454 FLX Titanium and the Illumina Genome Analyzer (GA) II, on the same DNA sample obtained from a complex freshwater planktonic community. Despite the substantial differences in read length and sequencing protocols, the platforms provided a comparable view of the community sampled. For instance, derived assemblies overlapped in ~90% of their total sequences and in situ abundances of genes and genotypes (estimated based on sequence coverage) correlated highly between the two platforms (R(2)>0.9). Evaluation of base-call error, frameshift frequency, and contig length suggested that Illumina offered equivalent, if not better, assemblies than Roche 454. The results from metagenomic samples were further validated against DNA samples of eighteen isolate genomes, which showed a range of genome sizes and G+C% content. We also provide quantitative estimates of the errors in gene and contig sequences assembled from datasets characterized by different levels of complexity and G+C% content. For instance, we noted that homopolymer-associated, single-base errors affected ~1% of the protein sequences recovered in Illumina contigs of 10× coverage and 50% G+C; this frequency increased to ~3% when non-homopolymer errors were also considered. Collectively, our results should serve as a useful practical guide for choosing proper sampling strategies and data possessing protocols for future metagenomic studies.

Measurement of oxidative DNA damage by gas chromatography-mass spectrometry: ethanethiol prevents artifactual generation of oxidized DNA bases.

PubMed Central

Jenner, A; England, T G; Aruoma, O I; Halliwell, B

1998-01-01

Analysis of oxidative damage to DNA bases by GC-MS enables identification of a range of base oxidation products, but requires a derivatization procedure. However, derivatization at high temperature in the presence of air can cause 'artifactual' oxidation of some undamaged bases, leading to an overestimation of their oxidation products, including 8-hydroxyguanine. Therefore derivatization conditions that could minimize this problem were investigated. Decreasing derivatization temperature to 23 degrees C lowered levels of 8-hydroxyguanine, 8-hydroxyadenine, 5-hydroxycytosine and 5-(hydroxymethyl)uracil measured by GC-MS in hydrolysed calf thymus DNA. Addition of the reducing agent ethanethiol (5%, v/v) to DNA samples during trimethylsilylation at 90 degrees C also decreased levels of these four oxidized DNA bases as well as 5-hydroxyuracil. Removal of guanine from hydrolysed DNA samples by treatment with guanase, prior to derivatization, resulted in 8-hydroxyguanine levels (54-59 pmol/mg of DNA) that were significantly lower than samples not pretreated with guanase, independent of the derivatization conditions used. Only hydrolysed DNA samples that were derivatized at 23 degrees C in the presence of ethanethiol produced 8-hydroxyguanine levels (56+/-8 pmol/mg of DNA) that were as low as those of guanase-pretreated samples. Levels of other oxidized bases were similar to samples derivatized at 23 degrees C without ethanethiol, except for 5-hydroxycytosine and 5-hydroxyuracil, which were further decreased by ethanethiol. Levels of 8-hydroxyguanine, 8-hydroxyadenine and 5-hydroxycytosine measured in hydrolysed calf thymus DNA by the improved procedures described here were comparable with those reported previously by HPLC with electrochemical detection and by GC-MS with prepurification to remove undamaged base. We conclude that artifactual oxidation of DNA bases during derivatization can be prevented by decreasing the temperature to 23 degrees C, removing air from the

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers.

PubMed

Koester, Meike; Gergs, René

2017-10-05

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (δ 15 N and δ 13 C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Hb Midnapore [β53(D4)Ala→Val; HBB: c.161C>T]: A Novel Hemoglobin Variant with a Structural Abnormality Associated with IVS-I-5 (G>C) (HBB: c.92+5G>C) Found in a Bengali Indian Family.

PubMed

Panja, Amrita; Chowdhury, Prosanto; Basu, Anupam

2016-09-01

We describe a novel C>T substitution at codon 53 of the HBB gene (HBB: c.161C>T). The proband was a transfusion-dependent β-thalassemia major (β-TM) patient. DNA was extracted and subsequently, DNA sequencing was done to detect the mutations on the HBB gene. Capillary zone electrophoresis (CZE) revealed the presence of an unknown peak. She inherited this mutation from her grandmother through her mother. This mutation exists in cis with the common β 0 mutation IVS-I-5 (G>C) (HBB: c.92+5G>C). The proband is homozygous for HBB: c.92+5G>C and needs monthly transfusions. On the other hand, her grandmother, mother and sister all possess this novel mutation cis with the heterozygous HBB: c.92+5G>C. They are carriers not thalassemic. This mutation produces the substitution β53(D4)Ala→Val; HBB: c.161C>T, a new structural hemoglobin (Hb) variant. As this variant was identified in a Bengali family from Paschim Midnapore district of West Bengal, India, it has been designated as Hb Midnapore. This variant has now been reported to the HbVar database.

TRX-LOGOS - a graphical tool to demonstrate DNA information content dependent upon backbone dynamics in addition to base sequence.

PubMed

Fortin, Connor H; Schulze, Katharina V; Babbitt, Gregory A

2015-01-01

It is now widely-accepted that DNA sequences defining DNA-protein interactions functionally depend upon local biophysical features of DNA backbone that are important in defining sites of binding interaction in the genome (e.g. DNA shape, charge and intrinsic dynamics). However, these physical features of DNA polymer are not directly apparent when analyzing and viewing Shannon information content calculated at single nucleobases in a traditional sequence logo plot. Thus, sequence logos plots are severely limited in that they convey no explicit information regarding the structural dynamics of DNA backbone, a feature often critical to binding specificity. We present TRX-LOGOS, an R software package and Perl wrapper code that interfaces the JASPAR database for computational regulatory genomics. TRX-LOGOS extends the traditional sequence logo plot to include Shannon information content calculated with regard to the dinucleotide-based BI-BII conformation shifts in phosphate linkages on the DNA backbone, thereby adding a visual measure of intrinsic DNA flexibility that can be critical for many DNA-protein interactions. TRX-LOGOS is available as an R graphics module offered at both SourceForge and as a download supplement at this journal. To demonstrate the general utility of TRX logo plots, we first calculated the information content for 416 Saccharomyces cerevisiae transcription factor binding sites functionally confirmed in the Yeastract database and matched to previously published yeast genomic alignments. We discovered that flanking regions contain significantly elevated information content at phosphate linkages than can be observed at nucleobases. We also examined broader transcription factor classifications defined by the JASPAR database, and discovered that many general signatures of transcription factor binding are locally more information rich at the level of DNA backbone dynamics than nucleobase sequence. We used TRX-logos in combination with MEGA 6.0 software

Analysis of residual monomers in dendritic methacrylate copolymers and composites by HPLC and headspace-GC/MS.

PubMed

Viljanen, Eeva K; Langer, Sarka; Skrifvars, Mikael; Vallittu, Pekka K

2006-09-01

The aim of this study was to analyze the residual monomer content of photopolymerized dendritic methacrylate copolymers and particulate filler composites. Headspace-gas chromatography/mass spectrometry (HS-GC/MS) was compared with high performance liquid chromatography (HPLC). The resin mixtures consisted of a dendritic methacrylate monomer, methyl methacrylate and acetoacetoxyethyl methacrylate in varied proportions. In addition, one of the composites contained 1,4-butanediol dimethacrylate. Camphorquinone and 2-(N,N-dimethylamino)ethyl methacrylate were used as the light-activated initiator system. The content of residual methyl methacrylate and acetoacetoxyethyl methacrylate after 40 s photopolymerization were analyzed with HPLC and HS-GC/MS. The content of residual methyl methacrylate decreased and residual acetoacetoxyethyl methacrylate increased with increasing concentration of acetoacetoxyethyl methacrylate in the resin mixture. The results with both methods had the same trend. The addition of acetoacetoxyethyl methacrylate enhanced the copolymerization of methyl methacrylate, but did not decrease the total residual monomer content. The HS-GC/MS method was found to be a feasible method in the analysis of low-boiling residuals in dental polymers.

Detection of the Spermicide Nonoxynol-9 Via GC-MS

NASA Astrophysics Data System (ADS)

Musah, Rabi A.; Vuong, Angela L.; Henck, Colin; Shepard, Jason R. E.

2012-05-01

The spermicide nonoxynol-9 is actually a complex mixture of dozens of closely related amphiphilic compounds, and the chemical properties of this assortment significantly hamper its characterization by GC-MS. The inability to perform routine GC-MS testing on nonoxynol-9 has limited its evidentiary value in forensic casework, which relies heavily on this technique for analysis. A disturbing trend in sexual assault is the use of condoms by assailants, to avoid leaving behind DNA evidence that can connect a perpetrator to a victim. This observation necessitates the development of alternative methods for the analysis of trace evidence that can show causal links between a victim and a suspect. Detection of lubricants associated with sexual assault is one such way to establish this connection. The development of GC-MS methods that permit facile detection of both nonoxynol-9 alone and nonoxynol-9 extracted from other complex matrices that have potential as trace evidence in sexual assault is reported. A detection limit of 2.14 μg of nonoxynol-9 is demonstrated, and a detailed mass spectral profile that elaborates on what is known of its structure is provided.

Cellular content and biosynthesis of polyamines during rooster spermatogenesis.

PubMed Central

Oliva, R; Vidal, S; Mezquita, C

1982-01-01

The natural polyamines spermine and spermidine, and the diamine putrescine, were extracted from rooster testis cells separated by sedimentation at unit gravity, and from vas-deferens spermatozoa. The ratios spermine/DNA and spermidine/DNA were kept relatively constant throughout spermatogenesis, whereas the ratio putrescine/DNA rose in elongated spermatids. The cellular content of spermine, spermidine and putrescine decreased markedly in mature spermatozoa. Two rate-limiting enzymes in the biosynthetic pathway of polyamines, ornithine decarboxylase and S-adenosyl-L-methionine decarboxylase, showed their highest activities at the end of spermiogenesis and were not detectable in vas-deferens spermatozoa. A marked reduction in cell volume during spermiogenesis without a parallel decrease in the cellular content of polyamines suggests the possibility that the marked changes in chromatin composition and structure occurring in rooster late spermatids could take place in an ambience of high polyamine concentration. Images PLATE 1 PMID:7159401

Alu–based cell-free DNA: a novel biomarker for screening of gastric cancer

PubMed Central

Zhao, Jianmei; Shen, Xianjuan; Jing, Rongrong; Yu, Juan; Li, Li; Shi, Yingjuan; Zhang, Lurong; Wang, Zhiwei; Cong, Hui

2017-01-01

Gastric cancer (GC) is the fourth most common cancer and the second major cause of cancer-related deaths worldwide. In our previous study, a novel and sensitive method for quantifying cell-free DNA (CFD) in human blood was established and tested for its ability to predict patients with tumor. We want to investigate CFD expression in the sera of GC patients in an attempt to explore the clinical significance of CFD in improving the early screening of GC and monitoring GC progression by the branched DNA (bDNA)-based Alu assay. The concentration of CFD was quantitated by bDNA-based Alu assay. CEA, CA19-9, C72-4 and CA50 concentrations were determined by ABBOTT ARCHITECT I2000 SR. We found the CFD concentrations have significant differences between GC patients, benign gastric disease (BGD) patients and healthy controls (P < 0.05). CFD were weakly correlated with CEA (r = −0.197, P < 0.05) or CA50 (r = 0.206, P < 0.05), and no correlation with CA19-9 (r = −0.061, P > 0.05) or CA72-4 (r = 0.011, P > 0.05). In addition, CFD concentrations were significantly higher in stage I GC patients than BGD patients and healthy controls (P < 0.05), but there was no significant difference in CEA, CA19-9 and CA50 among the three traditional tumor markers (P > 0.05). Our analysis showed that CFD was more sensitive than CEA, CA19-9, CA72-4 or CA50 in early screening of GC. Compared with CEA, CA19-9, CA72-4 and CA50, CFD may prove to be a better biomarker for the screening of GC, thus providing a sensitive biomarker for screening and monitoring progression of GC. PMID:28903321

p53 improves aerobic exercise capacity and augments skeletal muscle mitochondrial DNA content.

PubMed

Park, Joon-Young; Wang, Ping-Yuan; Matsumoto, Takumi; Sung, Ho Joong; Ma, Wenzhe; Choi, Jeong W; Anderson, Stasia A; Leary, Scot C; Balaban, Robert S; Kang, Ju-Gyeong; Hwang, Paul M

2009-09-25

Exercise capacity is a physiological characteristic associated with protection from both cardiovascular and all-cause mortality. p53 regulates mitochondrial function and its deletion markedly diminishes exercise capacity, but the underlying genetic mechanism orchestrating this is unclear. Understanding the biology of how p53 improves exercise capacity may provide useful insights for improving both cardiovascular as well as general health. The purpose of this study was to understand the genetic mechanism by which p53 regulates aerobic exercise capacity. Using a variety of physiological, metabolic, and molecular techniques, we further characterized maximum exercise capacity and the effects of training, measured various nonmitochondrial and mitochondrial determinants of exercise capacity, and examined putative regulators of mitochondrial biogenesis. As p53 did not affect baseline cardiac function or inotropic reserve, we focused on the involvement of skeletal muscle and now report a wider role for p53 in modulating skeletal muscle mitochondrial function. p53 interacts with Mitochondrial Transcription Factor A (TFAM), a nuclear-encoded gene important for mitochondrial DNA (mtDNA) transcription and maintenance, and regulates mtDNA content. The increased mtDNA in p53(+/+) compared to p53(-/-) mice was more marked in aerobic versus glycolytic skeletal muscle groups with no significant changes in cardiac tissue. These in vivo observations were further supported by in vitro studies showing overexpression of p53 in mouse myoblasts increases both TFAM and mtDNA levels whereas depletion of TFAM by shRNA decreases mtDNA content. Our current findings indicate that p53 promotes aerobic metabolism and exercise capacity by using different mitochondrial genes and mechanisms in a tissue-specific manner.

Prenatal synthetic glucocorticoid treatment changes DNA methylation states in male organ systems: multigenerational effects.

PubMed

Crudo, Ariann; Petropoulos, Sophie; Moisiadis, Vasilis G; Iqbal, Majid; Kostaki, Alisa; Machnes, Ziv; Szyf, Moshe; Matthews, Stephen G

2012-07-01

Prenatal synthetic glucocorticoids (sGC) are administered to pregnant women at risk of delivering preterm, approximately 10% of all pregnancies. Animal studies have demonstrated that offspring exposed to elevated glucocorticoids, either by administration of sGC or as a result of maternal stress, are at increased risk of developing behavioral, endocrine, and metabolic abnormalities. DNA methylation is a covalent modification of DNA that plays a critical role in long-lasting programming of gene expression. Here we tested the hypothesis that prenatal sGC treatment has both acute and long-term effects on DNA methylation states in the fetus and offspring and that these effects extend into a subsequent generation. Pregnant guinea pigs were treated with sGC in late gestation, and methylation analysis by luminometric methylation assay was undertaken in organs from fetuses and offspring across two generations. Expression of genes that modify the epigenetic state were measured by quantitative real-time PCR. Results indicate that there are organ-specific developmental trajectories of methylation in the fetus and newborn. Furthermore, these trajectories are substantially modified by intrauterine exposure to sGC. These sGC-induced changes in DNA methylation remain into adulthood and are evident in the next generation. Furthermore, prenatal sGC exposure alters the expression of several genes encoding proteins that modulate the epigenetic state. Several of these changes are long lasting and are also present in the next generation. These data support the hypothesis that prenatal sGC exposure leads to broad changes in critical components of the epigenetic machinery and that these effects can pass to the next generation.

Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation.

PubMed

Stigliani, S; Anserini, P; Venturini, P L; Scaruffi, P

2013-10-01

Is the amount of cell-free DNA released by human embryos into culture medium correlated with embryo morphological features? The mitochondrial DNA (mtDNA) content of culture medium is significantly associated with the fragmentation rate on Days 2 and 3 of embryo development, whether the oocyte came from women ≤ 35 or >35 years old. Cellular fragmentation is often utilized as one of the morphological parameters for embryo quality assessment. The amount of cellular fragments is considered to be an important morphological parameter for embryo implantation potential. It has been hypothesized that fragments are apoptotic bodies or anuclear cytoplasmatic pieces of blastomeres, although no definitive conclusion has been drawn about their pathogenesis. Human fertilized oocytes were individually cultured from Day 1 to Days 2 and 3. A total of 800 samples (166 spent media from Day 2 and 634 from Day 3) were enrolled into the present study. Double-stranded DNA (dsDNA) was quantified in 800 spent embryo culture media by Pico Green dye fluorescence assay. After DNA purification, genomic DNA (gDNA) and mtDNA were profiled by specific quantitative PCR. Statistical analyses defined correlations among DNA contents, embryo morphology and maternal age. Different independent tests confirmed the presence of DNA into embryo culture medium and, for the first time, we demonstrate that both gDNA and mtDNA are detectable in the secretome. The amount of DNA is larger in embryos with bad quality cleavage compared with high-grade embryos, suggesting that the DNA profile of culture medium is an objective marker for embryo quality assessment. In particular, DNA profiles are significantly associated with fragmentation feature (total dsDNA: P = 0.0010; mtDNA; P = 0.0247) and advanced maternal age. It is necessary to establish whether DNA profiling of spent embryo culture medium is a robust onsite test that can improve the prediction of blastulation, implantation and/or pregnancy rate. The

Characterization of four species of Trichuris (Nematoda: Enoplida) by their second internal transcribed spacer ribosomal DNA sequence.

PubMed

Oliveros, R; Cutillas, C; De Rojas, M; Arias, P

2000-12-01

Adult worms of Trichuris ovis and T. globulosa were collected from Ovis aries (sheep) and Capra hircus (goats). T. suis was isolated from Sus scrofa domestica (swine) and T. leporis was isolated from Lepus europaeus (rabbits) in Spain. Genomic DNA was isolated and a ribosomal internal transcribed spacer (ITS2) was amplified and sequenced using polymerase-chain-reaction (PCR) techniques. The ITS2 of T. ovis and T. globulosa was 407 nucleotides in length and had a GC content of about 62%. Furthermore, the ITS2 of T. suis and T. leporis was 534 and 418 nucleotides in length and had a GC content of about 64.8% and 62.4%, respectively. There was evidence of slight variation in the sequence within individuals of all species analyzed, indicating intraindividual variation in the sequence of different copies of the ribosomal DNA. Furthermore, low-level intraspecific variation was detected. Sequence analyses of ITS2 products of T. ovis and T. globulosa demonstrated no sequence difference between them. Nevertheless, differences were detected between the ITS2 sequences of T. suis, T. leporis, and T. ovis, indicating that Trichuris species can reliably be differentiated by their ITS2 sequences and PCR-linked restriction-fragment-length polymorphism (RFLP).

Stacked-unstacked equilibrium at the nick site of DNA.

PubMed

Protozanova, Ekaterina; Yakovchuk, Peter; Frank-Kamenetskii, Maxim D

2004-09-17

Stability of duplex DNA with respect to separation of complementary strands is crucial for DNA executing its major functions in the cell and it also plays a central role in major biotechnology applications of DNA: DNA sequencing, polymerase chain reaction, and DNA microarrays. Two types of interaction are well known to contribute to DNA stability: stacking between adjacent base-pairs and pairing between complementary bases. However, their contribution into the duplex stability is yet to be determined. Now we fill this fundamental gap in our knowledge of the DNA double helix. We have prepared a series of 32, 300 bp-long DNA fragments with solitary nicks in the same position differing only in base-pairs flanking the nick. Electrophoretic mobility of these fragments in the gel has been studied. Assuming the equilibrium between stacked and unstacked conformations at the nick site, all 32 stacking free energy parameters have been obtained. Only ten of them are essential and they govern the stacking interactions between adjacent base-pairs in intact DNA double helix. A full set of DNA stacking parameters has been determined for the first time. From these data and from a well-known dependence of DNA melting temperature on G.C content, the contribution of base-pairing into duplex stability has been estimated. The obtained energy parameters of the DNA double helix are of paramount importance for understanding sequence-dependent DNA flexibility and for numerous biotechnology applications.

Codon optimization of antigen coding sequences improves the immune potential of DNA vaccines against avian influenza virus H5N1 in mice and chickens.

PubMed

Stachyra, Anna; Redkiewicz, Patrycja; Kosson, Piotr; Protasiuk, Anna; Góra-Sochacka, Anna; Kudla, Grzegorz; Sirko, Agnieszka

2016-08-26

Highly pathogenic avian influenza viruses are a serious threat to domestic poultry and can be a source of new human pandemic and annual influenza strains. Vaccination is the main strategy of protection against influenza, thus new generation vaccines, including DNA vaccines, are needed. One promising approach for enhancing the immunogenicity of a DNA vaccine is to maximize its expression in the immunized host. The immunogenicity of three variants of a DNA vaccine encoding hemagglutinin (HA) from the avian influenza virus A/swan/Poland/305-135V08/2006 (H5N1) was compared in two animal models, mice (BALB/c) and chickens (broilers and layers). One variant encoded the wild type HA while the other two encoded HA without proteolytic site between HA1 and HA2 subunits and differed in usage of synonymous codons. One of them was enriched for codons preferentially used in chicken genes, while in the other modified variant the third position of codons was occupied in almost 100 % by G or C nucleotides. The variant of the DNA vaccine containing almost 100 % of the GC content in the third position of codons stimulated strongest immune response in two animal models, mice and chickens. These results indicate that such modification can improve not only gene expression but also immunogenicity of DNA vaccine. Enhancement of the GC content in the third position of the codon might be a good strategy for development of a variant of a DNA vaccine against influenza that could be highly effective in distant hosts, such as birds and mammals, including humans.

Vertebrate codon bias indicates a highly GC-rich ancestral genome.

PubMed

Nabiyouni, Maryam; Prakash, Ashwin; Fedorov, Alexei

2013-04-25

Two factors are thought to have contributed to the origin of codon usage bias in eukaryotes: 1) genome-wide mutational forces that shape overall GC-content and create context-dependent nucleotide bias, and 2) positive selection for codons that maximize efficient and accurate translation. Particularly in vertebrates, these two explanations contradict each other and cloud the origin of codon bias in the taxon. On the one hand, mutational forces fail to explain GC-richness (~60%) of third codon positions, given the GC-poor overall genomic composition among vertebrates (~40%). On the other hand, positive selection cannot easily explain strict regularities in codon preferences. Large-scale bioinformatic assessment, of nucleotide composition of coding and non-coding sequences in vertebrates and other taxa, suggests a simple possible resolution for this contradiction. Specifically, we propose that the last common vertebrate ancestor had a GC-rich genome (~65% GC). The data suggest that whole-genome mutational bias is the major driving force for generating codon bias. As the bias becomes prominent, it begins to affect translation and can result in positive selection for optimal codons. The positive selection can, in turn, significantly modulate codon preferences. Copyright © 2013 Elsevier B.V. All rights reserved.

Immunotherapy for Prostate Cancer with Gc Protein-Derived Macrophage-Activating Factor, GcMAF1

PubMed Central

Yamamoto, Nobuto; Suyama, Hirofumi; Yamamoto, Nobuyuki

2008-01-01

Serum Gc protein (known as vitamin D3-binding protein) is the precursor for the principal macrophage-activating factor (MAF). The MAF precursor activity of serum Gc protein of prostate cancer patients was lost or reduced because Gc protein was deglycosylated by serum α-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Therefore, macrophages of prostate cancer patients having deglycosylated Gc protein cannot be activated, leading to immunosuppression. Stepwise treatment of purified Gc protein with immobilized β-galactosidase and sialidase generated the most potent MAF (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages activated by GcMAF develop a considerable variation of receptors that recognize the abnormality in malignant cell surface and are highly tumoricidal. Sixteen nonanemic prostate cancer patients received weekly administration of 100 ng of GcMAF. As the MAF precursor activity increased, their serum Nagalase activity decreased. Because serum Nagalase activity is proportional to tumor burden, the entire time course analysis for GcMAF therapy was monitored by measuring the serum Nagalase activity. After 14 to 25 weekly administrations of GcMAF (100 ng/week), all 16 patients had very low serum Nagalase levels equivalent to those of healthy control values, indicating that these patients are tumor-free. No recurrence occurred for 7 years. PMID:18633461

Mitochondrial DNA and STR analyses for human DNA from maggots crop contents: a forensic entomology case from central-southern China.

PubMed

Li, X; Cai, J F; Guo, Y D; Xiong, F; Zhang, L; Feng, H; Meng, F M; Fu, Y; Li, J B; Chen, Y Q

2011-08-01

Insect larvae and adult insects found on human corpses can provide important forensic evidence however it is useful to be able to prove evidence of association. Without this, it could be claimed that the insect evidence was a contaminant or had been planted on the body. This paper describes how mitochondrial DNA (mtDNA) and STR analysis of the crop contents of larvae of the blowfly Aldrichina grahami collected from separated body parts was used to provide evidence of association.

Differential Mobility Spectrometry: Preliminary Findings on Determination of Fundamental Constants

NASA Technical Reports Server (NTRS)

Limero, Thomas; Cheng, Patti; Boyd, John

2007-01-01

The electron capture detector (ECD) has been used for 40+ years (1) to derive fundamental constants such as a compound's electron affinity. Given this historical perspective, it is not surprising that differential mobility spectrometry (DMS) might be used in a like manner. This paper will present data from a gas chromatography (GC)-DMS instrument that illustrates the potential capability of this device to derive fundamental constants for electron-capturing compounds. Potential energy curves will be used to provide possible explanation of the data.

Prenatal Ambient Air Pollution, Placental Mitochondrial DNA Content, and Birth Weight in the INMA (Spain) and ENVIRONAGE (Belgium) Birth Cohorts.

PubMed

Clemente, Diana B P; Casas, Maribel; Vilahur, Nadia; Begiristain, Haizea; Bustamante, Mariona; Carsin, Anne-Elie; Fernández, Mariana F; Fierens, Frans; Gyselaers, Wilfried; Iñiguez, Carmen; Janssen, Bram G; Lefebvre, Wouter; Llop, Sabrina; Olea, Nicolás; Pedersen, Marie; Pieters, Nicky; Santa Marina, Loreto; Souto, Ana; Tardón, Adonina; Vanpoucke, Charlotte; Vrijheid, Martine; Sunyer, Jordi; Nawrot, Tim S

2016-05-01

Mitochondria are sensitive to environmental toxicants due to their lack of repair capacity. Changes in mitochondrial DNA (mtDNA) content may represent a biologically relevant intermediate outcome in mechanisms linking air pollution and fetal growth restriction. We investigated whether placental mtDNA content is a possible mediator of the association between prenatal nitrogen dioxide (NO2) exposure and birth weight. We used data from two independent European cohorts: INMA (n = 376; Spain) and ENVIRONAGE (n = 550; Belgium). Relative placental mtDNA content was determined as the ratio of two mitochondrial genes (MT-ND1 and MTF3212/R3319) to two control genes (RPLP0 and ACTB). Effect estimates for individual cohorts and the pooled data set were calculated using multiple linear regression and mixed models. We also performed a mediation analysis. Pooled estimates indicated that a 10-μg/m3 increment in average NO2 exposure during pregnancy was associated with a 4.9% decrease in placental mtDNA content (95% CI: -9.3, -0.3%) and a 48-g decrease (95% CI: -87, -9 g) in birth weight. However, the association with birth weight was significant for INMA (-66 g; 95% CI: -111, -23 g) but not for ENVIRONAGE (-20 g; 95% CI: -101, 62 g). Placental mtDNA content was associated with significantly higher mean birth weight (pooled analysis, interquartile range increase: 140 g; 95% CI: 43, 237 g). Mediation analysis estimates, which were derived for the INMA cohort only, suggested that 10% (95% CI: 6.6, 13.0 g) of the association between prenatal NO2 and birth weight was mediated by changes in placental mtDNA content. Our results suggest that mtDNA content can be one of the potential mediators of the association between prenatal air pollution exposure and birth weight. Clemente DB, Casas M, Vilahur N, Begiristain H, Bustamante M, Carsin AE, Fernández MF, Fierens F, Gyselaers W, Iñiguez C, Janssen BG, Lefebvre W, Llop S, Olea N, Pedersen M, Pieters N, Santa Marina L, Souto A, Tardón A

Prenatal Ambient Air Pollution, Placental Mitochondrial DNA Content, and Birth Weight in the INMA (Spain) and ENVIRONAGE (Belgium) Birth Cohorts

PubMed Central

Clemente, Diana B.P.; Casas, Maribel; Vilahur, Nadia; Begiristain, Haizea; Bustamante, Mariona; Carsin, Anne-Elie; Fernández, Mariana F.; Fierens, Frans; Gyselaers, Wilfried; Iñiguez, Carmen; Janssen, Bram G.; Lefebvre, Wouter; Llop, Sabrina; Olea, Nicolás; Pedersen, Marie; Pieters, Nicky; Santa Marina, Loreto; Souto, Ana; Tardón, Adonina; Vanpoucke, Charlotte; Vrijheid, Martine; Sunyer, Jordi; Nawrot, Tim S.

2015-01-01

Background: Mitochondria are sensitive to environmental toxicants due to their lack of repair capacity. Changes in mitochondrial DNA (mtDNA) content may represent a biologically relevant intermediate outcome in mechanisms linking air pollution and fetal growth restriction. Objective: We investigated whether placental mtDNA content is a possible mediator of the association between prenatal nitrogen dioxide (NO2) exposure and birth weight. Methods: We used data from two independent European cohorts: INMA (n = 376; Spain) and ENVIRONAGE (n = 550; Belgium). Relative placental mtDNA content was determined as the ratio of two mitochondrial genes (MT-ND1 and MTF3212/R3319) to two control genes (RPLP0 and ACTB). Effect estimates for individual cohorts and the pooled data set were calculated using multiple linear regression and mixed models. We also performed a mediation analysis. Results: Pooled estimates indicated that a 10-μg/m3 increment in average NO2 exposure during pregnancy was associated with a 4.9% decrease in placental mtDNA content (95% CI: –9.3, –0.3%) and a 48-g decrease (95% CI: –87, –9 g) in birth weight. However, the association with birth weight was significant for INMA (–66 g; 95% CI: –111, –23 g) but not for ENVIRONAGE (–20 g; 95% CI: –101, 62 g). Placental mtDNA content was associated with significantly higher mean birth weight (pooled analysis, interquartile range increase: 140 g; 95% CI: 43, 237 g). Mediation analysis estimates, which were derived for the INMA cohort only, suggested that 10% (95% CI: 6.6, 13.0 g) of the association between prenatal NO2 and birth weight was mediated by changes in placental mtDNA content. Conclusion: Our results suggest that mtDNA content can be one of the potential mediators of the association between prenatal air pollution exposure and birth weight. Citation: Clemente DB, Casas M, Vilahur N, Begiristain H, Bustamante M, Carsin AE, Fernández MF, Fierens F, Gyselaers W, Iñiguez C, Janssen BG

An association analysis between mitochondrial DNA content, G10398A polymorphism, HPV infection, and the prognosis of cervical cancer in the Chinese Han population.

PubMed

Feng, Dali; Xu, Hui; Li, Xin; Wei, Yuehua; Jiang, Huangang; Xu, Hong; Luo, Aihua; Zhou, Fuxiang

2016-04-01

The aim was to analyze quantitative (mitochondrial DNA (mtDNA) content) and qualitative (G10398A polymorphism) mtDNA alterations as well as human papillomavirus (HPV) infection in cervical cancer prognosis. One hundred and twenty-two cases of formalin-fixed paraffin-embedded cervical carcinoma specimens were collected from the Yichang Tumor Hospital and Zhongnan Hospital of Wuhan University in the recent 10 years together with medical records. A quantitative real-time PCR (RT-PCR) was used to determine the copy number of the mitochondrial DNA and HPV expression levels. G10398A polymorphism was determined by PCR-RFLP assay. The overall survival of patients with higher mtDNA content was significantly reduced compared with lower mtDNA content patients (P = 0.029). But there was no difference of prognosis between the mtDNA 10398 A allele and G allele. However, the Kaplan-Meier survival curve illustrated a significantly reduced overall survival in the patients with 10398A plus high mtDNA copy number compared with the other groups (P < 0.05). Although no association between HPV expression level and cervical cancer prognosis was observed, 10398A got increased mtDNA content compared with 10398G (P < 0.05) and 10398G displayed an increased HPV-positive rate compared with 10398A. Furthermore, HPV-18 and mtDNA content were positively related in the younger subgroup (≤45 years) (correlation coefficient = 0.456, P = 0.022). This study indicated that mtDNA content and HPV infection status are associated with cervical cancer prognosis. High mitochondrial DNA content plus 10398 A may be a marker of poor prognosis in cervical cancer. And mtDNA variation may potentially influence the predisposition to HPV infection and cervical carcinogenesis.

Preparation of Gc protein-derived macrophage activating factor (GcMAF) and its structural characterization and biological activities.

PubMed

Mohamad, Saharuddin Bin; Nagasawa, Hideko; Uto, Yoshihiro; Hori, Hitoshi

2002-01-01

Gc protein has been reported to be a precursor of Gc protein-derived macrophage activation factor (GcMAF) in the inflammation-primed macrophage activation cascade. An inducible beta-galactosidase of B cells and neuraminidase of T cells convert Gc protein to GcMAF. Gc protein from human serum was purified using 25(OH)D3 affinity column chromatography and modified to GcMAF using immobilized glycosidases (beta-galactosidase and neuraminidase) The sugar moiety structure of GcMAF was characterized by lectin blotting by Helix pomatia agglutinin. The biological activities of GcMAF were evaluated by a superoxide generation assay and a phagocytosis assay. We successfully purified Gc protein from human serum. GcMAF was detected by lectin blotting and showed a high biological activity. Our results support the importance of the terminal N-acetylgalactosamine moiety in the GcMAF-mediated macrophage activation cascade, and the existence of constitutive GcMAF in human serum. These preliminary data are important for designing small molecular GcMAF mimics.

Cytophotometric and biochemical analyses of DNA in pentaploid and diploid Agave species.

PubMed

Cavallini, A; Natali, L; Cionini, G; Castorena-Sanchez, I

1996-04-01

Nuclear DNA content, chromatin structure, and DNA composition were investigated in four Agave species: two diploid, Agave tequilana Weber and Agave angustifolia Haworth var. marginata Hort., and two pentaploid, Agave fourcroydes Lemaire and Agave sisalana Perrine. It was determined that the genome size of pentaploid species is nearly 2.5 times that of diploid ones. Cytophotometric analyses of chromatin structure were performed following Feulgen or DAPI staining to determine optical density profiles of interphase nuclei. Pentaploid species showed higher frequencies of condensed chromatin (heterochromatin) than diploid species. On the other hand, a lower frequency of A-T rich (DAPI stained) heterochromatin was found in pentaploid species than in diploid ones, indicating that heterochromatin in pentaploid species is made up of sequences with base compositions different from those of diploid species. Since thermal denaturation profiles of extracted DNA showed minor variations in the base composition of the genomes of the four species, it is supposed that, in pentaploid species, the large heterochromatin content is not due to an overrepresentation of G-C repetitive sequences but rather to the condensation of nonrepetitive sequences, such as, for example, redundant gene copies switched off in the polyploid complement. It is suggested that speciation in the genus Agave occurs through point mutations and minor DNA rearrangements, as is also indicated by the relative stability of the karyotype of this genus. Key words : Agave, DNA cytophotometry, DNA melting profiles, chromatin structure, genome size.

The experimental and theoretical QM/MM study of interaction of chloridazon herbicide with ds-DNA

NASA Astrophysics Data System (ADS)

Ahmadi, F.; Jamali, N.; Jahangard-Yekta, S.; Jafari, B.; Nouri, S.; Najafi, F.; Rahimi-Nasrabadi, M.

2011-09-01

We report a multispectroscopic, voltammetric and theoretical hybrid of QM/MM study of the interaction between double-stranded DNA containing both adenine-thymine and guanine-cytosine alternating sequences and chloridazon (CHL) herbicide. The electrochemical behavior of CHL was studied by cyclic voltammetry on HMDE, and the interaction of ds-DNA with CHL was investigated by both cathodic differential pulse voltammetry (CDPV) at a hanging mercury drop electrode (HMDE) and anodic differential pulse voltammetry (ADPV) at a glassy carbon electrode (GCE). The constant bonding of CHL-DNA complex that was obtained by UV/vis, CDPV and ADPV was 2.1 × 10 4, 5.1 × 10 4 and 2.6 × 10 4, respectively. The competition fluorescence studies revealed that the CHL quenches the fluorescence of DNA-ethidium bromide complex significantly and the apparent Stern-Volmer quenching constant has been estimated to be 1.71 × 10 4. Thermal denaturation study of DNA with CHL revealed the Δ Tm of 8.0 ± 0.2 °C. Thermodynamic parameters, i.e., enthalpy (Δ H), entropy (Δ S°), and Gibbs free energy (Δ G) were 98.45 kJ mol -1, 406.3 J mol -1 and -22.627 kJ mol -1, respectively. The ONIOM, based on the hybridization of QM/MM (DFT, 6.31++G(d,p)/UFF) methodology, was also performed using Gaussian 2003 package. The results revealed that the interaction is base sequence dependent, and the CHL has more interaction with ds-DNA via the GC base sequence. The results revealed that CHL may have an interaction with ds-DNA via the intercalation mode.

Effects of Particulate Matter on Genomic DNA Methylation Content and iNOS Promoter Methylation

PubMed Central

Tarantini, Letizia; Bonzini, Matteo; Apostoli, Pietro; Pegoraro, Valeria; Bollati, Valentina; Marinelli, Barbara; Cantone, Laura; Rizzo, Giovanna; Hou, Lifang; Schwartz, Joel; Bertazzi, Pier Alberto; Baccarelli, Andrea

2009-01-01

Background Altered patterns of gene expression mediate the effects of particulate matter (PM) on human health, but mechanisms through which PM modifies gene expression are largely undetermined. Objectives We aimed at identifying short- and long-term effects of PM exposure on DNA methylation, a major genomic mechanism of gene expression control, in workers in an electric furnace steel plant with well-characterized exposure to PM with aerodynamic diameters < 10 μm (PM10). Methods We measured global genomic DNA methylation content estimated in Alu and long interspersed nuclear element-1 (LINE-1) repeated elements, and promoter DNA methylation of iNOS (inducible nitric oxide synthase), a gene suppressed by DNA methylation and induced by PM exposure in blood leukocytes. Quantitative DNA methylation analysis was performed through bisulfite PCR pyrosequencing on blood DNA obtained from 63 workers on the first day of a work week (baseline, after 2 days off work) and after 3 days of work (postexposure). Individual PM10 exposure was between 73.4 and 1,220 μg/m3. Results Global methylation content estimated in Alu and LINE-1 repeated elements did not show changes in postexposure measures compared with baseline. PM10 exposure levels were negatively associated with methylation in both Alu [β = −0.19 %5-methylcytosine (%5mC); p = 0.04] and LINE-1 [β = −0.34 %5mC; p = 0.04], likely reflecting long-term PM10 effects. iNOS promoter DNA methylation was significantly lower in postexposure blood samples compared with baseline (difference = −0.61 %5mC; p = 0.02). Conclusions We observed changes in global and gene specific methylation that should be further characterized in future investigations on the effects of PM. PMID:19270791

Raman study of CaDNA films as a function of water content and excess CaCl2 concentration: Stability of the B conformation.

NASA Astrophysics Data System (ADS)

Schwenker, Megan; Marlowe, Robert; Lee, Scott; Rupprecht, Allan

2006-03-01

Highly oriented, wet-spun films of CaDNA expand in the direction perpendicular to the helical axis as the hydration of the film is increased. CaDNA films with a high CaCl2 content show an unexpected shrinkage at a relative humidity of about 93%. We have performed Raman experiments on CaDNA films as a function of both water content and excess CaCl2 concentration in order to determine if this unexpected shrinkage might be related to a conformational transition of the DNA molecules. We find that the DNA molecules remain in the B conformation for all salt contents down to a relative humidity of 59%.

[A study on the relationship between postmortem interval and the changes of DNA content in the kidney cellule of rat].

PubMed

Liu, L; Peng, D B; Liu, Y; Deng, W N; Liu, Y L; Li, J J

2001-05-01

To study changes of DNA content in the kidney cellule of rats and relationship with the postmortem interval. This experiment chose seven parameter of cell nuclear, including the area and integral optical density, determined the changes of DNA content in the kidney cellule of 15 rats at different intervals between 0 and 48 h postmortem with auto-TV-image system. The degradation rate of DNA in nuclear has a certainty relationship to early PMI(in 48 h) of rat, and get binomial regress equation. Determining the quantity of DNA in nuclear should be an objective and exact way to estimate the PMI.

Library Construction from Subnanogram DNA for Pelagic Sea Water and Deep-Sea Sediments

PubMed Central

Hirai, Miho; Nishi, Shinro; Tsuda, Miwako; Sunamura, Michinari; Takaki, Yoshihiro; Nunoura, Takuro

2017-01-01

Shotgun metagenomics is a low biased technology for assessing environmental microbial diversity and function. However, the requirement for a sufficient amount of DNA and the contamination of inhibitors in environmental DNA leads to difficulties in constructing a shotgun metagenomic library. We herein examined metagenomic library construction from subnanogram amounts of input environmental DNA from subarctic surface water and deep-sea sediments using two library construction kits: the KAPA Hyper Prep Kit and Nextera XT DNA Library Preparation Kit, with several modifications. The influence of chemical contaminants associated with these environmental DNA samples on library construction was also investigated. Overall, shotgun metagenomic libraries were constructed from 1 pg to 1 ng of input DNA using both kits without harsh library microbial contamination. However, the libraries constructed from 1 pg of input DNA exhibited larger biases in GC contents, k-mers, or small subunit (SSU) rRNA gene compositions than those constructed from 10 pg to 1 ng DNA. The lower limit of input DNA for low biased library construction in this study was 10 pg. Moreover, we revealed that technology-dependent biases (physical fragmentation and linker ligation vs. tagmentation) were larger than those due to the amount of input DNA. PMID:29187708

Fabrication of flower-like direct Z-scheme β-Bi2O3/g-C3N4 photocatalyst with enhanced visible light photoactivity for Rhodamine B degradation

NASA Astrophysics Data System (ADS)

Zhang, Liping; Wang, Guohong; Xiong, Zhenzhong; Tang, Hua; Jiang, Chuanjia

2018-04-01

A combined hydrothermal-calcination approach is developed to synthesize hierarchical β-Bi2O3/g-C3N4 direct Z-scheme photocatalyst with enhanced visible light photoactivity for Rhodamine B (RhB) degradation. First, Bi2O2CO3 microflowers were hydrothermally prepared using Bi(NO3)3·5H2O as feedstocks, and then a series of β-Bi2O3/g-C3N4 direct Z-scheme photocatalysts were synthesized via a facile calcination method using Bi2O2CO3 and g-C3N4 as precursors. The samples were systematically characterized by various characterization technologies including X-ray diffraction, scanning and transmission electron microscopes, Fourier transform infrared spectroscopy and N2 absorption-desorption equipment. It was found that the g-C3N4 content in the precursors played a key role in affecting the photocatalytic activity of the final products. The β-Bi2O3/g-C3N4 heterojunction exhibited higher photocatalytic activity than single active components (β-Bi2O3 and g-C3N4), indicating the presence of a synergistic effect between two active components in β-Bi2O3/g-C3N4 heterojunction. Among all as-prepared catalysts, the 70 wt.% g-C3N4/Bi2O2CO3 exhibits the highest activity for RhB degradation, and the apparent reaction rate constant k (42.2 × 10-3 min-1) is 3.1 and 1.7 times as high as that of pure β-Bi2O3 (13.5 × 10-3 min-1) and g-C3N4 (25.2 × 10-3 min-1), respectively. The enhanced photocatalytic performance of β-Bi2O3/g-C3N4 heterostructure photocatalysts is mainly due to the high surface area, closely contacted interfaces between the β-Bi2O3 and g-C3N4 component, and the formation of direct Z-scheme structure in the β-Bi2O3/g-C3N4 composites.

Drosophila Melanogaster Mitochondrial DNA: Gene Organization and Evolutionary Considerations

PubMed Central

Garesse, R.

1988-01-01

The sequence of a 8351-nucleotide mitochondrial DNA (mtDNA) fragment has been obtained extending the knowledge of the Drosophila melanogaster mitochondrial genome to 90% of its coding region. The sequence encodes seven polypeptides, 12 tRNAs and the 3' end of the 16S rRNA and CO III genes. The gene organization is strictly conserved with respect to the Drosophila yakuba mitochondrial genome, and different from that found in mammals and Xenopus. The high A + T content of D. melanogaster mitochondrial DNA is reflected in a reiterative codon usage, with more than 90% of the codons ending in T or A, G + C rich codons being practically absent. The average level of homology between the D. melanogaster and D. yakuba sequences is very high (roughly 94%), although insertion and deletions have been detected in protein, tRNA and large ribosomal genes. The analysis of nucleotide changes reveals a similar frequency for transitions and transversions, and reflects a strong bias against G+C on both strands. The predominant type of transition is strand specific. PMID:3130291

Enhanced photocatalytic activity of electrospun nanofibrous TiO2/g-C3N4 heterojunction photocatalyst under simulated solar light

NASA Astrophysics Data System (ADS)

Wang, Chunlei; Hu, Liming; Chai, Bo; Yan, Juntao; Li, Jianfen

2018-02-01

Electrospun nanofibrous TiO2/g-C3N4 heterojunction photocatalysts with different TiO2 content have been synthesized via a facile electrospinning and subsequent in situ evaporation and calcination process for the first time, which are examined in terms of morphology, component content, optical properties, PL spectra, photocurrent response, EIS measurement, photocatalytic activity and mechanism. SEM images exhibit TiO2/g-C3N4-4 heterojunction photocatalyst possesses the excellent 1D structure. HRTEM and element mapping images confirm the formation of heterojunction structure. DRS tests identify that TiO2/g-C3N4-4 heterojunction exhibits the intensitive absorption in both UV and visible light region. The photoelectrochemical tests prove that the recombination between electrons and holes are effectively inhibited. Based on TG analysis and photodegradation experiments, TiO2/g-C3N4-4 heterojunction photocatalyst with TiO2 content of 29.30 wt% possesses the best photocatalytic degradation efficiency for the RhB among the g-C3N4, TiO2 and their mixture under simulated sunlight irradiation. Moreover, 1D morphology of TiO2/g-C3N4-4 heterojunction photocatalyst is in favor of separating from solution for reuse and transferring the electrons, and maintains a very high photocatalytic degradation efficiency of 96% even after four recycles experiments, which is beneficial for practical application.

Large-scale DNA Barcode Library Generation for Biomolecule Identification in High-throughput Screens.

PubMed

Lyons, Eli; Sheridan, Paul; Tremmel, Georg; Miyano, Satoru; Sugano, Sumio

2017-10-24

High-throughput screens allow for the identification of specific biomolecules with characteristics of interest. In barcoded screens, DNA barcodes are linked to target biomolecules in a manner allowing for the target molecules making up a library to be identified by sequencing the DNA barcodes using Next Generation Sequencing. To be useful in experimental settings, the DNA barcodes in a library must satisfy certain constraints related to GC content, homopolymer length, Hamming distance, and blacklisted subsequences. Here we report a novel framework to quickly generate large-scale libraries of DNA barcodes for use in high-throughput screens. We show that our framework dramatically reduces the computation time required to generate large-scale DNA barcode libraries, compared with a naїve approach to DNA barcode library generation. As a proof of concept, we demonstrate that our framework is able to generate a library consisting of one million DNA barcodes for use in a fragment antibody phage display screening experiment. We also report generating a general purpose one billion DNA barcode library, the largest such library yet reported in literature. Our results demonstrate the value of our novel large-scale DNA barcode library generation framework for use in high-throughput screening applications.

Uncovering Trophic Interactions in Arthropod Predators through DNA Shotgun-Sequencing of Gut Contents

PubMed Central

Paula, Débora P.; Linard, Benjamin; Crampton-Platt, Alex; Srivathsan, Amrita; Timmermans, Martijn J. T. N.; Sujii, Edison R.; Pires, Carmen S. S.; Souza, Lucas M.; Andow, David A.; Vogler, Alfried P.

2016-01-01

Characterizing trophic networks is fundamental to many questions in ecology, but this typically requires painstaking efforts, especially to identify the diet of small generalist predators. Several attempts have been devoted to develop suitable molecular tools to determine predatory trophic interactions through gut content analysis, and the challenge has been to achieve simultaneously high taxonomic breadth and resolution. General and practical methods are still needed, preferably independent of PCR amplification of barcodes, to recover a broader range of interactions. Here we applied shotgun-sequencing of the DNA from arthropod predator gut contents, extracted from four common coccinellid and dermapteran predators co-occurring in an agroecosystem in Brazil. By matching unassembled reads against six DNA reference databases obtained from public databases and newly assembled mitogenomes, and filtering for high overlap length and identity, we identified prey and other foreign DNA in the predator guts. Good taxonomic breadth and resolution was achieved (93% of prey identified to species or genus), but with low recovery of matching reads. Two to nine trophic interactions were found for these predators, some of which were only inferred by the presence of parasitoids and components of the microbiome known to be associated with aphid prey. Intraguild predation was also found, including among closely related ladybird species. Uncertainty arises from the lack of comprehensive reference databases and reliance on low numbers of matching reads accentuating the risk of false positives. We discuss caveats and some future prospects that could improve the use of direct DNA shotgun-sequencing to characterize arthropod trophic networks. PMID:27622637

Direct evidence for sequence-dependent attraction between double-stranded DNA controlled by methylation.

PubMed

Yoo, Jejoong; Kim, Hajin; Aksimentiev, Aleksei; Ha, Taekjip

2016-03-22

Although proteins mediate highly ordered DNA organization in vivo, theoretical studies suggest that homologous DNA duplexes can preferentially associate with one another even in the absence of proteins. Here we combine molecular dynamics simulations with single-molecule fluorescence resonance energy transfer experiments to examine the interactions between duplex DNA in the presence of spermine, a biological polycation. We find that AT-rich DNA duplexes associate more strongly than GC-rich duplexes, regardless of the sequence homology. Methyl groups of thymine acts as a steric block, relocating spermine from major grooves to interhelical regions, thereby increasing DNA-DNA attraction. Indeed, methylation of cytosines makes attraction between GC-rich DNA as strong as that between AT-rich DNA. Recent genome-wide chromosome organization studies showed that remote contact frequencies are higher for AT-rich and methylated DNA, suggesting that direct DNA-DNA interactions that we report here may play a role in the chromosome organization and gene regulation.

Direct evidence for sequence-dependent attraction between double-stranded DNA controlled by methylation

NASA Astrophysics Data System (ADS)

Yoo, Jejoong; Kim, Hajin; Aksimentiev, Aleksei; Ha, Taekjip

2016-03-01

Although proteins mediate highly ordered DNA organization in vivo, theoretical studies suggest that homologous DNA duplexes can preferentially associate with one another even in the absence of proteins. Here we combine molecular dynamics simulations with single-molecule fluorescence resonance energy transfer experiments to examine the interactions between duplex DNA in the presence of spermine, a biological polycation. We find that AT-rich DNA duplexes associate more strongly than GC-rich duplexes, regardless of the sequence homology. Methyl groups of thymine acts as a steric block, relocating spermine from major grooves to interhelical regions, thereby increasing DNA-DNA attraction. Indeed, methylation of cytosines makes attraction between GC-rich DNA as strong as that between AT-rich DNA. Recent genome-wide chromosome organization studies showed that remote contact frequencies are higher for AT-rich and methylated DNA, suggesting that direct DNA-DNA interactions that we report here may play a role in the chromosome organization and gene regulation.

Large-scale oscillation of structure-related DNA sequence features in human chromosome 21

NASA Astrophysics Data System (ADS)

Li, Wentian; Miramontes, Pedro

2006-08-01

Human chromosome 21 is the only chromosome in the human genome that exhibits oscillation of the (G+C) content of a cycle length of hundreds kilobases (kb) ( 500kb near the right telomere). We aim at establishing the existence of a similar periodicity in structure-related sequence features in order to relate this (G+C)% oscillation to other biological phenomena. The following quantities are shown to oscillate with the same 500kb periodicity in human chromosome 21: binding energy calculated by two sets of dinucleotide-based thermodynamic parameters, AA/TT and AAA/TTT bi- and tri-nucleotide density, 5'-TA-3' dinucleotide density, and signal for 10- or 11-base periodicity of AA/TT or AAA/TTT. These intrinsic quantities are related to structural features of the double helix of DNA molecules, such as base-pair binding, untwisting or unwinding, stiffness, and a putative tendency for nucleosome formation.

Silver(I) complexes with DNA and RNA studied by Fourier transform infrared spectroscopy and capillary electrophoresis.

PubMed Central

Arakawa, H; Neault, J F; Tajmir-Riahi, H A

2001-01-01

Ag(I) is a strong nucleic acids binder and forms several complexes with DNA such as types I, II, and III. However, the details of the binding mode of silver(I) in the Ag-polynucleotides remains unknown. Therefore, it was of interest to examine the binding of Ag(I) with calf-thymus DNA and bakers yeast RNA in aqueous solutions at pH 7.1-6.6 with constant concentration of DNA or RNA and various concentrations of Ag(I). Fourier transform infrared spectroscopy and capillary electrophoresis were used to analyze the Ag(I) binding mode, the binding constant, and the polynucleotides' structural changes in the Ag-DNA and Ag-RNA complexes. The spectroscopic results showed that in the type I complex formed with DNA, Ag(I) binds to guanine N7 at low cation concentration (r = 1/80) and adenine N7 site at higher concentrations (r = 1/20 to 1/10), but not to the backbone phosphate group. At r = 1/2, type II complexes formed with DNA in which Ag(I) binds to the G-C and A-T base pairs. On the other hand, Ag(I) binds to the guanine N7 atom but not to the adenine and the backbone phosphate group in the Ag-RNA complexes. Although a minor alteration of the sugar-phosphate geometry was observed, DNA remained in the B-family structure, whereas RNA retained its A conformation. Scatchard analysis following capillary electrophoresis showed two binding sites for the Ag-DNA complexes with K(1) = 8.3 x 10(4) M(-1) for the guanine and K(2) = 1.5 x 10(4) M(-1) for the adenine bases. On the other hand, Ag-RNA adducts showed one binding site with K = 1.5 x 10(5) M(-1) for the guanine bases. PMID:11509371

Characterization of nonstarch polysaccharides content from different edible organs of some vegetables, determined by GC and HPLC: comparative study.

PubMed

Villanueva-Suárez, M J; Redondo-Cuenca, A; Rodríguez-Sevilla, M D; de las Heras Martínez, M

2003-09-24

Content and composition of dietary fiber as nonstarch polysaccharides (NSP) was determined in vegetables belonging to different types of edible organs, using GC and HPLC. Samples analyzed were subterranean organs (radish and leek), leaves (celery, swiss chard, and lettuce), stalks (celery, swiss chard, and asparagus), inflorescence (broccoli), and fruits (tomato, green pepper, and marrow). The results indicate that though the monomeric profile is similar in all these samples quantitative differences were found for neutral sugars and uronic acids among samples of the same type of vegetal organ. The NSP values determined using CG method were in good agreement with HPLC method (R(2) = 0.9005). However, arabinose, mannose, and galactose plus rhamnose are more influenced by the analytical method used than the rest of the monomers in nearly all the samples analyzed. Final values of NSP depend on the method used in celery stalks, broccoli, and green pepper.

[Study on supercritical CO2 extraction of xiaoyaosan and its GC-MS fingerprint].

PubMed

Zuo, Ya-Mei; Tian, Jun-Sheng; Guo, Xiao-Qing; Zhou, Yu-Zhi; Gao, Xiao-Xia; Qin, Xue-Mei

2014-02-01

To determine the optimum conditions of supercritical CO2 extraction of Xiaoyaosan, and establish its fingerprint by gas chromatography-mass spectrometry (GC-MS), the yield of extract were investigated, an orthogonal test was used to quantify the effects of extraction temperature, pressure, CO2 flow rate and time, and fingerprint analysis of different batches of extracts were by GC-MS. The optimal extraction conditions were determined as follows: extraction pressure 20 MPa, extraction temperature 50 degrees C, CO2 flow rate 25 kg x h(-1), extraction time 3 h, and average yield 2.2%. The GC-MS fingerprint was established and 27 common peaks were found, whose contents add up to 81.89% of the total peak area. Among them, 21 compounds were identified, accounting for 53.20% of the total extract. The extraction process is reasonable and favorable for industrial production. The GC-MS method is accurate, reliable, reproducible, and can be used for quality control of supercritical CO2 extract from Xiaoyaosan.

DNA Content Differences Between Male and Female Chicken (Gallus gallus domesticus) Nuclei and Z and W Chromosomes Resolved by Image Cytometry

PubMed Central

Mendonça, Maria Andréia Corrêa; Carvalho, Carlos Roberto; Clarindo, Wellington Ronildo

2010-01-01

Chicken red blood cells (CRBCs) are widely used as standards for DNA content determination. Cytogenetic data have shown that the Z sex chromosome is approximately twice as large as the W, so that the DNA content differs to some extent between male (ZZ) and female (ZW) chickens. Despite this fact, male and female CRBCs have been indiscriminately used in absolute genome size determination. Our work was conducted to verify whether the DNA content differences between male and female Gallus gallus domesticus “Leghorn” nuclei and ZZ/ZW chromosomes can be resolved by image cytometry (ICM). Air-dried smears stained by Feulgen reaction were used for nuclei analysis. Chicken metaphase spreads upon Feulgen staining were analyzed for obtaining quantitative information on the Z and W chromosomes. Before each capture session, we conducted quality control of the ICM instrumentation. Our results from nuclear measurements showed that the 2C value is 0.09 pg higher in males than in females. In chromosomes, we found that the Z chromosome shows 200% more DNA content than does the W chromosome. ICM demonstrated resolution power to discriminate low DNA content differences in genomes. We suggest prudence in the general use of CRBC 2C values as standards in comparative cytometric analysis. (J Histochem Cytochem 58:229–235, 2010) PMID:19875846

Non-linear optical measurement of the twist elastic constant in thermotropic and DNA lyotropic chiral nematics.

PubMed

Lucchetti, Liana; Fraccia, Tommaso P; Ciciulla, Fabrizio; Bellini, Tommaso

2017-07-10

Throughout the whole history of liquid crystals science, the balancing of intrinsic elasticity with coupling to external forces has been the key strategy for most application and investigation. While the coupling of the optical field to the nematic director is at the base of a wealth of thoroughly described optical effects, a significant variety of geometries and materials have not been considered yet. Here we show that by adopting a simple cell geometry and measuring the optically induced birefringence, we can readily extract the twist elastic coefficient K 22 of thermotropic and lyotropic chiral nematics (N*). The value of K 22 we obtain for chiral doped 5CB thermotropic N* well matches those reported in the literature. With this same strategy, we could determine for the first time K 22 of the N* phase of concentrated aqueous solutions of DNA oligomers, bypassing the limitations that so far prevented measuring the elastic constants of this class of liquid crystalline materials. The present study also enlightens the significant nonlinear optical response of DNA liquid crystals.

Disentangling oil weathering using GC x GC. 1. chromatogram analysis.

PubMed

Arey, J Samuel; Nelson, Robert K; Reddy, Christopher M

2007-08-15

Historically, the thousands of compounds found in oils constituted an "unresolved complex mixture" that frustrated efforts to analyze oil weathering. Moreover, different weathering processes inflict rich and diverse signatures of compositional change in oil, and conventional methods do not effectively decode this elaborate record. Using comprehensive two-dimensional gas chromatography (GC x GC), we can separate thousands of hydrocarbon components and simultaneously estimate their chemical properties. We investigated 13 weathered field samples collected from the Bouchard 120 heavy fuel oil spill in Buzzards Bay, Massachusetts in 2003. We first mapped hydrocarbon vapor pressures and aqueous solubilities onto the compositional space explored by GC x GC chromatograms of weathered samples. Then we developed methods to quantitatively decouple mass loss patterns associated with evaporation and dissolution. The compositional complexity of oil, traditionally considered an obstacle, was now an advantage. We exploited the large inventory of chemical information encoded in oil to robustly differentiate signatures of mass transfer to air and water. With this new approach, we can evaluate mass transfer models (the Part 2 companion to this paper) and more properly account for evaporation, dissolution, and degradation of oil in the environment.

Studies on sildenafil citrate (Viagra) interaction with DNA using electrochemical DNA biosensor.

PubMed

Rauf, Sakandar; Nawaz, Haq; Akhtar, Kalsoom; Ghauri, Muhammad A; Khalid, Ahmad M

2007-05-15

The interaction of sildenafil citrate (Viagra) with DNA was studied by using an electrochemical DNA biosensor. The binding mechanism of sildenafil citrate was elucidated by using constant current potentiometry and differential pulse voltammetry at DNA-modified glassy carbon electrode. The decrease in the guanine oxidation peak area or peak current was used as an indicator for the interaction in 0.2M acetate buffer (pH 5). The binding constant (K) values obtained were 2.01+/-0.05 x 10(5) and 1.97+/-0.01 x 10(5)M(-1) with constant current potentiometry and differential pulse voltammetry, respectively. A linear dependence of the guanine peak area or peak current was observed within the range of 1-40 microM sildenafil citrate with slope=-2.74 x 10(-4)s/microM, r=0.989 and slope=-2.78 x 10(-3)microA/microM, r=0.995 by using constant current potentiometry and differential pulse voltammetry, respectively. Additionally, binding constant values for sildenafil citrate-DNA interaction were determined for the pH range of 4-8 and in biological fluids (serum and urine) at pH 5. The influence of sodium and calcium ions was also studied to elucidate the mechanism of sildenafil citrate-DNA interaction under different solution conditions. The present study may prove to be helpful in extending our understanding of the anticancer activity of sildenafil citrate from cellular to DNA level.

Protein Interactions in T7 DNA Replisome Facilitate DNA Damage Bypass.

PubMed

Zou, Zhenyu; Chen, Ze; Xue, Qizhen; Xu, Ying; Xiong, Jingyuan; Yang, Ping; Le, Shuai; Zhang, Huidong

2018-06-14

DNA replisome inevitably encounters DNA damage during DNA replication. T7 DNA replisome contains DNA polymerase (gp5), the processivity factor thioredoxin (trx), helicase-primase (gp4), and ssDNA binding protein (gp2.5). T7 protein interactions mediate this DNA replication. However, whether the protein interactions could promote DNA damage bypass is still little addressed. In this study, we investigated the strand-displacement DNA synthesis past 8-oxoG or O6-MeG at the synthetic DNA fork by T7 DNA replisome. DNA damage does not obviously affect the binding affinities among helicase, polymerase, and DNA fork. Relative to unmodified G, both 8-oxoG and O6-MeG, as well as GC-rich template sequence clusters, inhibit the strand-displacement DNA synthesis and produce partial extension products. Relative to gp4 ΔC-tail, gp4 promotes the DNA damage bypass. The presence of gp2.5 further promotes this bypass. Thus, the interactions of polymerase with helicase and ssDNA binidng protein faciliate the DNA damage bypass. Similarly, accessory proteins in other complicated DNA replisomes also facilitate the DNA damage bypass. This work provides the novel mechanism information of DNA damage bypass by DNA replisome. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Stability of nuclear DNA content among divergent and isolated populations of Fraser fir

Treesearch

L.D. Auckland; J.S. Johnston; H.J. Price; F.E. Bridgwater

2001-01-01

Fraser fir (Abies fraseri (Pursh) Poir.) is an endemic species consisting of six major disjunct populations in the Appalachian Mountains, U.S.A. Nuclear DNA content was measured with laser flow cytometry to determine if genome size differences could be detected among the disjunct populations of Fraser fir and its close relatives, balsam fir

Artifacts associated with the measurement of oxidized DNA bases.

PubMed Central

Cadet, J; Douki, T; Ravanat, J L

1997-01-01

In this paper we review recent aspects of the measurement of oxidized DNA bases, currently a matter of debate. There has long been an interest in the determination of the level of oxidized bases in cellular DNA under both normal and oxidative stress conditions. In this respect, the situation is confusing because variations that may be as large as two orders of magnitude have been reported for the yield of the formation of 8-oxo-7,8-dihydroguanine (8-oxoGua) in similar DNA samples. However, recent findings clearly show that application of several assays like gas chromatography-mass spectrometry (GC-MS) and -32P--postlabeling may lead to a significant overestimation of the level of oxidized bases in cellular DNA. In particular, the silylation step, which is required to make the samples volatile for the GC-MS analysis, has been shown to induce oxidation of normal bases at the level of about one oxidized base per 10(4) normal bases. This has been found to be a general process that applies in particular to 8-oxoGua, 8-oxo-7, 8-dihydroadenine,5-hydroxycytosine, 5-(hydroxymethyl)uracil, and 5-formyluracil. Interestingly, prepurification of the oxidized bases from DNA hydrolysate prior to the derivatization reaction prevents artefactual oxidation. Under these conditions, the level of oxidized bases measured by GC-MS is similar to that obtained by HPLC associated with electrochemical detection (HPLC-EC). It should be added that the level of 8-oxo-7,8-dihydro-2;-deoxyguanosine in control cellular DNA has been found to be about fivefold lower than in earlier HPLC-EC measurements by using appropriate conditions of extraction and enzymatic digestion of DNA. Similar conclusions were reached by measuring formamidopyrimidine-DNA glycosylase sensitive sites as revealed by the single cell gel electrophoresis (comet) assay. Images Figure 1. PMID:9349826

Ultrasonic-assisted pyrolyzation fabrication of reduced SnO2–x /g-C3N4 heterojunctions: Enhance photoelectrochemical and photocatalytic activity under visible LED light irradiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Kai; Zeng, Xiaoqiao; Gao, Shanmin

Novel SnO 2–x/g-C 3N 4 heterojunction nanocomposites composed of reduced SnO 2–x nanoparticles and exfoliated g-C 3N 4 nanosheets were prepared by a convenient one-step pyrolysis method. The structural, morphological, and optical properties of the as-prepared nanocomposites were characterized in detail, indicating that the aggregation of g-C 3N 4 nanosheets was prevented by small, well-dispersed SnO 2–x nanoparticles. The ultraviolet–visible spectroscopy absorption bands of the nanocomposites were shifted to a longer wavelength region than those exhibited by pure SnO 2 or g-C 3N 4. The charge transfer and recombination processes occurring in the nanocomposites were investigated using linear scan voltammetrymore » and electrochemical impedance spectroscopy. Under 30-W visible-light-emitting diode irradiation, the heterojunction containing 27.4 wt.% SnO 2–x exhibited the highest photocurrent density of 0.0468 mA·cm–2, which is 33.43 and 5.64 times larger than that of pure SnO 2 and g-C 3N 4, respectively. The photocatalytic activity of the heterojunction material was investigated by degrading rhodamine B under irradiation from the same light source. Kinetic study revealed a promising degradation rate constant of 0.0226 min-1 for the heterojunction containing 27.4 wt.% SnO 2–x, which is 32.28 and 5.79 times higher than that of pure SnO 2 and g-C 3N 4, respectively. The enhanced photoelectrochemical and photocatalytic performances of the nanocomposite may be due to its appropriate SnO 2–x content and the compact structure of the junction between the SnO 2–x nanoparticles and the g-C 3N 4 nanosheets, which inhibits the recombination of photogenerated electrons and holes.« less

The relationships among IGF-1, DNA content, and protein accumulation during skeletal muscle hypertrophy

NASA Technical Reports Server (NTRS)

Adams, G. R.; Haddad, F.

1996-01-01

Insulin-like growth factor-1 (IGF-1) is known to have anabolic effects on skeletal muscle cells. This study examined the time course of muscle hypertrophy and associated IGF-1 peptide and mRNA expression. Data were collected at 3, 7, 14, and 28 days after surgical removal of synergistic muscles of both normal and hypophysectomized (HX) animals. Overloading increased the plantaris (Plant) mass, myofiber size, and protein-to-body weight ratio in both groups (normal and HX; P < 0.05). Muscle IGF-1 peptide levels peaked at 3 (normal) and 7 (HX) days of overloading with maximum 4.1-fold (normal) and 6.2-fold (HX) increases. Increases in muscle IGF-1 preceded the hypertrophic response. Total DNA content of the overloaded Plant increased in both groups. There was a strong positive relationship between IGF-1 peptide and DNA content in the overloaded Plant from both groups. These results indicate that 1) the muscles from rats with both normal and severely depressed systemic levels of IGF-1 respond to functional overload with an increase in local IGF-1 expression and 2) this elevated IGF-1 may be contributing to the hypertrophy response, possibly via the mobilization of satellite cells to provide increases in muscle DNA.

An ingenious strategy of preparing TiO2/g-C3N4 heterojunction photocatalyst: In situ growth of TiO2 nanocrystals on g-C3N4 nanosheets via impregnation-calcination method

NASA Astrophysics Data System (ADS)

Zhang, Guanghui; Zhang, Tianyong; Li, Bin; Jiang, Shuang; Zhang, Xia; Hai, Li; Chen, Xingwei; Wu, Wubin

2018-03-01

An ingenious method was employed to design and fabricate the TiO2/g-C3N4 heterojunction photocatalysts in this study. The thermal oxidation etching of g-C3N4 nanosheets and the in situ growth of TiO2 nanocrystal on the surface of g-C3N4 nanosheets were completed simultaneously by the calcination process. The g-C3N4 nanosheets played a crucial role in regulating and assembling the structures and morphologies of TiO2. Furthermore, the thickness and content of g-C3N4, and the crystallinity of TiO2 in TiO2/g-C3N4 composites could be regulated and controlled by the calcination temperature. Among the resultant TiO2/g-C3N4 samples, the TiO2/g-C3N4 sample with 41.6 wt% g-C3N4 exhibited the highest photocatalytic activity. It could degrade almost all MO molecules under visible light irradiation within 3 h. Moreover, it displayed higher visible light photocatalytic performance for degrading MO solution than pure g-C3N4 and D-TiO2. The synergistic effect between TiO2 and g-C3N4 makes significant contributions to the enhancement of the visible light photocatalytic activity. In addition, the favorable photocatalytic performance of TiO2/g-C3N4 nanocomposites is also attributed to the porous structures and uniform morphologies, and large surface area. Furthermore, the resultant TiO2/g-C3N4 exhibits excellent photocatalytic stability. Radical trapping experiments indicated that rad O2- and h+ were the main reactive species during the photodegradation process under visible light irradiation. Hopefully, the results can offer new design and strategy for preparing other g-C3N4-based nanocomposites for environmental and energy applications.

DNA characterization and karyotypic evolution in the bee genus Melipona (Hymenoptera, Meliponini).

PubMed

Rocha, Marla Piumbini; Pompolo, Silvia Das Graças; Dergam, Jorge Abdala; Fernandes, Anderson; Campos, Lucio Antonio De Oliveira

2002-01-01

We analyzed patterns of heterochromatic bands in the Neotropical stingless bee genus Melipona (Hymenoptera, Meliponini). Group I species (Melipona bicolor bicolor, Melipona quadrifasciata, Melipona asilvae, Melipona marginata, Melipona subnitida) were characterized by low heterochromatic content. Group II species (Melipona capixaba, Melipona compressipes, Melipona crinita, Melipona seminigra fuscopilosa e Melipona scutellaris) had high heterochromatic content. All species had 2n = 18 and n = 9. In species of Group I heterochromatin was pericentromeric and located on the short arm of acrocentric chromosomes, while in Group II species heterochromatin was distributed along most of the chromosome length. The most effective sequential staining was quinacrine mustard (QM)/distamycin (DA)/chromomycin A3(CMA3)/4-6-diamidino-2-phenylindole (DAPI). Heterochromatic and euchromatic bands varied extensively within Group I. In Group II species euchromatin was restricted to the chromosome tips and it was uniformly GC+. Patterns of restriction enzymes (EcoRI, DraI, HindIII) showed that heterochromatin was heterogeneous. In all species the first pair of homologues was of unequal size and showed heteromorphism of a GC+ pericentromeric heterochromatin. In M. asilvae (Group I) this pair bore NOR and in M. compressipes (Group II) it hybridized with a rDNA FISH probe. As for Group I species the second pair was AT+ in M. subnitida and neutral for AT and GC in the remaining species of this group. Outgroup comparison indicates that high levels of heterochromatin represent a derived condition within Melipona. The pattern of karyotypic evolution sets Melipona in an isolated position within the Meliponini.

Correlation of DNA content and nucleomorphometric features with World Health Organization grading of meningiomas.

PubMed

Grunewald, J P; Röhl, F W; Kirches, E; Dietzmann, K

1998-02-01

Many studies dealing with extracranial cancer showed a strong correlation of DNA ploidy to a poor clinical outcome, recurrence, or malignancy. In brain tumors, analysis of DNA content did not always provided significant diagnostic information. In this study, DNA density and karyometric parameters of 50 meningiomas (26 Grade I, 10 Grade II, 14 Grade III) were quantitatively evaluated by digital cell image analyses of Feulgen-stained nuclei. In particular, the densitometric parameter SEXT, which describes nuclear DNA content, as well as the morphometric values LENG (a computer-assisted measurement of nuclear circumference), AREA (a computer-assisted measurement of nuclear area), FCON (a parameter that describes nuclear roundness), and CONC (a describing nuclear contour), evaluated with the software IMAGE C, were correlated to World Health Organization (WHO) grading using univariate and multivariate methods. AREA and LENG values showed significant differences between tumors of Grades I and III. FCON values were unable to distinguish WHO Grade III from Grade I/II but were useful in clearly separating Grade II from Grade I tumors. CONC values detected differences between WHO Grades II and I/III tumors but not between the latter. SEXT values clearly distinguished Grade III from Grade I/II tumors. The 1c, 2c, 2.5c, and 5c exceeding rates showed no predictive values. Only the 6c exceeding rate showed a significant difference between Grades I and III. These results outline the characteristic features of the atypical (Grade II) meningiomas, which make them a recognizable tumor entity distinct from benign and anaplastic meningiomas. The combination of DNA densitometric and morphometric findings seems to be a powerful addition to the histopathologic classification of meningiomas, as suggested by the WHO.

Effect of the Gc-derived macrophage-activating factor precursor (preGcMAF) on phagocytic activation of mouse peritoneal macrophages.

PubMed

Uto, Yoshihiro; Yamamoto, Syota; Takeuchi, Ryota; Nakagawa, Yoshinori; Hirota, Keiji; Terada, Hiroshi; Onizuka, Shinya; Nakata, Eiji; Hori, Hitoshi

2011-07-01

The 1f1f subtype of the Gc protein (Gc(1f1f) protein) was converted into Gc-derived macrophage-activating factor (GcMAF) by enzymatic processing in the presence of β-galactosidase of an activated B-cell and sialidase of a T-cell. We hypothesized that preGc(1f1f)MAF, the only Gc(1f1f) protein lacking galactose, can be converted to GcMAF in vivo because sialic acid is cleaved by residual sialidase. Hence, we investigated the effect of preGc(1f1f)MAF on the phagocytic activation of mouse peritoneal macrophages. We examined the sugar moiety of preGc(1f1f)MAF with a Western blot using peanut agglutinin (PNA) and Helix pomatia agglutinin (HPA) lectin. We also found that preGc(1f1f)MAF significantly enhanced phagocytic activity in mouse peritoneal macrophages but only in the presence of the mouse peritoneal fluid; the level of phagocytic activity was the same as that observed for GcMAF. PreGc(1f1f)MAF can be used as an effective macrophage activator in vivo.

Genome size and metabolic intensity in tetrapods: a tale of two lines

PubMed Central

Vinogradov, Alexander E; Anatskaya, Olga V

2005-01-01

We show the negative link between genome size and metabolic intensity in tetrapods, using the heart index (relative heart mass) as a unified indicator of metabolic intensity in poikilothermal and homeothermal animals. We found two separate regression lines of heart index on genome size for reptiles–birds and amphibians–mammals (the slope of regression is steeper in reptiles–birds). We also show a negative correlation between GC content and nucleosome formation potential in vertebrate DNA, and, consistent with this relationship, a positive correlation between genome GC content and nuclear size (independent of genome size). It is known that there are two separate regression lines of genome GC content on genome size for reptiles–birds and amphibians–mammals: reptiles–birds have the relatively higher GC content (for their genome sizes) compared to amphibians–mammals. Our results suggest uniting all these data into one concept. The slope of negative regression between GC content and nucleosome formation potential is steeper in exons than in non-coding DNA (where nucleosome formation potential is generally higher), which indicates a special role of non-coding DNA for orderly chromatin organization. The chromatin condensation and nuclear size are supposed to be key parameters that accommodate the effects of both genome size and GC content and connect them with metabolic intensity. Our data suggest that the reptilian–birds clade evolved special relationships among these parameters, whereas mammals preserved the amphibian-like relationships. Surprisingly, mammals, although acquiring a more complex general organization, seem to retain certain genome-related properties that are similar to amphibians. At the same time, the slope of regression between nucleosome formation potential and GC content is steeper in poikilothermal than in homeothermal genomes, which suggests that mammals and birds acquired certain common features of genomic organization. PMID:16519230

[In-situ measurement of background atmospheric HCFC-142b using GC-MS and GC-ECD method].

PubMed

Guo, Li-feng; Yao, Bo; Zhou, Ling-xi; Li, Pei-chang; Xu, Lin

2013-05-01

Custom-made GC-MS and GC-ECD in-situ measurement systems were established at the Shangdianzi GAW Regional station. From May 2010 to May 2011, the precisions for GC-MS and GC-ECD systems were 0.23% and 0.88%, respectively, and the HCFC-142b mole fraction during the observation period ranged from 21 x 10(-12) to 355 x 10(-12). The result of the independent-sample T test was P > 0.05, and there was no significant difference in HCFC-142b mole fraction measured by the two systems. The small difference of HCFC-142b mole fraction measured by GC-MS and GC-ECD might be associated with the different sampling time and precision of the two systems. A statistical filter of "robust local regression" was applied to separate HCFC-142b background and pollution data. The mean difference, median difference, 25 and 75 percent difference of background data measured by GC-MS and GC-ECD were all within the precisions. The pollution events captured by the two systems showed similar characters. Results from both systems showed a higher HCFC-142b level in summer and autumn than in winter. The pollution mole fraction of the two systems showed similar seasonal changes.

Interlaboratory Reproducibility of Droplet Digital Polymerase Chain Reaction Using a New DNA Reference Material Format.

PubMed

Pinheiro, Leonardo B; O'Brien, Helen; Druce, Julian; Do, Hongdo; Kay, Pippa; Daniels, Marissa; You, Jingjing; Burke, Daniel; Griffiths, Kate; Emslie, Kerry R

2017-11-07

Use of droplet digital PCR technology (ddPCR) is expanding rapidly in the diversity of applications and number of users around the world. Access to relatively simple and affordable commercial ddPCR technology has attracted wide interest in use of this technology as a molecular diagnostic tool. For ddPCR to effectively transition to a molecular diagnostic setting requires processes for method validation and verification and demonstration of reproducible instrument performance. In this study, we describe the development and characterization of a DNA reference material (NMI NA008 High GC reference material) comprising a challenging methylated GC-rich DNA template under a novel 96-well microplate format. A scalable process using high precision acoustic dispensing technology was validated to produce the DNA reference material with a certified reference value expressed in amount of DNA molecules per well. An interlaboratory study, conducted using blinded NA008 High GC reference material to assess reproducibility among seven independent laboratories demonstrated less than 4.5% reproducibility relative standard deviation. With the exclusion of one laboratory, laboratories had appropriate technical competency, fully functional instrumentation, and suitable reagents to perform accurate ddPCR based DNA quantification measurements at the time of the study. The study results confirmed that NA008 High GC reference material is fit for the purpose of being used for quality control of ddPCR systems, consumables, instrumentation, and workflow.

Targeted disruption of a novel gene contiguous to both glucocerebrodisidase (GC) and thrombospondin 3 (TSP3), results in an embryonic lethal phenotype in the mouse

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bornstein, P.; Shingu, T.; LaMarca, M.E.

1994-09-01

We have identified a new murine gene, termed gene X, that spans the 6 kb interval separating GC from TSP3. Mutations in GC result in Gaucher disease, the most common lysosomal storage disorder. Gene X and GC are transcribed convergently; their major polyadenylation sites are separated by only 431 bp. On the other hand, gene X and TSP3 are transcribed divergently and share a bidirectional promoter. The cDNA for gene X encodes a 317 amino acid protein, without either a signal sequence or N-linked glycosylation. Gene X is expressed ubiquitously in tissues of the young adult mouse, but no closemore » homologues have been found in the DNA or protein data bases. A targeted point mutation was introduced into the GC gene (Asn to Ser in exon 9) by homologous recombination in embryonic stem cells to establish a mouse model for a mild form of Gaucher disease. In the process, a PGK-neomycin gene cassette was inserted in the 3{prime} flanking region of GC as a selectable marker, in a sequence that was subsequently identified as exon 8 of gene X. Mice homozygous for the combined mutation die early in gestation. Since the amino acid mutation in humans is associated with milder type 1 Gaucher disease, we conclude that gene X is essential for embryonic development in mice. The locations of human and murine GC, gene X and TSP3 are similar, but the human genome includes a duplication that has produced GC and gene X pseudogenes. We are currently studying the possible functional interactions of GC, gene X and TSP3 in both mice and humans.« less

Chromosome numbers and DNA content in some species of Mecardonia (Gratiolae, Plantaginaceae)

PubMed Central

Sosa, María M.; Angulo, María B.; Greppi, Julián A.; Bugallo, Verónica

2016-01-01

Abstract Cytogenetic characterization and determination of DNA content by flow cytometry of five species of Mecardonia Ruiz et Pavon, 1798 (Gratiolae, Plantaginaceae) was performed. This is the first study of nuclear DNA content carried out in the genus. Mitotic analysis revealed a base chromosome number x = 11 for all entities and different ploidy levels, ranging from diploid (2n = 2x = 22) to hexaploid (2n = 6x = 66). The results include the first report of the chromosome numbers for Mecardonia flagellaris (Chamisso & Schlechtendal, 1827) (2n = 22), Mecardonia grandiflora (Bentham) Pennell, 1946 (2n = 22), Mecardonia kamogawae Greppi & Hagiwara, 2011 (2n = 66), and Mecardonia sp. (2n = 44). The three ploidy levels here reported suggest that polyploidy is common in Mecardonia and appear to be an important factor in the evolution of this genus. The 2C- and 1Cx-values were also estimated in all the species. The 2C-values ranged from 1.91 to 5.29 pg. The 1Cx-values ranged from 0.88 to 1.03 pg. The general tendency indicated a decrease in the 1Cx-value with increasing ploidy level. The significance of the results is discussed in relation to taxonomy of the genus. PMID:28123693

Interaction of Zn(II)bleomycin-A2 and Zn(II)peplomycin with a DNA hairpin containing the 5'-GT-3' binding site in comparison with the 5'-GC-3' binding site studied by NMR spectroscopy.

PubMed

Follett, Shelby E; Ingersoll, Azure D; Murray, Sally A; Reilly, Teresa M; Lehmann, Teresa E

2017-10-01

Bleomycins are a group of glycopeptide antibiotics synthesized by Streptomyces verticillus that are widely used for the treatment of various neoplastic diseases. These antibiotics have the ability to chelate a metal center, mainly Fe(II), and cause site-specific DNA cleavage. Bleomycins are differentiated by their C-terminal regions. Although this antibiotic family is a successful course of treatment for some types of cancers, it is known to cause pulmonary fibrosis. Previous studies have identified that bleomycin-related pulmonary toxicity is linked to the C-terminal region of these drugs. This region has been shown to closely interact with DNA. We examined the binding of Zn(II)peplomycin and Zn(II)bleomycin-A 2 to a DNA hairpin of sequence 5'-CCAGTATTTTTACTGG-3', containing the binding site 5'-GT-3', and compared the results with those obtained from our studies of the same MBLMs bound to a DNA hairpin containing the binding site 5'-GC-3'. We provide evidence that the DNA base sequence has a strong impact in the final structure of the drug-target complex.

GC-MS profile of antimicrobial and antioxidant fractions from Cordia rothii roots.

PubMed

Khan, Kehkashan; Firdous, Sadiqa; Ahmad, Aqeel; Fayyaz, Nida; Nadir, Muhammad; Rasheed, Munawwer; Faizi, Shaheen

2016-11-01

An ethnobotanical survey of Cordia rothii Roem. & Schult. (Boraginaceae) reveals it as a medicinal plant. Antimicrobial and antioxidant potential evaluation and identification of chemical constituents via GC-MS of C. rothii roots fractions. To the best of our knowledge, this is the first systematic investigation of the roots exploiting GC-MS. Extraction and fractionation of C. rothii roots furnished various fractions using solvents of varying polarity, i.e., n-hexane, chloroform, ethyl acetate, acetone and methanol. In vitro antimicrobial and antioxidant screening was performed using disk diffusion and DPPH methods, respectively. MIC of active fractions was also determined using disk diffusion method. GC-MS was used to identify constituents which may be responsible for these activities. Among various fractions from C. rothii roots, fraction KA-C showed strong antibacterial activity against 17 microorganisms tested, with MIC ranging from 250-31.25 μg/mL. Fractions KA-A, KM and KM-A exhibited significant antioxidant potential with EC 50 46.875 μg/mL, while fractions KEA-PE, KM-PE and KM-M were good with EC 50 93.750 μg/mL. Forty-five phytochemicals were identified in GC-MS studies including eight hydrocarbons, six free fatty acids, 11 fatty acids esters, two phenylpropanoids, four aromatics, four terpenoid quinones/hydroquinones, three triterpenes, four phytosterols, two hexose metabolites and a DNA base. Of these, 32 constituents have been reported for the first time from C. rothii, 24 from genus Cordia and 15 from Boraginaceae. Strong antibacterial and antioxidant potential of C. rothii roots may be due to the contribution of phytoconstituents identified through GC-MS studies.

[Replication of Streptomyces plasmids: the DNA nucleotide sequence of plasmid pSB 24.2].

PubMed

Bolotin, A P; Sorokin, A V; Aleksandrov, N N; Danilenko, V N; Kozlov, Iu I

1985-11-01

The nucleotide sequence of DNA in plasmid pSB 24.2, a natural deletion derivative of plasmid pSB 24.1 isolated from S. cyanogenus was studied. The plasmid amounted by its size to 3706 nucleotide pairs. The G-C composition was equal to 73 per cent. The analysis of the DNA structure in plasmid pSB 24.2 revealed the protein-encoding sequence of DNA, the continuity of which was significant for replication of the plasmid containing more than 1300 nucleotide pairs. The analysis also revealed two A-T-rich areas of DNA, the G-C composition of which was less than 55 per cent and a DNA area with a branched pin structure. The results may be of value in investigation of plasmid replication in actinomycetes and experimental cloning of DNA with this plasmid as a vector.

Reduction of Werner Syndrome Protein Enhances G:C → A:T Transition by O6-Methylguanine in Human Cells.

PubMed

Suzuki, Tetsuya; Kuramoto, Yoshie; Kamiya, Hiroyuki

2018-05-21

O 6 -Methylguanine ( O 6 -MeG) is a damaged base produced by methylating reagents. The Werner syndrome protein (WRN) is a cancer-related human DNA helicase. The effects of WRN reduction on O 6 -MeG-caused mutagenesis were assessed by an siRNA-mediated knockdown in human U2OS cells, using a shuttle plasmid with a single O 6 -MeG base in the supF gene. The plasmid DNA was replicated in the cells, isolated, and electroporated into an Escherichia coli indicator strain. The lowered amount of WRN increased the frequency of mutations induced by O 6 -MeG, mainly G:C → A:T substitution. The increased mutation rate suggested that the cancer-related WRN suppresses the G:C → A:T substitution by O 6 -MeG in human cells.

Relationship between radiation-induced aberrations in individual chromosomes and their DNA content: effects of interaction distance

NASA Technical Reports Server (NTRS)

Wu, H.; Durante, M.; Lucas, J. N.

2001-01-01

PURPOSE: To study the effect of the interaction distance on the frequency of inter- and intrachromosome exchanges in individual chromosomes with respect to their DNA content. Assumptions: Chromosome exchanges are formed by misrejoining of two DNA double-strand breaks (DSB) induced within an interaction distance, d. It is assumed that chromosomes in G(0)/G(1) phase of the cell cycle occupy a spherical domain in a cell nucleus, with no spatial overlap between individual chromosome domains. RESULTS: Formulae are derived for the probability of formation of inter-, as well as intra-, chromosome exchanges relating to the DNA content of the chromosome for a given interaction distance. For interaction distances <1 microm, the relative frequency of interchromosome exchanges predicted by the present model is similar to that by Cigarran et al. (1998) based on the assumption that the probability of interchromosome exchanges is proportional to the "surface area" of the chromosome territory. The "surface area" assumption is shown to be a limiting case of d-->0 in the present model. The present model also predicts that the probability of intrachromosome exchanges occurring in individual chromosomes is proportional to their DNA content with correction terms. CONCLUSION: When the interaction distance is small, the "surface area" distribution for chromosome participation in interchromosome exchanges has been expected. However, the present model shows that for the interaction distance as large as 1 microm, the predicted probability of interchromosome exchange formation is still close to the surface area distribution. Therefore, this distribution does not necessarily rule out the formation of complex chromosomal aberrations by long-range misrejoining of DSB.

Regulation of sGC via hsp90, Cellular Heme, sGC Agonists, and NO: New Pathways and Clinical Perspectives

PubMed Central

Ghosh, Arnab

2017-01-01

Abstract Significance: Soluble guanylate cyclase (sGC) is an intracellular enzyme that plays a primary role in sensing nitric oxide (NO) and transducing its multiple signaling effects in mammals. Recent Advances: The chaperone heat shock protein 90 (hsp90) associates with signaling proteins in cells, including sGC, where it helps to drive heme insertion into the sGC-β1 subunit. This allows sGC-β1 to associate with a partner sGC-α1 subunit and mature into an NO-responsive active form. Critical Issues: In this article, we review evidence to date regarding the mechanisms that modulate sGC activity by a pathway where binding of hsp90 or sGC agonist to heme-free sGC dictates the assembly and fate of an active sGC heterodimer, both by NO and heme-dependent or heme-independent pathways. Future Directions: We discuss some therapeutic implications of the NO-sGC-hsp90 nexus and its potential as a marker of inflammatory disease. Antioxid. Redox Signal. 26, 182–190. PMID:26983679

Engineering of DNA polymerase I from Thermus thermophilus using compartmentalized self-replication.

PubMed

Aye, Seaim Lwin; Fujiwara, Kei; Ueki, Asuka; Doi, Nobuhide

2018-05-05

Although compartmentalized self-replication (CSR) and compartmentalized partnered replication (CPR) are powerful tools for directed evolution of proteins and gene circuits, limitations remain in the emulsion PCR process with the wild-type Taq DNA polymerase used so far, including long run times, low amounts of product, and false negative results due to inhibitors. In this study, we developed a high-efficiency mutant of DNA polymerase I from Thermus thermophilus HB27 (Tth pol) suited for CSR and CPR. We modified the wild-type Tth pol by (i) deletion of the N-terminal 5' to 3' exonuclease domain, (ii) fusion with the DNA-binding protein Sso7d, (iii) introduction of four known effective point mutations from other DNA polymerase mutants, and (iv) codon optimization to reduce the GC content. Consequently, we obtained a mutant that provides higher product yields than the conventional Taq pol without decreased fidelity. Next, we performed four rounds of CSR selection with a randomly mutated library of this modified Tth pol and obtained mutants that provide higher product yields in fewer cycles of emulsion PCR than the parent Tth pol as well as the conventional Taq pol. Copyright © 2018 Elsevier Inc. All rights reserved.

Prenatal ambient air pollution exposure, infant growth and placental mitochondrial DNA content in the INMA birth cohort.

PubMed

Clemente, Diana B P; Casas, Maribel; Janssen, Bram G; Lertxundi, Aitana; Santa-Marina, Loreto; Iñiguez, Carmen; Llop, Sabrina; Sunyer, Jordi; Guxens, Mònica; Nawrot, Tim S; Vrijheid, Martine

2017-08-01

The association between prenatal air pollution exposure and postnatal growth has hardly been explored. Mitochondrial DNA (mtDNA), as a marker of oxidative stress, and growth at birth can play an intermediate role in this association. In a subset of the Spanish birth cohort INMA we assessed first whether prenatal nitrogen dioxide (NO 2 ) exposure is associated with infant growth. Secondly, we evaluated whether growth at birth (length and weight) could play a mediating role in this association. Finally, the mediation role of placental mitochondrial DNA content in this association was assessed. In 336 INMA children, relative placental mtDNA content was measured. Land-use regression models were used to estimate prenatal NO 2 exposure. Infant growth (height and weight) was assessed at birth, at 6 months of age, and at 1 year of age. We used multiple linear regression models and performed mediation analyses. The proportion of mediation was calculated as the ratio of indirect effect to total effect. Prenatal NO 2 exposure was inversely associated with all infant growth parameters. A 10µg/m³ increment in prenatal NO 2 exposure during trimester 1 of pregnancy was significantly inversely associated with height at 6 months of age (-6.6%; 95%CI: -11.4, -1.9) and weight at 1 year of age (-4.2%; 95%CI: -8.3, -0.1). These associations were mediated by birth length (31.7%; 95%CI: 34.5, 14.3) and weight (53.7%; 95%CI: 65.3, -0.3), respectively. Furthermore, 5.5% (95%CI: 10.0, -0.2) of the association between trimester 1 NO 2 exposure and length at 6 months of age could be mediated by placental mtDNA content. Our results suggest that impaired fetal growth caused by prenatal air pollution exposure can lead to impaired infant growth during the first year of life. Furthermore, molecular adaptations in placental mtDNA are associated with postnatal consequences of air pollution induced alterations in growth. Copyright © 2017 Elsevier Inc. All rights reserved.

Henry`s law constant for selected volatile organic compounds in high-boiling oils

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poddar, T.K.; Sirkar, K.K.

Absorption systems are often used to remove and recover organic vapors from process air/gas streams. A high boiling and inert liquid like silicone oil is an excellent absorbent for volatile organic compounds in air. Henry`s law constants of four different volatile organic compounds, namely, acetone, methanol, methylene chloride, and toluene between air and high-boiling oils were determined experimentally by the headspace-GC technique over a temperature range. The Henry`s law constants were fitted as a function of temperature to an equation.

C3d enhanced DNA vaccination induced humoral immune response to glycoprotein C of pseudorabies virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tong Tiezhu; Provincial Key Laboratory of Preventive Veterinary Medicine, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070; Fan Huiying

2006-09-08

Murine C3d were utilized to enhance immunogenicity of pseudorabies virus (PrV) gC DNA vaccination. Three copies of C3d and four copies of CR2-binding domain M28{sub 4} were fused, respectively, to truncated gC gene encoding soluble glycoprotein C (sgC) in pcDNA3.1. BALB/c mice were, respectively, immunized with recombinant plasmids, blank vector, and inactivated vaccine. The antibody ELISA titer for sgC-C3d{sub 3} DNA was 49-fold more than that for sgC DNA, and the neutralizing antibody obtained 8-fold rise. Protection of mice from death after lethal PrV (316 LD{sub 5}) challenge was augmented from 25% to 100%. Furthermore, C3d fusion increased Th2-biased immunemore » response by inducing IL-4 production. The IL-4 level for sgC-C3d{sub 3} DNA immunization approached that for the inactivated vaccine. Compared to C3d, M28 enhanced sgC DNA immunogenicity to a lesser extent. In conclusion, we demonstrated that murine C3d fusion significantly enhanced gC DNA immunity by directing Th1-biased to a balanced and more effective Th1/Th2 response.« less

GC-FID coupled with chemometrics for quantitative and chemical fingerprinting analysis of Alpinia oxyphylla oil.

PubMed

Miao, Qing; Kong, Weijun; Zhao, Xiangsheng; Yang, Shihai; Yang, Meihua

2015-01-01

Analytical methods for quantitative analysis and chemical fingerprinting of volatile oils from Alpinia oxyphylla were established. The volatile oils were prepared by hydrodistillation, and the yields were between 0.82% and 1.33%. The developed gas chromatography-flame ionization detection (GC-FID) method showed good specificity, linearity, reproducibility, stability and recovery, and could be used satisfactorily for quantitative analysis. The results showed that the volatile oils contained 2.31-77.30 μL/mL p-cymene and 12.38-99.34 mg/mL nootkatone. A GC-FID fingerprinting method was established, and the profiles were analyzed using chemometrics. GC-MS was used to identify the principal compounds in the GC-FID profiles. The profiles of almost all the samples were consistent and stable. The harvesting time and source were major factors that affected the profile, while the volatile oil yield and the nootkatone content had minor secondary effects. Copyright © 2014 Elsevier B.V. All rights reserved.

Switches in Genomic GC Content Drive Shifts of Optimal Codons under Sustained Selection on Synonymous Sites

PubMed Central

Sun, Yu; Tamarit, Daniel

2017-01-01

Abstract The major codon preference model suggests that codons read by tRNAs in high concentrations are preferentially utilized in highly expressed genes. However, the identity of the optimal codons differs between species although the forces driving such changes are poorly understood. We suggest that these questions can be tackled by placing codon usage studies in a phylogenetic framework and that bacterial genomes with extreme nucleotide composition biases provide informative model systems. Switches in the background substitution biases from GC to AT have occurred in Gardnerella vaginalis (GC = 32%), and from AT to GC in Lactobacillus delbrueckii (GC = 62%) and Lactobacillus fermentum (GC = 63%). We show that despite the large effects on codon usage patterns by these switches, all three species evolve under selection on synonymous sites. In G. vaginalis, the dramatic codon frequency changes coincide with shifts of optimal codons. In contrast, the optimal codons have not shifted in the two Lactobacillus genomes despite an increased fraction of GC-ending codons. We suggest that all three species are in different phases of an on-going shift of optimal codons, and attribute the difference to a stronger background substitution bias and/or longer time since the switch in G. vaginalis. We show that comparative and correlative methods for optimal codon identification yield conflicting results for genomes in flux and discuss possible reasons for the mispredictions. We conclude that switches in the direction of the background substitution biases can drive major shifts in codon preference patterns even under sustained selection on synonymous codon sites. PMID:27540085

β-Galactosidase treatment is a common first-stage modification of the three major subtypes of Gc protein to GcMAF.

PubMed

Uto, Yoshihiro; Yamamoto, Syota; Mukai, Hirotaka; Ishiyama, Noriko; Takeuchi, Ryota; Nakagawa, Yoshinori; Hirota, Keiji; Terada, Hiroshi; Onizuka, Shinya; Hori, Hitoshi

2012-06-01

The 1f1f subtype of the group-specific component (Gc) protein is converted into Gc protein-derived macrophage-activating factor (GcMAF) by enzymatic processing with β-galactosidase and sialidase. We previously demonstrated that preGc(1f1f)MAF, a full Gc(1f1f) protein otherwise lacking a galactosyl moiety, can be converted to GcMAF by treatment with mouse peritoneal fluid. Here, we investigated the effects of the β-galactosidase-treated 1s1s and 22 subtypes of Gc protein (preGc(1s1s)MAF and preGc₂₂MAF) on the phagocytic activation of mouse peritoneal macrophages. We demonstrated the presence of Gal-GalNAc disaccharide sugar structures in the Gc(1s1s) protein by western blotting using peanut agglutinin and Helix pomatia agglutinin lectin. We also found that preGc(1s1s)MAF and preGc₂₂MAF significantly enhanced the phagocytic activity of mouse peritoneal macrophages in the presence and absence of mouse peritoneal fluid. We demonstrate that preGc(1s1s)MAF and preGc₂₂MAF proteins can be used as effective macrophage activators.

Results of the First Mars Organic Molecule Analyzer (MOMA) GC-MS Coupling

NASA Astrophysics Data System (ADS)

Buch, Arnaud; Pinnick, Veronica; Szopa, Cyril; Danell, Ryan; Grand, Noel; Van Amerom, Friso; Glavin, Daniel; Freissinet, Caroline; Humeau, Olivier; Coll, Patrice; Arevalo, Ricardo; Stalport, Fabien; Brinckerhoff, William; Steininger, Harald; Goesmann, Fred; Mahaffy, Paul; Raulin, Francois

2014-11-01

The Mars Organic Molecule Analyzer (MOMA) aboard the ExoMars rover will be a key analytical tool in providing chemical (molecular) information from the solid samples collected by the rover, with a particular focus on the char-acterization of the organic content. The core of the MOMA instrument is a gas chromatograph coupled with a mass spectrometer (GC-MS) which provides the unique capability to characterize a broad range of compounds, including both of volatile and non-volatile species. Samples will be crushed and deposited into sample cups seated in a rotating carousel. Soil samples will be analyzed either by UV laser desorption / ionization (LDI) or pyrolysis gas chromatography ion trap mass spectrometry (pyr-GC-ITMS).The French GC brassboard was coupled to the US ion trap mass spectrometer brassboard in a flight-like con-figuration for several coupling campains. The MOMA GC setup is based on the SAM heritage design with a He reservoir and 4 separate analytical modules including traps, columns and Thermal Conductivity Detectors. Solid samples are sealed and heated in this setup using a manual tapping station, designed and built at MPS in Germany, for GC-MS analysis. The gaseous species eluting from the GC are then ionized by an electron impact ionization source in the MS chamber and analyzed by the linear ion trap mass spectrometer. Volatile and non-volatile compounds were injected in the MOMA instrumental suite. Both of these compounds classes were detected by the TCD and by the MS. MS signal (total ion current) and single mass spectra by comparison with the NIST library, gave us an unambiguous confirmation of these identifications. The mass spectra arise from an average of 10 mass spectra averaged around a given time point in the total ion chromatogram.Based on commercial instrument, the MOMA requirement for sensitivity in the GC-MS mode for organic molecules is 1 pmol. In this test, sensitivity was determined for the GC TCD and MS response to a dilution

Microwave modification of surface hydroxyl density for g-C3N4 with enhanced photocatalytic activity

NASA Astrophysics Data System (ADS)

An, Na; Zhao, Yang; Mao, Zhiyong; Agrawal, Dinesh Kumar; Wang, Dajian

2018-03-01

Microwave modification was performed on graphitic carbon nitride (g-C3N4) photocatalysts to tail the surface hydroxyl content for enhanced photocatalytic activity in this work. The influence of microwave heating on the surface hydroxyl density was investigated by a suite of characterization methods. The microwave treated g-C3N4 (MT-g-C3N4) delivered a higher photocatalytic activity in degradation of Rhodamine B (RhB) under visible light irradiation than pristine g-C3N4 due to its improved separation efficiency of photogenerated charge carries and promoted absorption capacity of RhB reactants on surface, which resulted from the increased surface hydroxyl density induced by microwave treatment. This study provides a simple and convenient method to modify g-C3N4 materials with enhanced photocatalytic activity for the potential application in photocatalytic elimination of environmental pollutants.

Ultrasensitive determination of DNA oxidation products by gas chromatography-tandem mass spectrometry and the role of antioxidants in the prevention of oxidative damage.

PubMed

Dawbaa, Sam; Aybastıer, Önder; Demir, Cevdet

2017-04-15

Oxidative stress is considered as one of the significant causes of DNA damage which in turn contributes to cell death through a series of intermediate processes such as cancer formation, mutation, and aging. Natural sources such as plant and fruit products have provided us with interesting substances of antioxidant activity that could be recruited in protecting the genetic materials of the cells. This study is an effort to discover some of those antioxidants effects in their standard and natural forms by performing an ultrasensitive determination of the products of DNA oxidation using GC-MS/MS. Experiments were used to determine the direct antioxidant activity of the substances contained in the tendrils of Vitis vinifera (var. alphonse) by extracting them and achieving Folin-Ciocalteau and CHROMAC analyses to determine the total phenolic content (TPC) and the antioxidant capacity of the extract, respectively; results revealed a phenolic content of 11.39±0.30mg Gallic Acid Equivalent (GAE)/g of the plant's fresh weight (FW) by Folin-Ciocalteau and 8.17±0.49mg Trolox Equivalent (TE)/g FW by CHROMAC assays. The qualitative analysis of the plant extract by HPLC-DAD technique revealed that two flavonoid glycosides namely rutin and isoquercitrin in addition to chlorogenic acid were contained in the extract. The determination of the DNA oxidation products was performed after putting DNA, rutin and isoquercitrin standard samples with different concentration, and the extract's sample under oxidative stress. Eighteen DNA oxidation products were traced using GC-MS/MS with ultra-sensitivity and the experiments proved a significant decrease in the concentration of the DNA oxidation products when the extract was used as a protectant against the oxidative stress. It is believed by conclusion that the extract of V. vinifera's (var. alphonse) tendrils has a good antioxidant activity; hence it is recommended to be used as a part of the daily healthy food list if possible

ANTIMICROBIAL AND ANTIBIOFILM EFFECTS OF EXTRACTS FROM TRAPA NATANS L., EVALUATION OF TOTAL PHENOLIC AND FLAVONOID CONTENTS AND GC-MS ANALYSIS.

PubMed

Radojevic, Ivana D; Vasic, Sava M; Dekic, Milan S; Radulovic, Niko S; Delic, Gorica T; Durdevic, Jelena S; Comic, Ljiljana R

2016-11-01

Research conducted in this study shows the applied in vitro antimicrobial and antibiofilm activity of the four extracts isolated from Trapa natans L. leaves. In this study, different methods were used (microdilution, tissue culture plate, different colorimetric methods, GC-FID and GC-MS analysis). While the water extract didn't show antibacterial activity, the acetone extract showed the strongest one. The same activity in the case of Pseudomonas aeruginosa (MIC was 313 μg/mL) was better than the activity of controls and it matched with antibiofilm activity. The effect of extracts was better on G+ bacteria (MICs were <78-625 μg/mL). For ethanol and ethyl acetate extracts all BIC values were better than MICs. Extracts showed a significant effect on Aspergillus restrictus (MICs were < 78/156 μg/mL). The GC and GC-MS analysis of the ethyl acetate extract revealed the identification of 22 compounds with (all E)-squalene (20.2%), n-alkanes and norlignan hinokiresinol among the most abundant ones. This is the first time that T. natans was studied using these methods.

Sequence-dependent nanometer-scale conformational dynamics of individual RecBCD–DNA complexes

PubMed Central

Carter, Ashley R.; Seaberg, Maasa H.; Fan, Hsiu-Fang; Sun, Gang; Wilds, Christopher J.; Li, Hung-Wen; Perkins, Thomas T.

2016-01-01

RecBCD is a multifunctional enzyme that possesses both helicase and nuclease activities. To gain insight into the mechanism of its helicase function, RecBCD unwinding at low adenosine triphosphate (ATP) (2–4 μM) was measured using an optical-trapping assay featuring 1 base-pair (bp) precision. Instead of uniformly sized steps, we observed forward motion convolved with rapid, large-scale (∼4 bp) variations in DNA length. We interpret this motion as conformational dynamics of the RecBCD–DNA complex in an unwinding-competent state, arising, in part, by an enzyme-induced, back-and-forth motion relative to the dsDNA that opens and closes the duplex. Five observations support this interpretation. First, these dynamics were present in the absence of ATP. Second, the onset of the dynamics was coupled to RecBCD entering into an unwinding-competent state that required a sufficiently long 5′ strand to engage the RecD helicase. Third, the dynamics were modulated by the GC-content of the dsDNA. Fourth, the dynamics were suppressed by an engineered interstrand cross-link in the dsDNA that prevented unwinding. Finally, these dynamics were suppressed by binding of a specific non-hydrolyzable ATP analog. Collectively, these observations show that during unwinding, RecBCD binds to DNA in a dynamic mode that is modulated by the nucleotide state of the ATP-binding pocket. PMID:27220465

The Hydroxyl Radical Reaction Rate Constant and Products of Dimethyl Succinate

DTIC Science & Technology

2008-03-01

phase reaction of OH + DMS, 10 liters of chamber content were flowed over 2,4- dinitrophenylhydrazine (DNPH) impregnated cartridges. Hydrazones formed... dinitrophenylhydrazine (DNPH) derivatization (method described in experimental methods), and GC/MS/MS (where samples were directly introduced into the mass

Separation of fatty acid methyl esters by GC-online hydrogenation × GC.

PubMed

Delmonte, Pierluigi; Fardin-Kia, Ali Reza; Rader, Jeanne I

2013-02-05

The separation of fatty acid methyl esters (FAME) provided by a 200 m × 0.25 mm SLB-IL111 capillary column is enhanced by adding a second dimension of separation ((2)D) in a GC × GC design. Rather than employing two GC columns of different polarities or using different elution temperatures, the separation in the two-dimensional space is achieved by altering the chemical structure of selected analytes between the two dimensions of separation. A capillary tube coated with palladium is added between the first dimension of separation ((1)D) column and the cryogenic modulator, providing the reduction of unsaturated FAMEs to their fully saturated forms. The (2)D separation is achieved using a 2.5 m × 0.10 mm SLB-IL111 capillary column and separates FAMEs based solely on their carbon skeleton. The two-dimensional separation can be easily interpreted based on the principle that all the saturated FAMEs lie on a straight diagonal line bisecting the separation plane, while the FAMEs with the same carbon skeleton but differing in the number, geometric configuration or position of double bonds lie on lines parallel to the (1)D time axis. This technique allows the separation of trans fatty acids (FAs) and polyunsaturated FAs (PUFAs) in a single experiment and eliminates the overlap between PUFAs with different chain lengths. To our knowledge, this the first example of GC × GC in which a chemical change is instituted between the two dimensions to alter the relative retentions of components and identify unsaturated FAMEs.

Antioxidant capacities, phenolic contents, and GC/MS analysis of Rhodiola imbricata Edgew. root extracts from Trans-Himalaya.

PubMed

Tayade, A B; Dhar, P; Sharma, M; Chauhan, R S; Chaurasia, O P; Srivastava, R B

2013-03-01

Our aim was to assess the antioxidant capacities and phenolic constituents of methanol and aqueous extracts of Rhodiola imbricata Edgew. root from Trans-Himalayan cold desert of Ladakh. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical scavenging capacity of the root extracts increased in a dose-dependent manner (up to 0.1 mg/mL) and root extract concentrations required for 50% inhibition of radical scavenging effect (IC50 ) were recorded as 0.013 and 0.014 mg/mL (for DPPH) and 0.016 and 0.017 mg/mL (for ABTS) for methanol and aqueous extracts, respectively. The total antioxidant power of the extract was determined by ferric reducing antioxidant power (FRAP) assay. Total polyphenol and phenolic acid content of methanol and aqueous extracts were 112.24, 59.06, 39.02, and 16.95 mg gallic acid equivalent/g of extract, respectively. Total flavonoid and flavonol contents were estimated to be 30.2, 17.67, 20.68, and 7.38 mg quercetin equivalent/g of extract, respectively. In all antioxidant capacity assays, the methanol extract exhibited significantly higher antioxidant capacity than that of aqueous extract due to the presence of significantly higher amount of vital phytoconstitiuents, viz. polyphenol, phenolic acid, and flavonol. GC/MS analysis showed that phytosterols, alkyl halide, phenols, and fatty acid esters were major phytochemical clusters. On the other hand, monoterpenes, fatty acids, tocopherols, aliphatic hydrocarbons, and ethers were found to be present in comparatively less amount in the methanol extract. Hence, our study signifies that this high-altitude medicinal herb could be used as the natural source of antioxidants and supports its use in traditional system of medicine to ameliorate oxidative stress and high-altitude maladies. © 2013 Institute of Food Technologists®

Imidazopyridine/Pyrrole and hydroxybenzimidazole/pyrrole pairs for DNA minor groove recognition.

PubMed

Renneberg, Dorte; Dervan, Peter B

2003-05-14

The DNA binding properties of fused heterocycles imidazo[4,5-b]pyridine (Ip) and hydroxybenzimidazole (Hz) paired with pyrrole (Py) in eight-ring hairpin polyamides are reported. The recognition profile of Ip/Py and Hz/Py pairs were compared to the five-membered ring pairs Im/Py and Hp/Py on a DNA restriction fragment at four 6-base pair recognition sites which vary at a single position 5'-TGTNTA-3', where N = G, C, T, A. The Ip/Py pair distinguishes G.C from C.G, T.A, and A.T, and the Hz/Py pair distinguishes T.A from A.T, G.C, and C.G, affording a new set of heterocycle pairs to target the four Watson-Crick base pairs in the minor groove of DNA.

The DBP Phenotype Gc-1f/Gc-1f Is Associated with Reduced Risk of Cancer. The Tromsø Study

PubMed Central

Jorde, Rolf; Schirmer, Henrik; Wilsgaard, Tom; Bøgeberg Mathiesen, Ellisiv; Njølstad, Inger; Løchen, Maja-Lisa; Joakimsen, Ragnar Martin; Grimnes, Guri

2015-01-01

Background and Objective In addition to its role as a transport protein, the vitamin D binding protein (DBP) may also affect lipid metabolism, inflammation and carcinogenesis. There are three common variants of the DBP, Gc1s (1s), Gc1f (1f), Gc2 (2) that result in six common phenotypes (1s/1s, 1s/1f, 1s/2, 1f/1f, 1f/2, and 2/2). These phenotypes can be identified by genotyping for the two single nucleotide polymorphisms rs7041 and rs4588 in the GC gene. The DBP variants have different binding coefficients for the vitamin D metabolites, and accordingly there may be important relations between DBP phenotypes and health. Methods DNA was prepared from subjects who participated in the fourth survey of the Tromsø Study in 1994-1995 and who were registered with the endpoints myocardial infarction (MI), type 2 diabetes (T2DM), cancer or death as well as a randomly selected control group. The endpoint registers were complete up to 2010- 2013. Genotyping was performed for rs7041 and rs4588 and serum 25-hydroxyvitamin D (25(OH)D) was measured. Results Genotyping for rs7041 and rs4588 was performed successfully in 11 704 subjects. Among these, 1660 were registered with incident MI, 958 with T2DM, 2410 with cancer and 4318 had died. Subjects with the DBP phenotype 1f/1f had 23 – 26 % reduced risk of incident cancer compared to the 1s/1s and 2/2 phenotypes (P < 0.02, Cox regression with gender as covariate). Differences in serum 25(OH)D levels could not explain the apparent cancer protective effect of the DBP variant 1f. In addition to cancer and 25(OH)D, there were significant associations between DBP phenotype and body height, hip circumference and serum calcium. Conclusion There are important biological differences between the common DBP phenotypes. If the relation between the DBP variant 1f and cancer is confirmed in other studies, determination of DBP phenotype may have clinical importance. PMID:25993554

Ionizing Radiation-Induced DNA Damage and Its Repair in Human Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dizdaroglu, Miral

DNA damage in mammalian chromatin in vitro and in cultured mammalian cells including human cells was studied. In the first phase of these studies, a cell culture laboratory was established. Necessary equipment including an incubator, a sterile laminar flow hood and several centrifuges was purchased. We have successfully grown several cell lines such as murine hybridoma cells, V79 cells and human K562 leukemia cells. This was followed by the establishment of a methodology for the isolation of chromatin from cells. This was a very important step, because a routine and successful isolation of chromatin was a prerequisite for the successmore » of the further studies in this project, the aim of which was the measurement of DNA darnage in mammalian chromatin in vitro and in cultured cells. Chromatin isolation was accomplished using a slightly modified procedure of the one described by Mee & Adelstein (1981). For identification and quantitation of DNA damage in cells, analysis of chromatin was preferred over the analysis of "naked DNA" for the following reasons: i. DNA may not be extracted efficiently from nucleoprotein in exposed cells, due to formation of DNA-protein cross-links, ii. the extractability of DNA is well known to decrease with increasing doses of radiation, iii. portions of DNA may not be extracted due to fragmentation, iv. unextracted DNA may contain a significant portion of damaged DNA bases and DNA-protein cross-links. The technique of gas chromatography/mass spectrometry (GC/MS), which was used in the present project, permits the identification and quantitation of modified DNA bases in chromatin in the presence of proteins without the necessity of first isolating DNA from chromatin. This has been demonstrated previously by the results from our laboratory and by the results obtained during the course of the present project. The quality of isolated chromatin was tested by measurement of its content of DNA, proteins, and RNA, by analysis of its protein

Is chondroitin sulfate responsible for the biological effects attributed to the GC protein-derived Macrophage Activating Factor (GcMAF)?

PubMed

Ruggiero, Marco; Reinwald, Heinz; Pacini, Stefania

2016-09-01

We hypothesize that a plasma glycosaminoglycan, chondroitin sulfate, may be responsible for the biological and clinical effects attributed to the Gc protein-derived Macrophage Activating Factor (GcMAF), a protein that is extracted from human blood. Thus, Gc protein binds chondroitin sulfate on the cell surface and such an interaction may occur also in blood, colostrum and milk. This interpretation would solve the inconsistencies encountered in explaining the effects of GcMAF in vitro and in vivo. According to our model, the Gc protein or the GcMAF bind to chondroitin sulfate both on the cell surface and in bodily fluids, and the resulting multimolecular complexes, under the form of oligomers trigger a transmembrane signal or, alternatively, are internalized and convey the signal directly to the nucleus thus eliciting the diverse biological effects observed for both GcMAF and chondroitin sulfate. Copyright © 2016 Elsevier Ltd. All rights reserved.

Genomics dataset of unidentified disclosed isolates.

PubMed

Rekadwad, Bhagwan N

2016-09-01

Analysis of DNA sequences is necessary for higher hierarchical classification of the organisms. It gives clues about the characteristics of organisms and their taxonomic position. This dataset is chosen to find complexities in the unidentified DNA in the disclosed patents. A total of 17 unidentified DNA sequences were thoroughly analyzed. The quick response codes were generated. AT/GC content of the DNA sequences analysis was carried out. The QR is helpful for quick identification of isolates. AT/GC content is helpful for studying their stability at different temperatures. Additionally, a dataset on cleavage code and enzyme code studied under the restriction digestion study, which helpful for performing studies using short DNA sequences was reported. The dataset disclosed here is the new revelatory data for exploration of unique DNA sequences for evaluation, identification, comparison and analysis.

Vibronic dephasing model for coherent-to-incoherent crossover in DNA

NASA Astrophysics Data System (ADS)

Karasch, Patrick; Ryndyk, Dmitry A.; Frauenheim, Thomas

2018-05-01

In this paper, we investigate the interplay between coherent and incoherent charge transport in cytosine-guanine (GC-) rich DNA molecules. Our objective is to introduce a physically grounded approach to dephasing in large molecules and to understand the length-dependent charge transport characteristics, and especially the crossover from the coherent tunneling to incoherent hopping regime at different temperatures. Therefore, we apply the vibronic dephasing model and compare the results to the Büttiker probe model which is commonly used to describe decoherence effects in charge transport. Using the full ladder model and simplified one-dimensional model of DNA, we consider molecular junctions with alternating and stacked GC sequences and compare our results to recent experimental measurements.

Constant fields and constant gradients in open ionic channels.

PubMed Central

Chen, D P; Barcilon, V; Eisenberg, R S

1992-01-01

Ions enter cells through pores in proteins that are holes in dielectrics. The energy of interaction between ion and charge induced on the dielectric is many kT, and so the dielectric properties of channel and pore are important. We describe ionic movement by (three-dimensional) Nemst-Planck equations (including flux and net charge). Potential is described by Poisson's equation in the pore and Laplace's equation in the channel wall, allowing induced but not permanent charge. Asymptotic expansions are constructed exploiting the long narrow shape of the pore and the relatively high dielectric constant of the pore's contents. The resulting one-dimensional equations can be integrated numerically; they can be analyzed when channels are short or long (compared with the Debye length). Traditional constant field equations are derived if the induced charge is small, e.g., if the channel is short or if the total concentration gradient is zero. A constant gradient of concentration is derived if the channel is long. Plots directly comparable to experiments are given of current vs voltage, reversal potential vs. concentration, and slope conductance vs. concentration. This dielectric theory can easily be tested: its parameters can be determined by traditional constant field measurements. The dielectric theory then predicts current-voltage relations quite different from constant field, usually more linear, when gradients of total concentration are imposed. Numerical analysis shows that the interaction of ion and channel can be described by a mean potential if, but only if, the induced charge is negligible, that is to say, the electric field is spatially constant. Images FIGURE 1 PMID:1376159

High GC Content Cas9-Mediated Genome-Editing and Biosynthetic Gene Cluster Activation in Saccharopolyspora erythraea.

PubMed

Liu, Yong; Wei, Wen-Ping; Ye, Bang-Ce

2018-05-18

The overexpression of bacterial secondary metabolite biosynthetic enzymes is the basis for industrial overproducing strains. Genome editing tools can be used to further improve gene expression and yield. Saccharopolyspora erythraea produces erythromycin, which has extensive clinical applications. In this study, the CRISPR-Cas9 system was used to edit genes in the S. erythraea genome. A temperature-sensitive plasmid containing the PermE promoter, to drive Cas9 expression, and the Pj23119 and PkasO promoters, to drive sgRNAs, was designed. Erythromycin esterase, encoded by S. erythraea SACE_1765, inactivates erythromycin by hydrolyzing the macrolactone ring. Sequencing and qRT-PCR confirmed that reporter genes were successfully inserted into the SACE_1765 gene. Deletion of SACE_1765 in a high-producing strain resulted in a 12.7% increase in erythromycin levels. Subsequent PermE- egfp knock-in at the SACE_0712 locus resulted in an 80.3% increase in erythromycin production compared with that of wild type. Further investigation showed that PermE promoter knock-in activated the erythromycin biosynthetic gene clusters at the SACE_0712 locus. Additionally, deletion of indA (SACE_1229) using dual sgRNA targeting without markers increased the editing efficiency to 65%. In summary, we have successfully applied Cas9-based genome editing to a bacterial strain, S. erythraea, with a high GC content. This system has potential application for both genome-editing and biosynthetic gene cluster activation in Actinobacteria.

Combined quantum-mechanics/molecular-mechanics dynamics simulation of A-DNA double strands irradiated by ultra-low-energy carbon ions

NASA Astrophysics Data System (ADS)

Ngaojampa, C.; Nimmanpipug, P.; Yu, L. D.; Anuntalabhochai, S.; Lee, V. S.

2011-02-01

In order to promote understanding of the fundamentals of ultra-low-energy ion interaction with DNA, molecular dynamics simulations using combined quantum-mechanics/molecular-mechanics of poly-AT and poly-GC A-DNA double strands irradiated by <200 eV carbon ions were performed to investigate the molecular implications of mutation bias. The simulations were focused on the responses of the DNA backbones and nitrogenous bases to irradiation. Analyses of the root mean square displacements of the backbones and non-hydrogen atoms of base rings of the simulated DNA structure after irradiation revealed a potential preference of DNA double strand separation, dependent on the irradiating energy. The results show that for the backbones, the large difference in the displacement between poly-GC and poly-AT in the initial time period could be the reason for the backbone breakage; for the nitrogenous base pairs, A-T is 30% more sensitive or vulnerable to ion irradiation than G-C, demonstrating a preferential, instead of random, effect of irradiation-induced mutation.

Effects of glass fiber mesh with different fiber content and structures on the compressive properties of complete dentures.

PubMed

Yu, Sang-Hui; Cho, Hye-Won; Oh, Seunghan; Bae, Ji-Myung

2015-06-01

No study has yet evaluated the strength of complete dentures reinforced with glass fiber meshes with different content and structures. The purpose of this study was to compare the reinforcing effects of glass fiber mesh with different content and structures with that of metal mesh in complete dentures. Two types of glass fiber mesh were used: SES mesh (SES) and glass cloth (GC2, GC3, and GC4). A metal mesh was used for comparison. The complete dentures were made by placing the reinforcement 1 mm away from the tissue surface. A control group was prepared without any reinforcement (n=10). The compressive properties were measured by a universal testing machine at a crosshead speed of 5 mm/min. The results were analyzed with the Kruskal-Wallis test and the Duncan multiple range test (α=.05). The fracture resistance of the SES group was significantly higher than that of the control, GC4, and metal groups (asymptotic P=.004), but not significantly different from the GC2 and GC3 groups. The toughness of the SES and GC3 groups was significantly higher than that of the others (asymptotic P<.001), but not significantly different from that of the GC4 group. SES and GC3, which have different structures but similar volume content, were the most effective in reinforcing complete dentures. The content of the glass fiber mesh seemed more important than the structures. Copyright © 2015 Editorial Council for the Journal of Prosthetic Dentistry. Published by Elsevier Inc. All rights reserved.

Immunization with a Vaccine Combining Herpes Simplex Virus 2 (HSV-2) Glycoprotein C (gC) and gD Subunits Improves the Protection of Dorsal Root Ganglia in Mice and Reduces the Frequency of Recurrent Vaginal Shedding of HSV-2 DNA in Guinea Pigs Compared to Immunization with gD Alone ▿

PubMed Central

Awasthi, Sita; Lubinski, John M.; Shaw, Carolyn E.; Barrett, Shana M.; Cai, Michael; Wang, Fushan; Betts, Michael; Kingsley, Susan; DiStefano, Daniel J.; Balliet, John W.; Flynn, Jessica A.; Casimiro, Danilo R.; Bryan, Janine T.; Friedman, Harvey M.

2011-01-01

Attempts to develop a vaccine to prevent genital herpes simplex virus 2 (HSV-2) disease have been only marginally successful, suggesting that novel strategies are needed. Immunization with HSV-2 glycoprotein C (gC-2) and gD-2 was evaluated in mice and guinea pigs to determine whether adding gC-2 to a gD-2 subunit vaccine would improve protection by producing antibodies that block gC-2 immune evasion from complement. Antibodies produced by gC-2 immunization blocked the interaction between gC-2 and complement C3b, and passive transfer of gC-2 antibody protected complement-intact mice but not C3 knockout mice against HSV-2 challenge, indicating that gC-2 antibody is effective, at least in part, because it prevents HSV-2 evasion from complement. Immunization with gC-2 also produced neutralizing antibodies that were active in the absence of complement; however, the neutralizing titers were higher when complement was present, with the highest titers in animals immunized with both antigens. Animals immunized with the gC-2-plus-gD-2 combination had robust CD4+ T-cell responses to each immunogen. Multiple disease parameters were evaluated in mice and guinea pigs immunized with gC-2 alone, gD-2 alone, or both antigens. In general, gD-2 outperformed gC-2; however, the gC-2-plus-gD-2 combination outperformed gD-2 alone, particularly in protecting dorsal root ganglia in mice and reducing recurrent vaginal shedding of HSV-2 DNA in guinea pigs. Therefore, the gC-2 subunit antigen enhances a gD-2 subunit vaccine by stimulating a CD4+ T-cell response, by producing neutralizing antibodies that are effective in the absence and presence of complement, and by blocking immune evasion domains that inhibit complement activation. PMID:21813597

Methylene blue binding to DNA with alternating AT base sequence: minor groove binding is favored over intercalation.

PubMed

Rohs, Remo; Sklenar, Heinz

2004-04-01

The results presented in this paper on methylene blue (MB) binding to DNA with AT alternating base sequence complement the data obtained in two former modeling studies of MB binding to GC alternating DNA. In the light of the large amount of experimental data for both systems, this theoretical study is focused on a detailed energetic analysis and comparison in order to understand their different behavior. Since experimental high-resolution structures of the complexes are not available, the analysis is based on energy minimized structural models of the complexes in different binding modes. For both sequences, four different intercalation structures and two models for MB binding in the minor and major groove have been proposed. Solvent electrostatic effects were included in the energetic analysis by using electrostatic continuum theory, and the dependence of MB binding on salt concentration was investigated by solving the non-linear Poisson-Boltzmann equation. We find that the relative stability of the different complexes is similar for the two sequences, in agreement with the interpretation of spectroscopic data. Subtle differences, however, are seen in energy decompositions and can be attributed to the change from symmetric 5'-YpR-3' intercalation to minor groove binding with increasing salt concentration, which is experimentally observed for the AT sequence at lower salt concentration than for the GC sequence. According to our results, this difference is due to the significantly lower non-electrostatic energy for the minor groove complex with AT alternating DNA, whereas the slightly lower binding energy to this sequence is caused by a higher deformation energy of DNA. The energetic data are in agreement with the conclusions derived from different spectroscopic studies and can also be structurally interpreted on the basis of the modeled complexes. The simple static modeling technique and the neglect of entropy terms and of non-electrostatic solute

Relative stability of DNA as a generic criterion for promoter prediction: whole genome annotation of microbial genomes with varying nucleotide base composition.

PubMed

Rangannan, Vetriselvi; Bansal, Manju

2009-12-01

The rapid increase in genome sequence information has necessitated the annotation of their functional elements, particularly those occurring in the non-coding regions, in the genomic context. Promoter region is the key regulatory region, which enables the gene to be transcribed or repressed, but it is difficult to determine experimentally. Hence an in silico identification of promoters is crucial in order to guide experimental work and to pin point the key region that controls the transcription initiation of a gene. In this analysis, we demonstrate that while the promoter regions are in general less stable than the flanking regions, their average free energy varies depending on the GC composition of the flanking genomic sequence. We have therefore obtained a set of free energy threshold values, for genomic DNA with varying GC content and used them as generic criteria for predicting promoter regions in several microbial genomes, using an in-house developed tool PromPredict. On applying it to predict promoter regions corresponding to the 1144 and 612 experimentally validated TSSs in E. coli (50.8% GC) and B. subtilis (43.5% GC) sensitivity of 99% and 95% and precision values of 58% and 60%, respectively, were achieved. For the limited data set of 81 TSSs available for M. tuberculosis (65.6% GC) a sensitivity of 100% and precision of 49% was obtained.

Electron-transfer oxidation properties of DNA bases and DNA oligomers.

PubMed

Fukuzumi, Shunichi; Miyao, Hiroshi; Ohkubo, Kei; Suenobu, Tomoyoshi

2005-04-21

Kinetics for the thermal and photoinduced electron-transfer oxidation of a series of DNA bases with various oxidants having the known one-electron reduction potentials (E(red)) in an aqueous solution at 298 K were examined, and the resulting electron-transfer rate constants (k(et)) were evaluated in light of the free energy relationship of electron transfer to determine the one-electron oxidation potentials (E(ox)) of DNA bases and the intrinsic barrier of the electron transfer. Although the E(ox) value of GMP at pH 7 is the lowest (1.07 V vs SCE) among the four DNA bases, the highest E(ox) value (CMP) is only 0.19 V higher than that of GMP. The selective oxidation of GMP in the thermal electron-transfer oxidation of GMP results from a significant decrease in the pH dependent oxidation potential due to the deprotonation of GMP*+. The one-electron reduced species of the photosensitizer produced by photoinduced electron transfer are observed as the transient absorption spectra when the free energy change of electron transfer is negative. The rate constants of electron-transfer oxidation of the guanine moieties in DNA oligomers with Fe(bpy)3(3+) and Ru(bpy)3(3+) were also determined using DNA oligomers containing different guanine (G) sequences from 1 to 10 G. The rate constants of electron-transfer oxidation of the guanine moieties in single- and double-stranded DNA oligomers with Fe(bpy)3(2+) and Ru(bpy)3(3+) are dependent on the number of sequential guanine molecules as well as on pH.

Cytometric analysis of shape and DNA content in mammalian sperm

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gledhill, B.L.

1983-10-10

Male germ cells respond dramatically to a variety of insults and are important reproductive dosimeters. Semen analyses are very useful in studies on the effects of drugs, chemicals, and environmental hazards on testicular function, male fertility and heritable germinal mutations. Sperm were analyzed by flow cytometry and slit-scan flow analysis for injury following the exposure of testes to mutagens. The utility of flow cytometry in genotoxin screening and monitoring of occupational exposure was evaluated. The technique proved valuable in separation of X- and Y-chromosome bearing sperm and the potential applicability of this technique in artificial insemination and a solution, ofmore » accurately assessing the DNA content of sperm were evaluated-with reference to determination of X- and Y-chromosome bearing sperm.« less

Combined effects of metal complexation and size expansion in the electronic structure of DNA base pairs

NASA Astrophysics Data System (ADS)

Brancolini, Giorgia; Di Felice, Rosa

2011-05-01

Novel DNA derivatives have been recently investigated in the pursuit of modified DNA duplexes to tune the electronic structure of DNA-based assemblies for nanotechnology applications. Size-expanded DNAs (e.g., xDNA) and metalated DNAs (M-DNA) may enhance stacking interactions and induce metallic conductivity, respectively. Here we explore possible ways of tailoring the DNA electronic structure by combining the aromatic size expansion with the metal-doping. We select the salient structures from our recent study on natural DNA pairs complexed with transition metal ions and consider the equivalent model configurations for xDNA pairs. We present the results of density functional theory electronic structure calculations of the metalated expanded base-pairs with various localized basis sets and exchange-correlation functionals. Implicit solvent and coordination water molecules are also included. Our results indicate that the effect of base expansion is largest in Ag-xGC complexes, while Cu-xGC complexes are the most promising candidates for nanowires with enhanced electron transfer and also for on-purpose modification of the DNA double-helix for signal detection.

Telomere Length in Leukocyte DNA in Gastric Cancer Patients and its Association with Clinicopathological Features and Prognosis.

PubMed

Tahara, Tomomitsu; Tahara, Sayumi; Horiguchi, Noriyuki; Kawamura, Tomohiko; Okubo, Masaaki; Ishizuka, Takamitsu; Yamada, Hyuga; Yoshida, Dai; Ohmori, Takafumi; Maeda, Kohei; Komura, Naruomi; Ikuno, Hirokazu; Jodai, Yasutaka; Kamano, Toshiaki; Nagasaka, Mitsuo; Nakagawa, Yoshihito; Tuskamoto, Tetsuya; Urano, Makoto; Shibata, Tomoyuki; Kuroda, Makoto; Ohmiya, Naoki

2017-04-01

Telomere shortening in leukocytes has been thought to be associated with reduced immune response capacity and increased chromosome instability. Several studies indicate that telomere length in the peripheral blood leukocyte DNA can predict clinical outcome of several cancers. We evaluated the potential association between telomere shortening in the leukocyte DNA and clinicopathological features and prognosis of gastric cancer (GC) in Japanese patients. Telomere length in leukocyte DNA was measured using quantitative real-time polymerase chain reaction (PCR) in 207 GC patients. The association between telomere length and clinicopathological features and prognosis was evaluated. These short-telomere group was significantly associated with advanced stage (p=0.015), worse overall survival (OS) and progression-free survival (PFS) (p=0.046 and 0.026, respectively). The same group was also weakly associated with overall and peritoneal recurrences (p=0.052 and 0.059, respectively). Telomere shortening in leukocyte DNA is associated with advanced stage and poor prognosis of GC, which may reflect their reduced immune response capacity or increased chromosome instability. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

The mitochondrial genome of the pathogenic yeast Candida subhashii: GC-rich linear DNA with a protein covalently attached to the 5′ termini

PubMed Central

Fricova, Dominika; Valach, Matus; Farkas, Zoltan; Pfeiffer, Ilona; Kucsera, Judit; Tomaska, Lubomir; Nosek, Jozef

2010-01-01

As a part of our initiative aimed at a large-scale comparative analysis of fungal mitochondrial genomes, we determined the complete DNA sequence of the mitochondrial genome of the yeast Candida subhashii and found that it exhibits a number of peculiar features. First, the mitochondrial genome is represented by linear dsDNA molecules of uniform length (29 795 bp), with an unusually high content of guanine and cytosine residues (52.7 %). Second, the coding sequences lack introns; thus, the genome has a relatively compact organization. Third, the termini of the linear molecules consist of long inverted repeats and seem to contain a protein covalently bound to terminal nucleotides at the 5′ ends. This architecture resembles the telomeres in a number of linear viral and plasmid DNA genomes classified as invertrons, in which the terminal proteins serve as specific primers for the initiation of DNA synthesis. Finally, although the mitochondrial genome of C. subhashii contains essentially the same set of genes as other closely related pathogenic Candida species, we identified additional ORFs encoding two homologues of the family B protein-priming DNA polymerases and an unknown protein. The terminal structures and the genes for DNA polymerases are reminiscent of linear mitochondrial plasmids, indicating that this genome architecture might have emerged from fortuitous recombination between an ancestral, presumably circular, mitochondrial genome and an invertron-like element. PMID:20395267

Effect of proton transfer on the electronic coupling in DNA

NASA Astrophysics Data System (ADS)

Rak, Janusz; Makowska, Joanna; Voityuk, Alexander A.

2006-06-01

The effects of single and double proton transfer within Watson-Crick base pairs on donor-acceptor electronic couplings, Vda, in DNA are studied on the bases of quantum chemical calculations. Four dimers [AT,AT], [GC,GC], [GC,AT] and [GC,TA)] are considered. Three techniques - the generalized Mulliken-Hush scheme, the fragment charge method and the diabatic states method - are employed to estimate Vda for hole transfer between base pairs. We show that both single- and double proton transfer (PT) reactions may substantially affect the electronic coupling in DNA. The electronic coupling in [AT,AT] is predicted to be most sensitive to PT. Single PT within the first base pair in the dimer leads to increase in the hole transfer efficiency by a factor of 4, while proton transfer within the second pair should substantially, by 2.7 times, decrease the rate of charge transfer. Thus, directional asymmetry of the PT effects on the electronic coupling is predicted. The changes in the Vda matrix elements correlate with the topological properties of orbitals of donor and acceptor and can be qualitatively rationalized in terms of resonance structures of donor and acceptor. Atomic pair contributions to the Vda matrix elements are also analyzed.

Detection and quantification of ochratoxin A and deoxynivalenol in barley grains by GC-MS and electronic nose.

PubMed

Olsson, J; Börjesson, T; Lundstedt, T; Schnürer, J

2002-02-05

Mycotoxin contamination of cereal grains can be detected and quantified using complex extraction procedures and analytical techniques. Normally, the grain odour, i.e. the presence of non-grain volatile metabolites, is used for quality classification of grain. We have investigated the possibility of using fungal volatile metabolites as indicators of mycotoxins in grain. Ten barley samples with normal odour, and 30 with some kind of off-odour were selected from Swedish granaries. The samples were evaluated with regard to moisture content, fungal contamination, ergosterol content, and levels of ochratoxin A (OA) and deoxynivalenol (DON). Volatile compounds were also analysed using both an electronic nose and gas chromatography combined with mass spectrometry (GC-MS). Samples with normal odour had no detectable ochratoxin A and average DON contents of 16 microg kg(-1) (range 0-80), while samples with off-odour had average OA contents of 76 microg kg(-1) (range 0-934) and DON contents of 69 microg kg(-1) (range 0-857). Data were evaluated by multivariate data analysis using projection methods such as principal component analysis (PCA) and partial least squares (PLS). The results show that it was possible to classify the OA level as below or above the maximum limit of 5 microg kg(-1) cereal grain established by the Swedish National Food Administration, and that the DON level could be estimated using PLS. Samples with OA levels below 5 microg kg(-1) had higher concentration of aldehydes (nonanal, 2-hexenal) and alcohols (1-penten-3-ol, 1-octanol). Samples with OA levels above 5 microg kg(-1) had higher concentrations of ketones (2-hexanone, 3-octanone). The GC-MS system predicted OA concentrations with a higher accuracy than the electronic nose, since the GC-MS misclassified only 3 of 37 samples and the electronic nose 7 of 37 samples. No correlation was found between odour and OA level, as samples with pronounced or strong off-odours had OA levels both below and above 5

The GC-rich mitochondrial and plastid genomes of the green alga Coccomyxa give insight into the evolution of organelle DNA nucleotide landscape

DOE PAGES

Smith, David Roy; Burki, Fabien; Yamada, Takashi; ...

2011-08-26

Here, most of the available mitochondrial and plastid genome sequences are biased towards adenine and thymine (AT) over guanine and cytosine (GC). Examples of GC-rich organelle DNAs are limited to a small but eclectic list of species, including certain green algae. Here, to gain insight in the evolution of organelle nucleotide landscape, we present the GC-rich mitochondrial and plastid DNAs from the trebouxiophyte green alga Coccomyxa sp. C-169. We compare these sequences with other GC-rich organelle DNAs and argue that the forces biasing them towards G and C are nonadaptive and linked to the metabolic and/or life history features ofmore » this species. The Coccomyxa organelle genomes are also used for phylogenetic analyses, which highlight the complexities in trying to resolve the interrelationships among the core chlorophyte green algae, but ultimately favour a sister relationship between the Ulvophyceae and Chlorophyceae, with the Trebouxiophyceae branching at the base of the chlorophyte crown.« less

The GC-Rich Mitochondrial and Plastid Genomes of the Green Alga Coccomyxa Give Insight into the Evolution of Organelle DNA Nucleotide Landscape

DOE Office of Scientific and Technical Information (OSTI.GOV)

Smith, David Roy; Burki, Fabien; Yamada, Takashi

2011-05-13

Most of the available mitochondrial and plastid genome sequences are biased towards adenine and thymine (AT) over guanine and cytosine (GC). Examples of GC-rich organelle DNAs are limited to a small but eclectic list of species, including certain green algae. Here, to gain insight in the evolution of organelle nucleotide landscape, we present the GC-rich mitochondrial and plastid DNAs from the trebouxiophyte green alga Coccomyxa sp. C-169. We compare these sequences with other GC-rich organelle DNAs and argue that the forces biasing them towards G and C are nonadaptive and linked to the metabolic and/or life history features of thismore » species. The Coccomyxa organelle genomes are also used for phylogenetic analyses, which highlight the complexities in trying to resolve the interrelationships among the core chlorophyte green algae, but ultimately favour a sister relationship between the Ulvophyceae and Chlorophyceae, with the Trebouxiophyceae branching at the base of the chlorophyte crown.« less

A Constant Rate of Spontaneous Mutation in DNA-Based Microbes

NASA Astrophysics Data System (ADS)

Drake, John W.

1991-08-01

In terms of evolution and fitness, the most significant spontaneous mutation rate is likely to be that for the entire genome (or its nonfrivolous fraction). Information is now available to calculate this rate for several DNA-based haploid microbes, including bacteriophages with single- or double-stranded DNA, a bacterium, a yeast, and a filamentous fungus. Their genome sizes vary by ≈6500-fold. Their average mutation rates per base pair vary by ≈16,000-fold, whereas their mutation rates per genome vary by only ≈2.5-fold, apparently randomly, around a mean value of 0.0033 per DNA replication. The average mutation rate per base pair is inversely proportional to genome size. Therefore, a nearly invariant microbial mutation rate appears to have evolved. Because this rate is uniform in such diverse organisms, it is likely to be determined by deep general forces, perhaps by a balance between the usually deleterious effects of mutation and the physiological costs of further reducing mutation rates.

Photocatalytic degradation of clofibric acid by g-C3N4/P25 composites under simulated sunlight irradiation: The significant effects of reactive species.

PubMed

Chen, Ping; Wang, Fengliang; Zhang, Qianxin; Su, Yuehan; Shen, Lingzhi; Yao, Kun; Chen, Zhi-Feng; Liu, Yang; Cai, Zongwei; Lv, Wenying; Liu, Guoguang

2017-04-01

Pharmaceutically emerging micropollutants have become an environmental concern in recent years. In the present paper, the reactive species (RSs)-induced degradation mechanism of clofibric acid (CA) was investigated using a newly sunlight-driven g-C 3 N 4 /P25 photocatalyst. A very low g-C 3 N 4 content of 8.0 weight percent resulted in a 3.36 and a 2.29 times faster reaction rate for CA photodegradation than for pristine g-C 3 N 4 and P25, respectively. Electron spin resonance and quenching experiments demonstrated the participation of HO, h + , e - , 1 O 2 and O 2 ·- in the photocatalytic system, and the contribution rates were calculated to 73.3%, 15.3%, 5.1%, 6.7% and 33.1%, respectively. According to the pulse radiolysis measurements and the competitive kinetics approaches, the bimolecular reaction rate constants for HO, e - , and 1 O 2 with CA were (8.47 ± 0.33) × 10 9  M -1 s -1 , (6.41 ± 0.48) × 10 9  M -1 s -1 and (6.6 ± 0.37) × 10 6  M -1 s -1 , respectively. RSs were found to significantly influence the degradation of CA, and the degradation pathways occurred primarily via e - reduction, HO addition and 1 O 2 attack reactions on the basis of mass spectrometry and theoretical calculations. Copyright © 2017 Elsevier Ltd. All rights reserved.

DNA vaccine expressing herpes simplex virus 1 glycoprotein C and D protects mice against herpes simplex keratitis

PubMed Central

Dong, Li-Li; Tang, Ru; Zhai, Yu-Jia; Malla, Tejsu; Hu, Kai

2017-01-01

AIM To investigate whether DNA vaccine encoding herpes simplex virus 1 (HSV-1) glycoprotein C (gC) and glycoprotein D (gD) will achieve better protective effect against herpes simplex keratitis (HSK) than DNA vaccine encoding gD alone. METHODS DNA vaccine expressing gD or gC combined gD (gD.gC) were constructed and carried by chitosan nanoparticle. The expression of fusion protein gD and gC were detected in DNA/nanoparticle transfected 293T cells by Western-blot. For immunization, mice were inoculated with DNA/nanoparticle for 3 times with 2wk interval, and two weeks after the final immunization, the specific immune responses and clinical degrees of primary HSK were evaluated. RESULTS Fusion protein gD.gC could be expressed successfully in cultured 293T cells. And, pRSC-gC.gD-IL21 DNA/chitosan nanoparticle could effectively elicit strongest humoral and cellular immune response in primary HSK mice evidenced by higher levels of specific neutralizing antibody and sIgA production, enhanced cytotoxicities of splenocytes and nature killer cells (NK), when compared with those of gD alone or mocked vaccine immunized mice. As a result, gC-based vaccine immunized mice showed least HSK disease. CONCLUSION gC-based DNA vaccine could effectively prevent the progress of primary HSK, suggesting that this DNA vaccine could be a promising vaccine for HSK treatment in the future. PMID:29181304

Persulfate enhanced photocatalytic degradation of bisphenol A by g-C3N4 nanosheets under visible light irradiation.

PubMed

Liu, Bochuan; Qiao, Meng; Wang, Yanbin; Wang, Lijuan; Gong, Yan; Guo, Tao; Zhao, Xu

2017-12-01

The enhancement of g-C 3 N 4 photocatalytic degradation of bisphenol A (BPA) via persulfate (PS) addition was investigated under visible light irradiation. The effects of various parameters on the BPA degradation were investigated, such as catalysts dosage, PS concentrations, initial pH value and BPA concentration. The results showed that g-C 3 N 4 nanosheets exhibited superior photocatalytic activity toward BPA degradation as compared with bulk g-C 3 N 4 . The addition of PS can further improve the g-C 3 N 4 photocatalytic performance for BPA degradation. With 5 mM PS, the degradation rate of BPA was increased from 72.5% to 100% at 90 min, and the corresponding first-order kinetic constants were increased from 0.0028 to 0.0140 min -1 . The removal efficiency of BPA increased with the decrease of solution pH value. The active radicals in the reaction system were tested by electron spin resonance (ESR) and radicals quenching experiments. Instead of persulfate radicals' oxidation, it was proposed that the main active radicals for BPA degradation were superoxide radicals and the photogenerated holes. Copyright © 2017. Published by Elsevier Ltd.

Biodiesel production from microalgal isolates of southern Pakistan and quantification of FAMEs by GC-MS/MS analysis

PubMed Central

2012-01-01

Background Microalgae have attracted major interest as a sustainable source for biodiesel production on commercial scale. This paper describes the screening of six microalgal species, Scenedesmus quadricauda, Scenedesmus acuminatus, Nannochloropsis sp., Anabaena sp., Chlorella sp. and Oscillatoria sp., isolated from fresh and marine water resources of southern Pakistan for biodiesel production and the GC-MS/MS analysis of their fatty acid methyl esters (FAMEs). Results Growth rate, biomass productivity and oil content of each algal species have been investigated under autotrophic condition. Biodiesel was produced from algal oil by acid catalyzed transesterification reaction and resulting fatty acid methyl esters (FAMEs) content was analyzed by GC/MS. Fatty acid profiling of the biodiesel, obtained from various microalgal oils showed high content of C-16:0, C-18:0, cis-Δ9C-18:1, cis-Δ11C-18:1 (except Scenedesmus quadricauda) and 10-hydroxyoctadecanoic (except Scenedesmus acuminatus). Absolute amount of C-14:0, C-16:0 and C-18:0 by a validated GC-MS/MS method were found to be 1.5-1.7, 15.0-42.5 and 4.2-18.4 mg/g, respectively, in biodiesel obtained from various microalgal oils. Biodiesel was also characterized in terms of cetane number, kinematic viscosity, density and higher heating value and compared with the standard values. Conclusion Six microalgae of local origin were screened for biodiesel production. A method for absolute quantification of three important saturated fatty acid methyl esters (C-14, C-16 and C-18) by gas chromatography-tandem mass spectrometry (GC-MS/MS), using multiple reactions monitoring (MRM) mode, was employed for the identification and quantification of biodiesels obtained from various microalgal oils. The results suggested that locally found microalgae can be sustainably harvested for the production of biodiesel. This offers the tremendous economic opportunity for an energy-deficient nation. PMID:23216896

Rapid quantitative detection of glucose content in glucose injection by reaction headspace gas chromatography.

PubMed

Xie, Wei-Qi; Gong, Yi-Xian; Yu, Kong-Xian

2017-10-20

This work investigates an automated technique for rapid detecting the glucose content in glucose injection by reaction headspace gas chromatography (HS-GC). This method is based on the oxidation reaction of glucose in glucose injection with potassium dichromate. The carbon dioxide (CO 2 ) formed from the oxidation reaction can be quantitatively detected by GC. The results show that the relative standard deviation (RSD) of the present method was within 2.91%, and the measured glucose contents in glucose injection closely match those quantified by the reference method (relative differences <6.45%). The new HS-GC technique is rapid, practical and can be used to the batch detection of the glucose content in glucose injection related applications. Copyright © 2017 Elsevier B.V. All rights reserved.

Evaluation of volatiles from two subtropical strawberry cultivars using GC-olfactometry, GC-MS odor activity values, and sensory analysis

USDA-ARS?s Scientific Manuscript database

Flavor profiles of two Florida strawberry cultivars were determined using GC-olfactometry,GC-MS, odor activity values (OAVs) and sensory analysis. Thirty-six aroma active compounds were detected using GC-O. Thirty-four were identified. The major odor-active compounds in decreasing intensity were: me...

Modeling hole transport in wet and dry DNA.

PubMed

Pavanello, Michele; Adamowicz, Ludwik; Volobuyev, Maksym; Mennucci, Benedetta

2010-04-08

We present a DFT/classical molecular dynamics model of DNA charge conductivity. The model involves a temperature-driven, hole-hopping charge transfer and includes the time-dependent nonequilibrium interaction of DNA with its molecular environment. We validate our method against a variety of hole transport experiments. The method predicts a significant hole-transfer slowdown of approximately 35% from dry to wet DNA with and without electric field bias. In addition, in agreement with experiments, it also predicts an insulating behavior of (GC)(N) oligomers for 40 < N < 1000, depending on the experimental setup.

Ribosomal DNA, tri- and bi-partite pericentromeres in the permanent translocation heterozygote Rhoeo spathacea.

PubMed

Golczyk, Hieronim; Hasterok, Robert; Szklarczyk, Marek

2010-12-01

High- and low-stringency FISH and base-specific fluorescence were performed on the permanent translocation heterozygote Rhoeo spathacea (2n = 12). Our results indicate that 45S rDNA arrays, rDNA-related sequences and other GC-rich DNA fraction(s) are located within the pericentromeric regions of all twelve chromosomes, usually colocalizing with the chromomycin A(3)-positive bands. Homogenization of the pericentromeric regions appears to result from the concerted spread of GC-rich sequences, with differential amplification likely. We found new 5S rDNA patterns, which suggest a variability in the breakpoints and in the consequent chromosome reorganizations. It was found that the large 5S rDNA locus residing on each of the 8E and 9E arms consisted of two smaller loci. On each of the two chromosome arms 3b and 4b, in addition to the major subtelomeric 5S rDNA locus, a new minor locus was found interstitially about 40% along the arm length. The arrangement of cytotogenetic landmarks and chromosome arm measurements are discussed with regard to genome repatterning in Rhoeo.

Diversity and distribution of single-stranded DNA phages in the North Atlantic Ocean

PubMed Central

Tucker, Kimberly P; Parsons, Rachel; Symonds, Erin M; Breitbart, Mya

2011-01-01

Knowledge of marine phages is highly biased toward double-stranded DNA (dsDNA) phages; however, recent metagenomic surveys have also identified single-stranded DNA (ssDNA) phages in the oceans. Here, we describe two complete ssDNA phage genomes that were reconstructed from a viral metagenome from 80 m depth at the Bermuda Atlantic Time-series Study (BATS) site in the northwestern Sargasso Sea and examine their spatial and temporal distributions. Both genomes (SARssφ1 and SARssφ2) exhibited similarity to known phages of the Microviridae family in terms of size, GC content, genome organization and protein sequence. PCR amplification of the replication initiation protein (Rep) gene revealed narrow and distinct depth distributions for the newly described ssDNA phages within the upper 200 m of the water column at the BATS site. Comparison of Rep gene sequences obtained from the BATS site over time revealed changes in the diversity of ssDNA phages over monthly time scales, although some nearly identical sequences were recovered from samples collected 4 years apart. Examination of ssDNA phage diversity along transects through the North Atlantic Ocean revealed a positive correlation between genetic distance and geographic distance between sampling sites. Together, the data suggest fundamental differences between the distribution of these ssDNA phages and the distribution of known marine dsDNA phages, possibly because of differences in host range, host distribution, virion stability, or viral evolution mechanisms and rates. Future work needs to elucidate the host ranges for oceanic ssDNA phages and determine their ecological roles in the marine ecosystem. PMID:21124487

The Variation Analysis of DNA Methylation in Wheat Carrying Gametocidal Chromosome 3C from Aegilops triuncialis

PubMed Central

Wang, Dan; Zhao, Jieyu; Bai, Yan; Ao, You; Guo, Changhong

2017-01-01

Gametocidal (Gc) chromosomes can ensure their preferential transmission by killing the gametes without themselves through causing chromosome breakage and therefore have been exploited as an effective tool for genetic breeding. However, to date very little is known about the molecular mechanism of Gc action. In this study, we used methylation-sensitive amplified polymorphism (MSAP) technique to assess the extent and pattern of cytosine methylation alterations at the whole genome level between two lines of wheat Gc addition line and their common wheat parent. The results indicated that the overall levels of cytosine methylation of two studied Gc addition lines (CS–3C and CS–3C3C, 48.68% and 48.65%, respectively) were significantly increased when compared to common wheat CS (41.31%) and no matter fully methylated or hemimethylated rates enhanced in Gc addition lines. A set of 30 isolated fragments that showed different DNA methylation or demethylation patterns between the three lines were sequenced and the results indicated that 8 fragments showed significant homology to known sequences, of which three were homologous to MITE transposon (Miniature inverted–repeat transposable elements), LTR-retrotransposon WIS-1p and retrotransposon Gypsy, respectively. Overall, our results showed that DNA methylation could play a role in the Gc action. PMID:28796162

The Variation Analysis of DNA Methylation in Wheat Carrying Gametocidal Chromosome 3C from Aegilops triuncialis.

PubMed

Wang, Dan; Zhao, Jieyu; Bai, Yan; Ao, You; Guo, Changhong

2017-08-10

Gametocidal (Gc) chromosomes can ensure their preferential transmission by killing the gametes without themselves through causing chromosome breakage and therefore have been exploited as an effective tool for genetic breeding. However, to date very little is known about the molecular mechanism of Gc action. In this study, we used methylation-sensitive amplified polymorphism (MSAP) technique to assess the extent and pattern of cytosine methylation alterations at the whole genome level between two lines of wheat Gc addition line and their common wheat parent. The results indicated that the overall levels of cytosine methylation of two studied Gc addition lines (CS-3C and CS-3C3C, 48.68% and 48.65%, respectively) were significantly increased when compared to common wheat CS (41.31%) and no matter fully methylated or hemimethylated rates enhanced in Gc addition lines. A set of 30 isolated fragments that showed different DNA methylation or demethylation patterns between the three lines were sequenced and the results indicated that 8 fragments showed significant homology to known sequences, of which three were homologous to MITE transposon (Miniature inverted-repeat transposable elements), LTR-retrotransposon WIS-1p and retrotransposon Gypsy , respectively. Overall, our results showed that DNA methylation could play a role in the Gc action.

Genome Calligrapher: A Web Tool for Refactoring Bacterial Genome Sequences for de Novo DNA Synthesis.

PubMed

Christen, Matthias; Deutsch, Samuel; Christen, Beat

2015-08-21

Recent advances in synthetic biology have resulted in an increasing demand for the de novo synthesis of large-scale DNA constructs. Any process improvement that enables fast and cost-effective streamlining of digitized genetic information into fabricable DNA sequences holds great promise to study, mine, and engineer genomes. Here, we present Genome Calligrapher, a computer-aided design web tool intended for whole genome refactoring of bacterial chromosomes for de novo DNA synthesis. By applying a neutral recoding algorithm, Genome Calligrapher optimizes GC content and removes obstructive DNA features known to interfere with the synthesis of double-stranded DNA and the higher order assembly into large DNA constructs. Subsequent bioinformatics analysis revealed that synthesis constraints are prevalent among bacterial genomes. However, a low level of codon replacement is sufficient for refactoring bacterial genomes into easy-to-synthesize DNA sequences. To test the algorithm, 168 kb of synthetic DNA comprising approximately 20 percent of the synthetic essential genome of the cell-cycle bacterium Caulobacter crescentus was streamlined and then ordered from a commercial supplier of low-cost de novo DNA synthesis. The successful assembly into eight 20 kb segments indicates that Genome Calligrapher algorithm can be efficiently used to refactor difficult-to-synthesize DNA. Genome Calligrapher is broadly applicable to recode biosynthetic pathways, DNA sequences, and whole bacterial genomes, thus offering new opportunities to use synthetic biology tools to explore the functionality of microbial diversity. The Genome Calligrapher web tool can be accessed at https://christenlab.ethz.ch/GenomeCalligrapher  .

Dynamic and spectroscopic studies of nano-micelles comprising dye in water/ dioctyl sodium sulfosuccinate /decane droplet microemulsion at constant water content

NASA Astrophysics Data System (ADS)

Rahdar, Abbas; Almasi-Kashi, Mohammad

2017-01-01

In the present work, the dynamic and spectroscopic properties of water-in-decane dioctyl sodium sulfosuccinate (AOT) microemulsions comprising dye, Rhodamine B (RB), were studied by varying content of decane at the constant water content (W = 20), by using dynamic light scattering (DLS), UV/visible, and fluorescence techniques. The characterization results of DLS of AOT micelles showed that by decreasing concentration of Rhodamine B in the water/AOT/decane microemulsion, the inter-droplet interactions changed from attractive to repulsive as the mass fraction of nano-droplets (MFD) increased. A deviation in the absorption spectra of Rhodamine B from the Beer's law at the high Rhodamine B concentration (0.001) was observed in the AOT reversed micelles. The Quenching in the emission intensity of AOT droplets comprising Rhodamine B and red shift in λmax of fluorescence of dye was observed as a function of concentration of RB in AOT RMs. The Stokes shift of AOT droplets containing the high concentration of RB, increased with mass fraction of nano-droplet (MFD), whereas at the low Rhodamine B concentration, its variation remained constant up to MFD = 0.07, and then increased.

Identification (GC and GC-MS) of unsaturated acetates in Elasmopalpus lignosellus and their biological activity (GC-EAD and EAG).

PubMed

Jham, Gulab N; da Silva, Alexsandro A; Lima, Eraldo R; Viana, Paulo

2005-02-01

Two insect colonies of Elasmopalpus lignosellus were reared in our laboratory, the first being initiated from pupae obtained from a cornfield in the region of Sete Lagoas, Minas Gerais and the second from a cornfield in the region of Goiânia, Goiás. From the two colonies, two extracts were prepared from the pheromone glands of virgin E. lignosellus females. The extract obtained from the first colony was designated as extract 1 while the extract obtained from the second colony was designated as extract 2. Extract 1 was analyzed by gas chromatography-mass spectrometry (GC-MS) with (Z)-9-hexadecenyl acetate [(Z)-9-HDA] and (Z)-11-hexadecenyl acetate [(Z)-11-HDA] being identified and confirmed by the formation of DMDS derivatives. In addition, a third acetate, which could be either (E)-8-hexadecenyl acetate [(E)-8-HDA] or (E)-9-hexadecenyl acetate [(E)-9-HDA] was detected by GC-MS. Extract 2 was analyzed by gas chromatography (GC) and gas chromatography-electroannetography (GC-EAD) revealing the presence of (Z)-11-HDA and (Z)-9-TDA. In addition, the same compounds elicited a response with the E. lignosellus male antenna obtained from the second insect colony. Electroantennography (EAG) screening with the male E. lignosellus antenna (obtained from the second insect colony) was conducted with the 23 possible tetradecenyl acetates (TDA) and 22 hexadecenyl acetates (HDA) as standards. Out of the 23 TDA isomers evaluated, only (Z)-9-TDA elicited a response and out of the 22 HDA [(Z) and (E) isomers gamma2 to delta13] evaluated only (Z)-11-HDA elicited a response. The acetate compositions of two extracts obtained from insects originating from the two states (Minas Gerais and Goiás) of Brazil were different from one another as well as from that obtained from insects in Tifton, GA, USA. The bioactivity data (GC-EAD) of the extract 2 differed from those reported for the Tifton, GA, USA population. These data suggest polymorphism in relation to the insect populations found in

Photocatalytic reductive dechlorination of 2-chlorodibenzo-p-dioxin by Pd modified g-C3N4 photocatalysts under UV-vis irradiation: Efficacy, kinetics and mechanism.

PubMed

Ding, Jiafeng; Long, Gaoyuan; Luo, Yang; Sun, Runze; Chen, Mengxia; Li, Yajun; Zhou, Yanfang; Xu, Xinhua; Zhao, Weirong

2018-05-09

Polychlorinated dibenzo-p-dioxins (PCDDs), as a group of notorious anthropogenic environmental toxicants, are arguably ubiquitous in nature. In this study, we investigated the photocatalytic reductive dechlorination of 2-chlorodibenzo-p-dioxin (2-CDD) over Pd/g-C 3 N 4 catalysts under UV-vis irradiation. The g-C 3 N 4 and a series of Pd/g-C 3 N 4 catalysts were prepared by thermal polymerization and mechanical mixing-illumination method and characterized by XRD, TEM, BET, SEM and UV-vis DRS analyses. Among all the samples, the Pd/g-C 3 N 4 (5 wt%) yielded the optimal dechlorination activity with a total 2-CDD conversion of 54% within 4 h, and 76% of those converted 2-CDD were evolved to dibenzo-p-dioxin (DD). The kinetics of dechlorination could be described as pseudo-first-order decay model (R 2  > 0.84). Corresponding rate constants (k) increased from 0.052 to 0.17 h -1 with Pd contents up to 5 wt% and decreased to 0.13 h -1 with a 10 wt% of Pd. The enhanced activities originated from the surface plasmonic resonance (SPR) effect of Pd nanoparticles and the formation of Schottky barrier between Pd and g-C 3 N 4 , which extend the spectrum responsive range and suppress the charge recombination of g-C 3 N 4 . This is the first report on the photocatalytic reductive removal of PCDDs and may provide a new approach for PCDDs pollution control. Copyright © 2018 Elsevier B.V. All rights reserved.

Genomics dataset on unclassified published organism (patent US 7547531).

PubMed

Khan Shawan, Mohammad Mahfuz Ali; Hasan, Md Ashraful; Hossain, Md Mozammel; Hasan, Md Mahmudul; Parvin, Afroza; Akter, Salina; Uddin, Kazi Rasel; Banik, Subrata; Morshed, Mahbubul; Rahman, Md Nazibur; Rahman, S M Badier

2016-12-01

Nucleotide (DNA) sequence analysis provides important clues regarding the characteristics and taxonomic position of an organism. With the intention that, DNA sequence analysis is very crucial to learn about hierarchical classification of that particular organism. This dataset (patent US 7547531) is chosen to simplify all the complex raw data buried in undisclosed DNA sequences which help to open doors for new collaborations. In this data, a total of 48 unidentified DNA sequences from patent US 7547531 were selected and their complete sequences were retrieved from NCBI BioSample database. Quick response (QR) code of those DNA sequences was constructed by DNA BarID tool. QR code is useful for the identification and comparison of isolates with other organisms. AT/GC content of the DNA sequences was determined using ENDMEMO GC Content Calculator, which indicates their stability at different temperature. The highest GC content was observed in GP445188 (62.5%) which was followed by GP445198 (61.8%) and GP445189 (59.44%), while lowest was in GP445178 (24.39%). In addition, New England BioLabs (NEB) database was used to identify cleavage code indicating the 5, 3 and blunt end and enzyme code indicating the methylation site of the DNA sequences was also shown. These data will be helpful for the construction of the organisms' hierarchical classification, determination of their phylogenetic and taxonomic position and revelation of their molecular characteristics.

Sensitivity of GC-EI/MS, GC-EI/MS/MS, LC-ESI/MS/MS, LC-Ag(+) CIS/MS/MS, and GC-ESI/MS/MS for analysis of anabolic steroids in doping control.

PubMed

Cha, Eunju; Kim, Sohee; Kim, Ho Jun; Lee, Kang Mi; Kim, Ki Hun; Kwon, Oh-Seung; Lee, Jaeick

2015-01-01

This study compared the sensitivity of various separation and ionization methods, including gas chromatography with an electron ionization source (GC-EI), liquid chromatography with an electrospray ionization source (LC-ESI), and liquid chromatography with a silver ion coordination ion spray source (LC-Ag(+) CIS), coupled to a mass spectrometer (MS) for steroid analysis. Chromatographic conditions, mass spectrometric transitions, and ion source parameters were optimized. The majority of steroids in GC-EI/MS/MS and LC-Ag(+) CIS/MS/MS analysis showed higher sensitivities than those obtained with other analytical methods. The limits of detection (LODs) of 65 steroids by GC-EI/MS/MS, 68 steroids by LC-Ag(+) CIS/MS/MS, 56 steroids by GC-EI/MS, 54 steroids by LC-ESI/MS/MS, and 27 steroids by GC-ESI/MS/MS were below cut-off value of 2.0 ng/mL. LODs of steroids that formed protonated ions in LC-ESI/MS/MS analysis were all lower than the cut-off value. Several steroids such as unconjugated C3-hydroxyl with C17-hydroxyl structure showed higher sensitivities in GC-EI/MS/MS analysis relative to those obtained using the LC-based methods. The steroids containing 4, 9, 11-triene structures showed relatively poor sensitivities in GC-EI/MS and GC-ESI/MS/MS analysis. The results of this study provide information that may be useful for selecting suitable analytical methods for confirmatory analysis of steroids. Copyright © 2015 John Wiley & Sons, Ltd.

Determination of Nitroaromatic, Nitramine, and Nitrate Ester Explosives in Soils Using GC-ECD

DTIC Science & Technology

1999-08-01

for supplying soils from minefields; and Dr. Paul H. Miyares, CRREL, for HPLC analysis of Fort Leonard Wood soil extracts. ii CONTENTS P reface...42 ILLUSTRATIONS Figure 1. Correlation analysis of GC-ECD concentration (mg/kg) estimates with those from HPLC -UV...kg) estimates with those from HPLC -UV analysis using splits of the same acetonitrile extract from archived soils

Systematic evaluation of bias in microbial community profiles induced by whole genome amplification.

PubMed

Direito, Susana O L; Zaura, Egija; Little, Miranda; Ehrenfreund, Pascale; Röling, Wilfred F M

2014-03-01

Whole genome amplification methods facilitate the detection and characterization of microbial communities in low biomass environments. We examined the extent to which the actual community structure is reliably revealed and factors contributing to bias. One widely used [multiple displacement amplification (MDA)] and one new primer-free method [primase-based whole genome amplification (pWGA)] were compared using a polymerase chain reaction (PCR)-based method as control. Pyrosequencing of an environmental sample and principal component analysis revealed that MDA impacted community profiles more strongly than pWGA and indicated that this related to species GC content, although an influence of DNA integrity could not be excluded. Subsequently, biases by species GC content, DNA integrity and fragment size were separately analysed using defined mixtures of DNA from various species. We found significantly less amplification of species with the highest GC content for MDA-based templates and, to a lesser extent, for pWGA. DNA fragmentation also interfered severely: species with more fragmented DNA were less amplified with MDA and pWGA. pWGA was unable to amplify low molecular weight DNA (< 1.5 kb), whereas MDA was inefficient. We conclude that pWGA is the most promising method for characterization of microbial communities in low-biomass environments and for currently planned astrobiological missions to Mars. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

GC/HRSIR as a Complementary Technique to GC/ECNIMS

EPA Science Inventory

Gas chromatography/electron capture negative ion mass spectrometry (GC/ECNIMS) is a highly selective and sensitive technique for the analysis of appropriate analytes in complex matrices. Its major drawback is often the lack of fragmentation indicative of structure that can be use...

Effect of treated-sewage contamination upon bacterial energy charge, adenine nucleotides, and DNA content in a sandy aquifer on cape cod

USGS Publications Warehouse

Metge, D.W.; Brooks, M.H.; Smith, R.L.; Harvey, R.W.

1993-01-01

Changes in adenylate energy charge (EC(A)) and in total adenine nucleotides (A(T)) and DNA content (both normalized to the abundance of free- living, groundwater bacteria) in response to carbon loading were determined for a laboratory-grown culture and for a contaminated aquifer. The latter study involved a 3-km-long transect through a contaminant plume resulting from continued on-land discharge of secondary sewage to a shallow, sandy aquifer on Cape Cod, Mass. With the exception of the most contaminated groundwater immediately downgradient from the contaminant source, DNA and adenylate levels correlated strongly with bacterial abundance and decreased exponentially with increasing distance downgradient. EC(A)s (0.53 to 0.60) and the ratios of ATP to DNA (0.001 to 0.003) were consistently low, suggesting that the unattached bacteria in this groundwater study are metabolically stressed, despite any eutrophication that might have occurred. Elevated EC(A)s (up to 0.74) were observed in glucose-amended groundwater, confirming that the metabolic state of this microbial community could be altered. In general, per-bacterium DNA and ATP contents were approximately twofold higher in the plume than in surrounding groundwater, although EC(A) and per-bacterium levels of A(T) differed little in the plume and the surrounding uncontaminated groundwater. However, per-bacterium levels of DNA and A(T) varied six- and threefold, respectively, during a 6-h period of decreasing growth rate for an unidentified pseudomonad isolated from contaminated groundwater and grown in batch culture. These data suggest that the DNA content of groundwater bacteria may be more sensitive than their A(T) to the degree of carbon loading, which may have significant ramifications in the use of nucleic acids and adenine nucleotides for estimating the metabolic status of bacterial communities within more highly contaminated aquifers.

Competition between B-Z and B-L transitions in a single DNA molecule: Computational studies

NASA Astrophysics Data System (ADS)

Kwon, Ah-Young; Nam, Gi-Moon; Johner, Albert; Kim, Seyong; Hong, Seok-Cheol; Lee, Nam-Kyung

2016-02-01

Under negative torsion, DNA adopts left-handed helical forms, such as Z-DNA and L-DNA. Using the random copolymer model developed for a wormlike chain, we represent a single DNA molecule with structural heterogeneity as a helical chain consisting of monomers which can be characterized by different helical senses and pitches. By Monte Carlo simulation, where we take into account bending and twist fluctuations explicitly, we study sequence dependence of B-Z transitions under torsional stress and tension focusing on the interaction with B-L transitions. We consider core sequences, (GC) n repeats or (TG) n repeats, which can interconvert between the right-handed B form and the left-handed Z form, imbedded in a random sequence, which can convert to left-handed L form with different (tension dependent) helical pitch. We show that Z-DNA formation from the (GC) n sequence is always supported by unwinding torsional stress but Z-DNA formation from the (TG) n sequence, which are more costly to convert but numerous, can be strongly influenced by the quenched disorder in the surrounding random sequence.

The Met Office Global Coupled Model 3.0 and 3.1 (GC3.0 and GC3.1) Configurations

NASA Astrophysics Data System (ADS)

Williams, K. D.; Copsey, D.; Blockley, E. W.; Bodas-Salcedo, A.; Calvert, D.; Comer, R.; Davis, P.; Graham, T.; Hewitt, H. T.; Hill, R.; Hyder, P.; Ineson, S.; Johns, T. C.; Keen, A. B.; Lee, R. W.; Megann, A.; Milton, S. F.; Rae, J. G. L.; Roberts, M. J.; Scaife, A. A.; Schiemann, R.; Storkey, D.; Thorpe, L.; Watterson, I. G.; Walters, D. N.; West, A.; Wood, R. A.; Woollings, T.; Xavier, P. K.

2018-02-01

The Global Coupled 3 (GC3) configuration of the Met Office Unified Model is presented. Among other applications, GC3 is the basis of the United Kingdom's submission to the Coupled Model Intercomparison Project 6 (CMIP6). This paper documents the model components that make up the configuration (although the scientific descriptions of these components are in companion papers) and details the coupling between them. The performance of GC3 is assessed in terms of mean biases and variability in long climate simulations using present-day forcing. The suitability of the configuration for predictability on shorter time scales (weather and seasonal forecasting) is also briefly discussed. The performance of GC3 is compared against GC2, the previous Met Office coupled model configuration, and against an older configuration (HadGEM2-AO) which was the submission to CMIP5. In many respects, the performance of GC3 is comparable with GC2, however, there is a notable improvement in the Southern Ocean warm sea surface temperature bias which has been reduced by 75%, and there are improvements in cloud amount and some aspects of tropical variability. Relative to HadGEM2-AO, many aspects of the present-day climate are improved in GC3 including tropospheric and stratospheric temperature structure, most aspects of tropical and extratropical variability and top-of-atmosphere and surface fluxes. A number of outstanding errors are identified including a residual asymmetric sea surface temperature bias (cool northern hemisphere, warm Southern Ocean), an overly strong global hydrological cycle and insufficient European blocking.

[Determination of DNA content in individual Yersinia pestis cells by using flow cytofluorimetry method: comparative analysis of inhomogeneity in cultures of strains with various biological properties].

PubMed

Kravtsov, A L; Liapin, M N; Shmel'kova, T P; Golovko, E M; Maliukova, T A; Kostiukova, T A; Ezhov, I N

2011-01-01

Comparative analysis of Yersinia pestis strains with various biological properties by DNA content in individual cells. Virulent strain 231, avirulent strain KM 260 (12) [231], that is its isogenic (no-plasmid) derivative, and vaccine strain EV NIIEG were used. 48-hour agar cultures of the studied strains reproduced at 28 degrees C and their subcultures obtained by cultivation of the initial cultures by aeration on liquid nutrient medium from 37 degrees C were prepared. DNA of the fixed bacteria was dyed by a mixture of ethidium bromide and mitramycin, and then the bacteria were studied by using flow cytofluorimeter for the determination of rates of cells with relatively low or high DNA content in the studied bacterial populations. The degree of inhomogeneity of a bacterial population was evaluated by DNA histogram variation coefficient value. In 6 hours of growth at 37 degrees C in optically non-dense bacterial cultures a high degree of DNA content per cell inhomogeneity was established that is related to the activation of DNA replication process in bacteria. In 48 hours of growth this inhomogeneity completely disappeared in the virulent strain cultures and remained in the avirulent strain cultures of the plague pathogen. Based on the studied parameters the vaccine strain held an intermediate position. Further studies of the plague culture DNA content per cell inhomogeneity may become a base for the operative strain differentiation based on pathogenicity level (hazard) for humans, and therefore the requirements for the management of safe working conditions with this microorganism.

[Increased content of nucleotide sequences in transcriptionally active DNA and poly(A)+-mRNA of the rat liver and a rise in its translation activity as affected by inducers].

PubMed

Dashkevich, V S; Vishnivetskiĭ, S N; Skobel'tsina, L M; Luk'ianchikova, N L; Kaledin, V I

1986-12-01

Cortisol and 3'-methyl-4-dimethyl-amino-azobenzene induce an increase in the content of repeated sequences (RS) in transcriptionally active (TA) DNA, while the content of respective RS in potentially active DNA fractions enriched with regulatory regions of the genome decreases. RS content in induced poly A+-mRNA also rises, as determined by the nature of hybridization of respective c DNA with total DNA. The translation of induced poly A+-mRNA rises essentially, with the qualitative distinctions in in vitro synthesized protein product spectrum being absent. Inducible RS with unstable chromatine conformation are thought to provide a universal system of rapid response of the genetic apparatus to extreme situations, serving as transcription intensifiers in TA DNA and as translation intensifiers in induced poly A+-mRNA.

Exploring the common molecular basis for the universal DNA mutation bias: Revival of Loewdin mutation model

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu, Liang-Yu; Center for Bioinformatics, Huazhong Agricultural University, Wuhan 430070; Wang, Guang-Zhong

2011-06-10

Highlights: {yields} There exists a universal G:C {yields} A:T mutation bias in three domains of life. {yields} This universal mutation bias has not been sufficiently explained. {yields} A DNA mutation model proposed by Loewdin 40 years ago offers a common explanation. -- Abstract: Recently, numerous genome analyses revealed the existence of a universal G:C {yields} A:T mutation bias in bacteria, fungi, plants and animals. To explore the molecular basis for this mutation bias, we examined the three well-known DNA mutation models, i.e., oxidative damage model, UV-radiation damage model and CpG hypermutation model. It was revealed that these models cannot providemore » a sufficient explanation to the universal mutation bias. Therefore, we resorted to a DNA mutation model proposed by Loewdin 40 years ago, which was based on inter-base double proton transfers (DPT). Since DPT is a fundamental and spontaneous chemical process and occurs much more frequently within GC pairs than AT pairs, Loewdin model offers a common explanation for the observed universal mutation bias and thus has broad biological implications.« less

[Comparative GC analysis of essential oil in imported sandalwood].

PubMed

Wang, Z; Hong, X

1991-01-01

The GC-fingerprint spectra of essential oils in imported sandalwood are established by the new technique of GC-relative retention value fingerprint spectrum (GC-FPS). According to the GC-FPS of samples, their chromatographic peaks, overlap ratio of peaks and eight strong peaks are studied comparatively.

Application of HS-SPME and GC-MS to characterization of volatile compounds emitted from Osmanthus flowers.

PubMed

Deng, Chunhui; Song, Guoxin; Hu, Yaoming

2004-12-01

Headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS) was developed for characterization of volatile compounds emitted from two varieties Osmanthus flowers of O. fragrans var. latifolius and O. fragrans var. thunbergii. The SPME parameters were studied, the optimum conditions of a 65 microm carbowax/divinylbenzene (CW/DVB), extraction temperature of 22 degrees C and extraction time of 10 min were obtained and applied to extraction of the volatile emissions. Fourteen compounds released from both varieties of Osmanthus flowers were separated and identified by GC-MS, which mainly included alpha-linalool, beta-linalool, trans-linalool oxide, cis-linalool oxide, alpha-lonone, beta-lonone, capraldehyde and decalactone. By comparing their peak areas, we found that the sums of the fourteen compounds from the two Osmanthus flowers were very close, while the relative contents of individual volatile compounds in the two emissions were very different. The relative content of alpha-linalool and beta-linalool in O. fragrans var. latifolius were 39.46% and 0.51%, while in O. fragrans var. thunbergii were 9.53% and 27.71%. Due to their different relative contents, the two varieties of flower have different fragrances.

Predicting stability of DNA duplexes in solutions containing magnesium and monovalent cations.

PubMed

Owczarzy, Richard; Moreira, Bernardo G; You, Yong; Behlke, Mark A; Walder, Joseph A

2008-05-13

Accurate predictions of DNA stability in physiological and enzyme buffers are important for the design of many biological and biochemical assays. We therefore investigated the effects of magnesium, potassium, sodium, Tris ions, and deoxynucleoside triphosphates on melting profiles of duplex DNA oligomers and collected large melting data sets. An empirical correction function was developed that predicts melting temperatures, transition enthalpies, entropies, and free energies in buffers containing magnesium and monovalent cations. The new correction function significantly improves the accuracy of predictions and accounts for ion concentration, G-C base pair content, and length of the oligonucleotides. The competitive effects of potassium and magnesium ions were characterized. If the concentration ratio of [Mg (2+)] (0.5)/[Mon (+)] is less than 0.22 M (-1/2), monovalent ions (K (+), Na (+)) are dominant. Effects of magnesium ions dominate and determine duplex stability at higher ratios. Typical reaction conditions for PCR and DNA sequencing (1.5-5 mM magnesium and 20-100 mM monovalent cations) fall within this range. Conditions were identified where monovalent and divalent cations compete and their stability effects are more complex. When duplexes denature, some of the Mg (2+) ions associated with the DNA are released. The number of released magnesium ions per phosphate charge is sequence dependent and decreases surprisingly with increasing oligonucleotide length.

Quantitative analysis of total starch content in wheat flour by reaction headspace gas chromatography.

PubMed

Xie, Wei-Qi; Gong, Yi-Xian; Yu, Kong-Xian

2017-09-01

This paper proposed a new reaction headspace gas chromatographic (HS-GC) method for efficiently quantifying the total starch content in wheat flours. A certain weight of wheat flour was oxidized by potassium dichromate in an acidic condition in a sealed headspace vial. The results show that the starch in wheat flour can be completely transferred to carbon dioxide at the given conditions (at 100 °C for 40 min) and the total starch content in wheat flour sample can be indirectly quantified by detecting the CO 2 formed from the oxidation reaction. The data showed that the relative standard deviation of the reaction HS-GC method in the precision test was less than 3.06%, and the relative differences between the new method and the reference method (titration method) were no more than 8.90%. The new reaction HS-GC method is automated, accurate, and can be a reliable tool for determining the total starch content in wheat flours in both laboratory and industrial applications. Graphical abstract The total starch content in wheat flour can be indirectly quantified by the GC detection of the CO 2 formed from the oxidation reaction between wheat flour and potassium dichromate in an acidic condition.

Effect of serotonin on the expression of antigens and DNA levels in Yersinia pestis cells with different plasmid content

NASA Astrophysics Data System (ADS)

Klueva, Svetlana N.; Korsukov, Vladimir N.; Schukovskaya, Tatyana N.; Kravtsov, Alexander L.

2004-08-01

Using flow cytometry (FCM) the influence of exogenous serotonin on culture growth, DNA content and fluorescence intensity of cells binding FITC-labelled plague polyclonal immunoglobulins was studied in Yersinia pestis EV (pFra+, pCad+, pPst+), Yersinia pestis KM218 (pFra-, pCad-, pPst-), Yersinia pestis KM 216 (pFra-, pCad-, pPst+). The results have been obtained by FCM showed serotonin accelerated Yersinia pestis EV (pFra+, pCad+, pPst+), Yersinia pestis KM218 (pFra-, pCad-, pPst-) culture growth during cultivation in Hottinger broth pH 7.2 at 28°C at concentration of 10-5 M. The presence of 10-5 M serotonin in nutrient broth could modulate DNA content in 37°C growing population of plague microbe independently of their plasmid content. Serotonin have been an impact on the distribution pattern of the cells according to their phenotypical characteristics, which was reflected in the levels of population heterogeneity in the intensity of specific immunofluorescence determined by FMC.

Studies on the interaction of a synthetic nitro-flavone derivative with DNA: A multi-spectroscopic and molecular docking approach.

PubMed

Mitra, A; Saikh, F; Das, J; Ghosh, S; Ghosh, R

2018-05-22

Interaction of a ligand with DNA is often the basis of drug action of many molecules. Flavones are important in this regard as their structural features confer them the ability to bind to DNA. 2-(4-Nitrophenyl)-4H-chromen-4-one (4NCO) is an important biologically active synthetic flavone derivative. We are therefore interested in studying its interaction with DNA. Absorption spectroscopy studies included standard and reverse titration, effect of ionic strength on titration, determination of stoichiometry of binding and thermal denaturation. Spectrofluorimetry techniques included fluorimetric titration, quenching studies and fluorescence displacement assay. Assessment of relative viscosity and estimation of thermodynamic parameters from CD spectral studies were also undertaken. Furthermore, molecular docking analyses were also done with different short DNA sequences. The fluorescent flavone 4NCO reversibly interacted with DNA through partial intercalation as well as minor-groove binding. The binding constant and the number of binding sites were of the order 10 4  M -1 and 1 respectively. The binding stoichiometry with DNA was found to be 1:1. The nature of the interaction of 4NCO with DNA was hydrophobic in nature and the process of binding was spontaneous, endothermic and entropy-driven. The flavone also showed a preference for binding to GC rich sequences. The study presents a profile for structural and thermodynamic parameters, for the binding of 4NCO with DNA. DNA is an important target for ligands that are effective against cell proliferative disorders. In this regard, the molecule 4NCO is important since it can exert its biological activity through its DNA binding ability and can be a potential drug candidate. Copyright © 2018 Elsevier B.V. All rights reserved.

The Dynamic Interplay Between DNA Topoisomerases and DNA Topology.

PubMed

Seol, Yeonee; Neuman, Keir C

2016-09-01

Topological properties of DNA influence its structure and biochemical interactions. Within the cell DNA topology is constantly in flux. Transcription and other essential processes including DNA replication and repair, alter the topology of the genome, while introducing additional complications associated with DNA knotting and catenation. These topological perturbations are counteracted by the action of topoisomerases, a specialized class of highly conserved and essential enzymes that actively regulate the topological state of the genome. This dynamic interplay among DNA topology, DNA processing enzymes, and DNA topoisomerases, is a pervasive factor that influences DNA metabolism in vivo . Building on the extensive structural and biochemical characterization over the past four decades that established the fundamental mechanistic basis of topoisomerase activity, the unique roles played by DNA topology in modulating and influencing the activity of topoisomerases have begun to be explored. In this review we survey established and emerging DNA topology dependent protein-DNA interactions with a focus on in vitro measurements of the dynamic interplay between DNA topology and topoisomerase activity.

The dynamic interplay between DNA topoisomerases and DNA topology.

PubMed

Seol, Yeonee; Neuman, Keir C

2016-11-01

Topological properties of DNA influence its structure and biochemical interactions. Within the cell, DNA topology is constantly in flux. Transcription and other essential processes, including DNA replication and repair, not only alter the topology of the genome but also introduce additional complications associated with DNA knotting and catenation. These topological perturbations are counteracted by the action of topoisomerases, a specialized class of highly conserved and essential enzymes that actively regulate the topological state of the genome. This dynamic interplay among DNA topology, DNA processing enzymes, and DNA topoisomerases is a pervasive factor that influences DNA metabolism in vivo. Building on the extensive structural and biochemical characterization over the past four decades that has established the fundamental mechanistic basis of topoisomerase activity, scientists have begun to explore the unique roles played by DNA topology in modulating and influencing the activity of topoisomerases. In this review we survey established and emerging DNA topology-dependent protein-DNA interactions with a focus on in vitro measurements of the dynamic interplay between DNA topology and topoisomerase activity.

Electrochemical DNA sensor for anthrax toxin activator gene atxA-detection of PCR amplicons.

PubMed

Das, Ritu; Goel, Ajay K; Sharma, Mukesh K; Upadhyay, Sanjay

2015-12-15

We report the DNA probe functionalized electrochemical genosensor for the detection of Bacillus anthracis, specific towards the regulatory gene atxA. The DNA sensor is fabricated on electrochemically deposited gold nanoparticle on self assembled layer of (3-Mercaptopropyl) trimethoxysilane (MPTS) on GC electrode. DNA hybridization is monitored by differential pulse voltammogram (DPV). The modified GC electrode is characterized by atomic force microscopy (AFM), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) method. We also quantified the DNA probe density on electrode surface by the chronocoulometric method. The detection is specific and selective for atxA gene by DNA probe on the electrode surface. No report is available for the detection of B. anthracis by using atxA an anthrax toxin activator gene. In the light of real and complex sample, we have studied the PCR amplicons of 303, 361 and 568 base pairs by using symmetric and asymmetric PCR approaches. The DNA probe of atxA gene efficiently hybridizes with different base pairs of PCR amplicons. The detection limit is found to be 1.0 pM (S/N ratio=3). The results indicate that the DNA sensor is able to detect synthetic target as well as PCR amplicons of different base pairs. Copyright © 2015 Elsevier B.V. All rights reserved.

Intranuclear DNA density affects chromosome condensation in metazoans

PubMed Central

Hara, Yuki; Iwabuchi, Mari; Ohsumi, Keita; Kimura, Akatsuki

2013-01-01

Chromosome condensation is critical for accurate inheritance of genetic information. The degree of condensation, which is reflected in the size of the condensed chromosomes during mitosis, is not constant. It is differentially regulated in embryonic and somatic cells. In addition to the developmentally programmed regulation of chromosome condensation, there may be adaptive regulation based on spatial parameters such as genomic length or cell size. We propose that chromosome condensation is affected by a spatial parameter called the chromosome amount per nuclear space, or “intranuclear DNA density.” Using Caenorhabditis elegans embryos, we show that condensed chromosome sizes vary during early embryogenesis. Of importance, changing DNA content to haploid or polyploid changes the condensed chromosome size, even at the same developmental stage. Condensed chromosome size correlates with interphase nuclear size. Finally, a reduction in nuclear size in a cell-free system from Xenopus laevis eggs resulted in reduced condensed chromosome sizes. These data support the hypothesis that intranuclear DNA density regulates chromosome condensation. This suggests an adaptive mode of chromosome condensation regulation in metazoans. PMID:23783035

A constant radius of curvature model for the organization of DNA in toroidal condensates.

PubMed Central

Hud, N V; Downing, K H; Balhorn, R

1995-01-01

Toroidal DNA condensates have received considerable attention for their possible relationship to the packaging of DNA in viruses and in general as a model of ordered DNA condensation. A spool-like model has primarily been supported for DNA organization within toroids. However, our observations suggest that the actual organization may be considerably different. We present an alternate model in which DNA for a given toroid is organized within a series of equally sized contiguous loops that precess about the toroid axis. A related model for the toroid formation process is also presented. This kinetic model predicts a distribution of toroid sizes for DNA condensed from solution that is in good agreement with experimental data. Images Fig. 1 Fig. 2 Fig. 3 Fig. 5 PMID:7724602

CpG Distribution and Methylation Pattern in Porcine Parvovirus

PubMed Central

Tóth, Renáta; Mészáros, István; Stefancsik, Rajmund; Bartha, Dániel; Bálint, Ádám; Zádori, Zoltán

2013-01-01

Based on GC content and the observed/expected CpG ratio (oCpGr), we found three major groups among the members of subfamily Parvovirinae: Group I parvoviruses with low GC content and low oCpGr values, Group II with low GC content and high oCpGr values and Group III with high GC content and high oCpGr values. Porcine parvovirus belongs to Group I and it features an ascendant CpG distribution by position in its coding regions similarly to the majority of the parvoviruses. The entire PPV genome remains hypomethylated during the viral lifecycle independently from the tissue of origin. In vitro CpG methylation of the genome has a modest inhibitory effect on PPV replication. The in vitro hypermethylation disappears from the replicating PPV genome suggesting that beside the maintenance DNMT1 the de novo DNMT3a and DNMT3b DNA methyltransferases can’t methylate replicating PPV DNA effectively either, despite that the PPV infection does not seem to influence the expression, translation or localization of the DNA methylases. SNP analysis revealed high mutability of the CpG sites in the PPV genome, while introduction of 29 extra CpG sites into the genome has no significant biological effects on PPV replication in vitro. These experiments raise the possibility that beyond natural selection mutational pressure may also significantly contribute to the low level of the CpG sites in the PPV genome. PMID:24392033

Yeast aconitase binds and provides metabolically coupled protection to mitochondrial DNA.

PubMed

Chen, Xin Jie; Wang, Xiaowen; Butow, Ronald A

2007-08-21

Aconitase (Aco1p) is a multifunctional protein: It is an enzyme of the tricarboxylic acid cycle. In animal cells, Aco1p also is a cytosolic protein binding to mRNAs to regulate iron metabolism. In yeast, Aco1p was identified as a component of mtDNA nucleoids. Here we show that yeast Aco1p protects mtDNA from excessive accumulation of point mutations and ssDNA breaks and suppresses reductive recombination of mtDNA. Aconitase binds to both ds- and ssDNA, with a preference for GC-containing sequences. Therefore, mitochondria are opportunistic organelles that seize proteins, such as metabolic enzymes, for construction of the nucleoid, an mtDNA maintenance/segregation apparatus.

Constant load and constant volume response of municipal solid waste in simple shear.

PubMed

Zekkos, Dimitrios; Fei, Xunchang

2017-05-01

Constant load and constant volume simple shear testing was conducted on relatively fresh municipal solid waste (MSW) from two landfills in the United States, one in Michigan and a second in Texas, at respective natural moisture content below field capacity. The results were assessed in terms of two failure strain criteria, at 10% and 30% shear strain, and two interpretations of effective friction angle. Overall, friction angle obtained assuming that the failure plane is horizontal and at 10% shear strain resulted in a conservative estimation of shear strength of MSW. Comparisons between constant volume and constant load simple shear testing results indicated significant differences in the shear response of MSW with the shear resistance in constant volume being lower than the shear resistance in constant load. The majority of specimens were nearly uncompacted during specimen preparation to reproduce the state of MSW in bioreactor landfills or in uncontrolled waste dumps. The specimens had identical percentage of <20mm material but the type of <20mm material was different. The <20mm fraction from Texas was finer and of high plasticity. MSW from Texas was overall weaker in both constant load and constant volume conditions compared to Michigan waste. The results of these tests suggest the possibility of significantly lower shear strength of MSW in bioreactor landfills where waste is placed with low compaction effort and constant volume, i.e., "undrained", conditions may occur. Compacted MSW specimens resulted in shear strength parameters that are higher than uncompacted specimens and closer to values reported in the literature. However, the normalized undrained shear strength in simple shear for uncompacted and compacted MSW was still higher than the normalized undrained shear strength reported in the literature for clayey and silty soils. Copyright © 2016 Elsevier Ltd. All rights reserved.

The comparison of solid phase microextraction-GC and static headspace-GC for determination of solvent residues in vegetable oils.

PubMed

Ligor, Magdalena; Buszewski, Bogusław

2008-02-01

The objective of these investigations has been the determination of volatile organic compounds including residue solvents present in vegetable oil samples. Some olive oil, rape oil, sunflower oil, soy-bean oil, pumpkin oil, grape oil, rice oil as well as hazel-nut oil samples were analysed. Among residue solvents the following compounds have been mentioned: acetone, n-hexane, benzene, and toluene. Some experiments for the solid phase microextraction (SPME)-GC-flame ionisation detection (FID) were performed to examine extraction conditions such as fiber exposure time, temperature of extraction, and temperature of desorption. Various SPME fibers such as polydimethylsiloxane, Carboxen/polydimethylsiloxane and polydimethylsiloxane/divinylbenzene coatings were used for the isolation of tested compounds from vegetable oil samples. After optimisation of SPME, real vegetable oil samples were examined using SPME-GC/MS. Based on preliminary experiments the qualitative and quantitative analyses for the determination of acetone, n-hexane, benzene and toluene were performed by SPME-GC-FID and static head-space (SHS)-GC-FID methods. The regression coefficients for calibration curves for the examined compounds were R(2) > or = 0.992. This shows that the used method is linear in the examined concentration range (0.005-0.119 mg/kg for SPME-GC-FID and 0.003-0.728 mg/kg for SHS-GC-FID). Chemical properties of analysed vegetable oils have been characterised by chemometric procedure (cluster analysis).

Enhanced visible light photocatalytic H2-production of g-C3N4/WS2 composite heterostructures

NASA Astrophysics Data System (ADS)

Akple, Maxwell Selase; Low, Jingxiang; Wageh, S.; Al-Ghamdi, Ahmed. A.; Yu, Jiaguo; Zhang, Jun

2015-12-01

As a clean and renewable solar H2-production system to address the increasing global environmental crisis and energy demand, photocatalytic hydrogen production from water splitting using earth abundant materials has received a lot of attention. In this study, WS2-graphitic carbon nitride (g-C3N4) composites were prepared using WO3 and thiourea as precursors through a gas-solid reaction. Different amount of WS2 were loaded on g-C3N4 to form the heterostructures and the composite samples exhibited enhanced photocatalytic activity for H2 production under visible light. The composite sample with 0.01 wt% WS2 exhibited the highest H2-production rate of 101 μmol g-1 h-1, which was even better than that of the Pt-C3N4 sample with the same loading content. The high photocatalytic activity was attributed to the formation of heterojunction between g-C3N4 and WS2 cocatalyst which allowed for effective separation of photogenerated charge carriers. This work showed the possibility for the utilization of low cost WS2 as an efficient cocatalyst to promote the photocatalytic H2 production of g-C3N4.

Astragalin from Cassia alata Induces DNA Adducts in Vitro and Repairable DNA Damage in the Yeast Saccharomyces cerevisiae

PubMed Central

Saito, Samuel; Silva, Givaldo; Santos, Regineide Xavier; Gosmann, Grace; Pungartnik, Cristina; Brendel, Martin

2012-01-01

Reverse phase-solid phase extraction from Cassia alata leaves (CaRP) was used to obtain a refined extract. Higher than wild-type sensitivity to CaRP was exhibited by 16 haploid Saccharomyces cerevisiae mutants with defects in DNA repair and membrane transport. CaRP had a strong DPPH free radical scavenging activity with an IC50 value of 2.27 μg mL−1 and showed no pro-oxidant activity in yeast. CaRP compounds were separated by HPLC and the three major components were shown to bind to DNA in vitro. The major HPLC peak was identified as kampferol-3-O-β-d-glucoside (astragalin), which showed high affinity to DNA as seen by HPLC-UV measurement after using centrifugal ultrafiltration of astragalin-DNA mixtures. Astragalin-DNA interaction was further studied by spectroscopic methods and its interaction with DNA was evaluated using solid-state FTIR. These and computational (in silico) docking studies revealed that astragalin-DNA binding occurs through interaction with G-C base pairs, possibly by intercalation stabilized by H-bond formation. PMID:22489129

Astragalin from Cassia alata induces DNA adducts in vitro and repairable DNA damage in the yeast Saccharomyces cerevisiae.

PubMed

Saito, Samuel; Silva, Givaldo; Santos, Regineide Xavier; Gosmann, Grace; Pungartnik, Cristina; Brendel, Martin

2012-01-01

Reverse phase-solid phase extraction from Cassia alata leaves (CaRP) was used to obtain a refined extract. Higher than wild-type sensitivity to CaRP was exhibited by 16 haploid Saccharomyces cerevisiae mutants with defects in DNA repair and membrane transport. CaRP had a strong DPPH free radical scavenging activity with an IC(50) value of 2.27 μg mL(-1) and showed no pro-oxidant activity in yeast. CaRP compounds were separated by HPLC and the three major components were shown to bind to DNA in vitro. The major HPLC peak was identified as kampferol-3-O-β-d-glucoside (astragalin), which showed high affinity to DNA as seen by HPLC-UV measurement after using centrifugal ultrafiltration of astragalin-DNA mixtures. Astragalin-DNA interaction was further studied by spectroscopic methods and its interaction with DNA was evaluated using solid-state FTIR. These and computational (in silico) docking studies revealed that astragalin-DNA binding occurs through interaction with G-C base pairs, possibly by intercalation stabilized by H-bond formation.

An oxygen-vacancy-rich Z-scheme g-C3N4/Pd/TiO2 heterostructure for enhanced visible light photocatalytic performance

NASA Astrophysics Data System (ADS)

Guo, Yanru; Xiao, Limin; Zhang, Min; Li, Qiuye; Yang, Jianjun

2018-05-01

An oxygen-vacancy-rich Z-scheme g-C3N4/Pd/TiO2 ternary nanocomposite was fabricated using nanotubular titanic acid as precursors via a simple photo-deposition of Pd nanoparticles and calcination process. The prepared nanocomposites were investigated by X-ray diffraction, transmission electron microscopy, X-ray photoelectron spectroscopy, and UV-visible diffuse reflectance spectroscopy, respectively. For g-C3N4/TiO2 binary nanocomposites, at the optimal content of g-C3N4 (2%), the apparent photocatalytic activity of 2%g-C3N4/TiO2 was 9 times higher than that of pure TiO2 under visible-light illumination. After deposition of Pd (1 wt%) at the contact interface between g-C3N4 and TiO2, the 2%g-C3N4/Pd/TiO2 ternary nanocomposites demonstrated the highest visible-light-driven photocatalytic activity for the degradation of gaseous propylene, which was 16- and 2-fold higher activities than pure TiO2 and 2%g-C3N4/TiO2, respectively. The mechanism for the enhanced photocatalytic performance of the g-C3N4/Pd/TiO2 photo-catalyst is proposed to be based on the efficient separation of photo-generated electron-hole pairs through Z-scheme system, in which uniform dispersity of Pd nanoparticles at contact interface between g-C3N4 and TiO2 and oxygen vacancies promote charge separation.

Solvent effect on the intermolecular proton transfer of the Watson and Crick guanine-cytosine and adenine-thymine base pairs: a polarizable continuum model study.

PubMed

Romero, Eduardo E; Hernandez, Florencio E

2018-01-03

Herein we present our results on the study of the double proton transfer (DPT) mechanism in the adenine-thymine (AT) and guanine-cytosine (GC) base pairs, both in gas phase and in solution. The latter was modeled using the polarizable continuum method (PCM) in different solvents. According to our DFT calculations, the DPT may occur for both complexes in a stepwise mechanism in condensate phase. In gas phase only the GC base pair exhibits a concerted DPT mechanism. Using the Wigner's tunneling corrections to the transition state theory we demonstrate that such corrections are important for the prediction of the rate constants of both systems in gas and in condensate phase. We also show that (i) as the polarity of the medium decreases the equilibrium constant of the DPT reaction increases in both complexes, and (ii) that the equilibrium constant in the GC complex is four orders of magnitude larger than in AT. This observation suggests that the spontaneous mutations in DNA base pairs are more probable in GC than in AT.

Uracil recognition by replicative DNA polymerases is limited to the archaea, not occurring with bacteria and eukarya.

PubMed

Wardle, Josephine; Burgers, Peter M J; Cann, Isaac K O; Darley, Kate; Heslop, Pauline; Johansson, Erik; Lin, Li-Jung; McGlynn, Peter; Sanvoisin, Jonathan; Stith, Carrie M; Connolly, Bernard A

2008-02-01

Family B DNA polymerases from archaea such as Pyrococcus furiosus, which live at temperatures approximately 100 degrees C, specifically recognize uracil in DNA templates and stall replication in response to this base. Here it is demonstrated that interaction with uracil is not restricted to hyperthermophilic archaea and that the polymerase from mesophilic Methanosarcina acetivorans shows identical behaviour. The family B DNA polymerases replicate the genomes of archaea, one of the three fundamental domains of life. This publication further shows that the DNA replicating polymerases from the other two domains, bacteria (polymerase III) and eukaryotes (polymerases delta and epsilon for nuclear DNA and polymerase gamma for mitochondrial) are also unable to recognize uracil. Uracil occurs in DNA as a result of deamination of cytosine, either in G:C base-pairs or, more rapidly, in single stranded regions produced, for example, during replication. The resulting G:U mis-pairs/single stranded uracils are promutagenic and, unless repaired, give rise to G:C to A:T transitions in 50% of the progeny. The confinement of uracil recognition to polymerases of the archaeal domain is discussed in terms of the DNA repair pathways necessary for the elimination of uracil.

Division-induced DNA double strand breaks in the chromosome terminus region of Escherichia coli lacking RecBCD DNA repair enzyme

PubMed Central

Durand, Adeline; Desfontaines, Jean-Michel; Iurchenko, Ielyzaveta; Auger, Hélène; Leach, David R. F.

2017-01-01

Marker frequency analysis of the Escherichia coli recB mutant chromosome has revealed a deficit of DNA in a specific zone of the terminus, centred on the dif/TerC region. Using fluorescence microscopy of a marked chromosomal site, we show that the dif region is lost after replication completion, at the time of cell division, in one daughter cell only, and that the phenomenon is transmitted to progeny. Analysis by marker frequency and microscopy shows that the position of DNA loss is not defined by the replication fork merging point since it still occurs in the dif/TerC region when the replication fork trap is displaced in strains harbouring ectopic Ter sites. Terminus DNA loss in the recB mutant is also independent of dimer resolution by XerCD at dif and of Topo IV action close to dif. It occurs in the terminus region, at the point of inversion of the GC skew, which is also the point of convergence of specific sequence motifs like KOPS and Chi sites, regardless of whether the convergence of GC skew is at dif (wild-type) or a newly created sequence. In the absence of FtsK-driven DNA translocation, terminus DNA loss is less precisely targeted to the KOPS convergence sequence, but occurs at a similar frequency and follows the same pattern as in FtsK+ cells. Importantly, using ftsIts, ftsAts division mutants and cephalexin treated cells, we show that DNA loss of the dif region in the recB mutant is decreased by the inactivation of cell division. We propose that it results from septum-induced chromosome breakage, and largely contributes to the low viability of the recB mutant. PMID:28968392

GC-MS Analysis: In Vivo Hepatoprotective and Antioxidant Activities of the Essential Oil of Achillea biebersteinii Afan. Growing in Saudi Arabia.

PubMed

Al-Said, Mansour S; Mothana, Ramzi A; Al-Yahya, Mohammed M; Rafatullah, Syed; Al-Sohaibani, Mohammed O; Khaled, Jamal M; Alatar, Abdulrahman; Alharbi, Naiyf S; Kurkcuoglu, Mine; Baser, Husnu C

2016-01-01

Liver disease is a worldwide problem. It represents one of the main causes of morbidity and mortality in humans. Achillea biebersteinii is used as herbal remedy for various ailments including liver diseases. But the scientific basis for its medicinal use remains unknown. Thus, this research was undertaken to evaluate the efficiency of A. biebersteinii essential oil (ABEO) (0.2 mL/kg) in the amelioration of CCl4-induced hepatotoxicity in rodent model. Moreover, the chemical content of the oil was investigated using GC and GC-MS. The following biochemical parameters were evaluated: serum glutamic oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), gamma-glutamyl-transpeptidase (γ-GGT), alkaline phosphatase (ALP), and total bilirubin. Furthermore, lipid profile, malondialdehyde (MDA), nonprotein sulfhydryl (NP-SH), and total protein (TP) contents in liver tissue were estimated. 44 components (92.0%) of the total oil have been identified by GC-MS analysis where α-terpinene and p-cymene were the most abundant. The high serum enzymatic (GOT, GPT, GGT, and ALP) and bilirubin concentrations as well as the level of MDA, NP-SH, and TP contents in liver tissues were significantly reinstated towards normalization by the ABEO. Histopathological study further confirmed these findings. In addition, ABEO showed mild antioxidant activity in 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and β-carotene-linoleic acid assays.

The glycosylation and characterization of the candidate Gc macrophage activating factor.

PubMed

Ravnsborg, Tina; Olsen, Dorthe T; Thysen, Anna Hammerich; Christiansen, Maja; Houen, Gunnar; Højrup, Peter

2010-04-01

The vitamin D binding protein, Gc globulin, has in recent years received some attention for its role as precursor for the extremely potent macrophage activating factor (GcMAF). An O-linked trisaccharide has been allocated to the threonine residue at position 420 in two of the three most common isoforms of Gc globulin (Gc1s and Gc1f). A substitution for a lysine residue at position 420 in Gc2 prevents this isoform from being glycosylated at that position. It has been suggested that Gc globulin subjected sequentially to sialidase and galactosidase treatment generates GcMAF in the form of Gc globulin with only a single GalNAc attached to T420. In this study we confirm the location of a linear trisaccharide on T420. Furthermore, we provide the first structural evidence of the generation of the proposed GcMAF by use of glycosidase treatment and mass spectrometry. Additionally the generated GcMAF candidate was tested for its effect on cytokine release from macrophages in human whole blood. Copyright 2010 Elsevier B.V. All rights reserved.

Content of gamma-oryzanol and composition of steryl ferulates in brown rice (Oryza sativa L.) of European origin.

PubMed

Miller, Andreas; Engel, Karl-Heinz

2006-10-18

The content of gamma-oryzanol and the composition of steryl ferulates were determined in brown rice of European origin using on-line coupled liquid chromatography-gas chromatography (LC-GC). Analysis of 30 brown rice samples of various cultivars, grown at different sites and in different seasons, revealed the gamma-oryzanol content to range from 26 to 63 mg/100 g. Cycloartenyl ferulate and 24-methylenecycloartanyl ferulate were the major components of gamma-oryzanol followed by campesteryl ferulate, campestanyl ferulate, and beta-sitosteryl ferulate. The proportions of individual steryl ferulates exhibited enormous variability. However, irrespectively of the great variations observed for single steryl ferulates, the proportions of the sum of 4,4'-dimethylsteryl ferulates (cycloartenyl ferulate, 24-methylenecycloartanyl ferulate) and the sum of 4-desmethylsteryl ferulates (campesteryl ferulate, campestanyl ferulate, and beta-sitosteryl ferulate) were rather constant. The significant natural variability observed for gamma-oryzanol content and composition of steryl ferulates were shown to be influenced by environmental conditions but not by the degree of maturity of rice grains.

Screening of Polish Fir Honeydew Honey Using GC/MS, HPLC-DAD, and Physical-Chemical Parameters: Benzene Derivatives and Terpenes as Chemical Markers.

PubMed

Kuś, Piotr M; Jerković, Igor; Marijanović, Zvonimir; Tuberoso, Carlo I G

2017-09-01

GC/MS of headspace solid phase micro extraction (HS-SPME) and solvent extractives along with targeted HPLC-DAD of Polish fir (Abies alba Mill.) honeydew honey (FHH), were used to determine the chemical profiles and potential markers of botanical origin. Additionally, typical physical-chemical parameters were also assigned. The values determined for FHH were: conductivity (1.2 mS/cm), water content (16.7 g/100 g), pH (4.5), and CIE chromaticity coordinates (L* = 48.4, a* = 20.6, b* = 69.7, C* = 72.9, and h° = 73.5). FHH contained moderate-high total phenolic content (533.2 mg GAE/kg) and antioxidant activity (1.1 mmol TEAC/kg) and (3.2 mmol Fe 2+ /kg) in DPPH and FRAP assays. The chemical profiles were dominated by source plant-originated benzene derivatives: 3,4-dihydroxybenzoic acid (up to 8.7 mg/kg, HPLC/honey solution), methyl syringate (up to 14.5%, GC/solvent extracts) or benzaldehyde (up to 43.7%, GC/headspace). Other markers were terpenes including norisoprenoid (4-hydroxy-3,5,6-trimethyl-4-(3-oxobut-1-enyl)cyclohex-2-en-1-one, up to 20.3%, GC/solvent extracts) and monoterpenes, mainly linalool derivatives (up to 49%, GC/headspace) as well as borneol (up to 5.9%, GC/headspace). The application of various techniques allowed comprehensive characterisation of FHH. 4-Hydroxy-3,5,6-trimethyl-4-(3-oxobut-1-enyl)cyclohex-2-en-1-one, coniferyl alcohol, borneol, and benzaldehyde were first time proposed for FHH screening. Protocatechuic acid may be a potential marker of FFH regardless of the geographical origin. © 2017 Wiley-VHCA AG, Zurich, Switzerland.

Kinetic studies of amino acid-based surfactant binding to DNA.

PubMed

Santhiya, Deenan; Dias, Rita S; Dutta, Sounak; Das, Prasanta Kumar; Miguel, Maria G; Lindman, Björn; Maiti, Souvik

2012-05-24

In this work, the binding kinetics of amino acid-based surfactants, presenting different linkers and head groups, with calf thymus (CT)-DNA was studied using stopped-flow fluorescence spectroscopy. The kinetic studies were carried out as a function of Na(+) concentration and surfactant-to-DNA charge ratio. The surfactant binding on DNA took place in two consecutive steps, for which the corresponding first and second relative rate constants (k(1) and k(2)) were determined. The fast step was attributed to the surfactant binding to DNA and micelle formation in its vicinity, the slower step to DNA condensation and possible rearrangement of the surfactant aggregates. In general, both relative rate constants increase with surfactant concentration and decrease with the ionic strength of the medium. The architecture of the surfactant was found to have a significant impact on the kinetics of the DNA-surfactant complexation. Surfactants with amide linkers showed larger relative rate constants than those with ester linkers. The variation of the relative rate constants with the head groups of the surfactants, alanine and proline, was found to be less obvious, being partially dependent on the surfactant concentration.

Crystal structure of a suicidal DNA repair protein: the Ada O6-methylguanine-DNA methyltransferase from E. coli.

PubMed

Moore, M H; Gulbis, J M; Dodson, E J; Demple, B; Moody, P C

1994-04-01

The mutagenic and carcinogenic effects of simple alkylating agents are mainly due to methylation at the O6 position of guanine in DNA. O6-methylguanine directs the incorporation of either thymine or cytosine without blocking DNA replication, resulting in GC to AT transition mutations. In prokaryotic and eukaryotic cells antimutagenic repair is effected by direct reversal of this DNA damage. A suicidal methyltransferase repair protein removes the methyl group from DNA to one of its own cysteine residues. The resulting self-methylation of the active site cysteine renders the protein inactive. Here we report the X-ray structure of the 19 kDa C-terminal domain of the Escherichia coli ada gene product, the prototype of these suicidal methyltransferases. In the crystal structure the active site cysteine is buried. We propose a model for the significant conformational change that the protein must undergo in order to bind DNA and effect methyl transfer.

Determination of D-lactide content in lactide stereoisomeric mixture using gas chromatography-polarimetry.

PubMed

Feng, Lidong; Bian, Xinchao; Chen, Zhiming; Xiang, Sheng; Liu, Yanlong; Sun, Bin; Li, Gao; Chen, Xuesi

2017-03-01

An analytical method has been proposed to quantify the D-lactide content in a lactide stereoisomeric mixture using combined gas chromatography and polarimetry (GC- polarimetry). As for a lactide stereoisomeric mixture, meso-lactide can be determined quantitatively using GC, but D- and L-lactides cannot be separated by the given GC system. The composition of a lactide stereoisomeric mixture is directly relative to its specific optical rotation. The specific optical rotations of neat L-lactide were obtained in different solutions, which were -266.3° and -298.8° in dichloromethane (DCM) and toluene solutions at 20°C, respectively. Therefore, for a lactide sample, the D-lactide content could be calculated based on the meso-lactide content obtained from GC and the specific optical rotations of the sample and neat L-lactide obtained from polarimetry. The effects of impurities and temperature on the test results were investigated, respectively. When the total content of impurities was not more than 1.0%, the absolute error for determining D-lactide content was less than 0.10% in DCM and toluene solutions. When the D-lactide content was calculated according to the specific optical rotation of neat L-lactide at 20°C, the absolute error caused by the variation in temperature of 20±15°C was not more than 0.2 and 0.7% in DCM and toluene solutions, respectively, and thus usually could be ignored in a DCM solution. When toluene was used as a solvent for the determination of D-lactide content, a temperature correction for specific optical rotations could be introduced and would ensure the accuracy of results. This method is applicable to the determination of D-lactide content in lactide stereoisomeric mixtures. The standard deviation (STDEV) of the measurements is less than 0.5%, indicating that the precision is suitable for this method. Copyright © 2016 Elsevier B.V. All rights reserved.

Variation in Pockels constants of silicate glass-ceramics prepared by perfect surface crystallization

NASA Astrophysics Data System (ADS)

Takano, Kazuya; Takahashi, Yoshihiro; Miyazaki, Takamichi; Terakado, Nobuaki; Fujiwara, Takumi

2018-01-01

We investigated the Pockels effect in polycrystalline materials consisting of highly oriented polar fresnoite-type Sr2TiSi2O8 fabricated using perfectly surface-crystallized glass-ceramics (PSC-GCs). The chemical composition of the precursor glass was shown to significantly affect the crystallized texture, e.g., the crystal orientation and appearance of amorphous nanoparasites in the domains, resulting in variations in the Pockels constants. Single crystals exhibiting spontaneous polarization possessed large structural anisotropy, leading to a strong dependence of the nonlinear-optical properties on the direction of polarized light. This study suggests that variations in the Pockels constants (r13 and r33) and tuning of the r13/r33 ratio can be realized in PSC-GC materials.

Using BPCA and pyrolysis-GC/MS patterns as a measure of charring intensity

NASA Astrophysics Data System (ADS)

Kaal, Joeri; Schneider, Maximilian P. W.; Schmidt, Michael W. I.

2010-05-01

Many questions remain on the molecular properties of Black C (organic fire residues such as charcoal and soot). Here we compare parameters from two methods that have recently shown to be related to the degree of thermal modification ("charring intensity") of charcoal-Black C: i) the proportion of mellitic acid (B6CA) among benzenepolycarboxylic acids in the BPCA method [1,2,3] and ii) the relative proportions and degree of alkylation of pyrolysis products from Black C in pyrolysis-GC/MS [4]. For that purpose we used laboratory chars from rice straw (grass) and chestnut wood (wood) produced at 200-1000 °C under N2 flow. The chars obtained at 450 °C are reference materials of the Black Carbon Ring Trial [5]. Positive correlations between the charring temperature and BPCA and pyrolysis patterns confirm that these methods can be used to study the degree of thermal impact of charred remains. Pyrolysis-GC/MS allowed us to track the thermal degradation of the major biocomponents lignin, polysaccharides, tannin, aliphatic chain lipids, triterpenoids, chlorophyll and proteins, mostly between 250 and 450 °C. The proportions of the pyrolysis products of Black C (benzene, toluene, benzonitrile, PAHs, etc.) and also the ratios that reflect the abundance of aliphatic cross-linkages between aromatic moieties (benzene/toluene, naphthalene/alkylnaphthalenes, benzofuran/alkylbenzofurans), increase with charring intensity. Nonetheless, chars obtained at T > 600 °C (especially for wood) gave low quality pyrograms and poor reproducibility because of high thermal stability. The relative contributions of B6CA, one of the molecular markers used for the BPCA method, are indicative for the degree of condensation of the chars. The BPCA approach showed a clear increase in the relative contribution of B6CA from ca. 5 % at 200 °C to ca. 95 % at 1000 °C, confirming the ability of this parameter to assess charring intensity. The relative contribution of B6CA remains almost constant at ca

A facile, branched DNA assay to quantitatively measure glucocorticoid receptor auto-regulation in T-cell acute lymphoblastic leukemia

PubMed Central

Schwartz, Jason R.; Sarvaiya, Purvaba J.; Leiva, Lily E.; Velez, Maria C.; Singleton, Tammuella C.; Yu, Lolie C.; Vedeckis, Wayne V.

2012-01-01

Glucocorticoid (GC) steroid hormones are used to treat acute lymphoblastic leukemia (ALL) because of their pro-apoptotic effects in hematopoietic cells. However, not all leukemia cells are sensitive to GC, and no assay to stratify patients is available. In the GC-sensitive T-cell ALL cell line CEM-C7, auto-up-regulation of RNA transcripts for the glucocorticoid receptor (GR) correlates with increased apoptotic response. This study aimed to determine if a facile assay of GR transcript levels might be promising for stratifying ALL patients into hormone-sensitive and hormone-resistant populations. The GR transcript profiles of various lymphoid cell lines and 4 bone marrow samples from patients with T-cell ALL were analyzed using both an optimized branched DNA (bDNA) assay and a real-time quantitative reverse transcription-polymerase chain reaction assay. There were significant correlations between both assay platforms when measuring total GR (exon 5/6) transcripts in various cell lines and patient samples, but not for a probe set that detects a specific, low abundance GR transcript (exon 1A3). Our results suggest that the bDNA platform is reproducible and precise when measuring total GR transcripts and, with further development, may ultimately offer a simple clinical assay to aid in the prediction of GC-sensitivity in ALL patients. PMID:22739263

[Study on the DNA vaccine against foot-and-mouth disease virus using the heavy chain constant region of swine IgG as the carrier for peptide epitopes].

PubMed

Li, G J; Yan, W Y; Xu, Q X; Sheng, Z T; Zheng, Z X

2001-05-01

The peptide of amino acids 141-160 of VP1 protein of foot-and-mouth disease virus (FMDV) is a major B cell epitope and the peptide of amino acids 21-40 is an important T cell epitope. In this study, the DNA fragments of 141-160 and 21-40 peptide epitopes of a strain of type O FMDV was chemically synthesized and arranged into a tandem repeat 141-160 (20AA)-21-40 (20AA)-141-160 (20AA). This tandem sequence was fused to the 3' end of the heavy chain constant region gene of swine immunoglobulin G and was then cloned into mammalian expression vector pCDM8 to form a recombinant plasmid pCDM8FZ3. After pCDM8FZ3 was inoculated intramuscularly into guinea pigs, it elicited a neutralizing antibody response and a specific spleen T cell proliferative response, and 66% of the vaccinated animals were protected from viral challenge. Our study indicated that the heavy chain constant region of swine IgG can act as the carrier protein for FMDV peptide epitopes, and pC-DM8FZ3 is a potential DNA vaccine candidate to prevent FMDV infection.

Altered mitochondrial genome content signals worse pathology and prognosis in prostate cancer.

PubMed

Kalsbeek, Anton M F; Chan, Eva K F; Grogan, Judith; Petersen, Desiree C; Jaratlerdsiri, Weerachai; Gupta, Ruta; Lyons, Ruth J; Haynes, Anne-Maree; Horvath, Lisa G; Kench, James G; Stricker, Phillip D; Hayes, Vanessa M

2018-01-01

Mitochondrial genome (mtDNA) content is depleted in many cancers. In prostate cancer, there is intra-glandular as well as inter-patient mtDNA copy number variation. In this study, we determine if mtDNA content can be used as a predictor for prostate cancer staging and outcomes. Fresh prostate cancer biopsies from 115 patients were obtained at time of surgery. All cores underwent pathological review, followed by isolation of cancer and normal tissue. DNA was extracted and qPCR performed to quantify the total amount of mtDNA as a ratio to genomic DNA. Differences in mtDNA content were compared for prostate cancer pathology features and disease outcomes. We showed a significantly reduced mtDNA content in prostate cancer compared with normal adjacent prostate tissue (mean difference 1.73-fold, P-value <0.001). Prostate cancer with increased mtDNA content showed unfavorable pathologic characteristics including, higher disease stage (PT2 vs PT3 P-value = 0.018), extracapsular extension (P-value = 0.02) and a trend toward an increased Gleason score (P-value = 0.064). No significant association was observed between changes in mtDNA content and biochemical recurrence (median follow up of 107 months). Contrary to other cancer types, prostate cancer tissue shows no universally depleted mtDNA content. Rather, the change in mtDNA content is highly variable, mirroring known prostate cancer genome heterogeneity. Patients with high mtDNA content have an unfavorable pathology, while a high mtDNA content in normal adjacent prostate tissue is associated with worse prognosis. © 2017 Wiley Periodicals, Inc.

Patterns of DNA barcode variation in Canadian marine molluscs.

PubMed

Layton, Kara K S; Martel, André L; Hebert, Paul D N

2014-01-01

Molluscs are the most diverse marine phylum and this high diversity has resulted in considerable taxonomic problems. Because the number of species in Canadian oceans remains uncertain, there is a need to incorporate molecular methods into species identifications. A 648 base pair segment of the cytochrome c oxidase subunit I gene has proven useful for the identification and discovery of species in many animal lineages. While the utility of DNA barcoding in molluscs has been demonstrated in other studies, this is the first effort to construct a DNA barcode registry for marine molluscs across such a large geographic area. This study examines patterns of DNA barcode variation in 227 species of Canadian marine molluscs. Intraspecific sequence divergences ranged from 0-26.4% and a barcode gap existed for most taxa. Eleven cases of relatively deep (>2%) intraspecific divergence were detected, suggesting the possible presence of overlooked species. Structural variation was detected in COI with indels found in 37 species, mostly bivalves. Some indels were present in divergent lineages, primarily in the region of the first external loop, suggesting certain areas are hotspots for change. Lastly, mean GC content varied substantially among orders (24.5%-46.5%), and showed a significant positive correlation with nearest neighbour distances. DNA barcoding is an effective tool for the identification of Canadian marine molluscs and for revealing possible cases of overlooked species. Some species with deep intraspecific divergence showed a biogeographic partition between lineages on the Atlantic, Arctic and Pacific coasts, suggesting the role of Pleistocene glaciations in the subdivision of their populations. Indels were prevalent in the barcode region of the COI gene in bivalves and gastropods. This study highlights the efficacy of DNA barcoding for providing insights into sequence variation across a broad taxonomic group on a large geographic scale.

Determination of Henry’s Law Constants Using Internal Standards with Benchmark Values

EPA Science Inventory

It is shown that Henry’s law constants can be experimentally determined by comparing headspace content of compounds with known constants to interpolate the constants of other compounds. Studies were conducted over a range of water temperatures to identify temperature dependence....

Fundamental Insight on Developing Low Dielectric Constant Polyimides

NASA Technical Reports Server (NTRS)

Simpson, J. O.; SaintClair, A. K.

1997-01-01

Thermally stable, durable, insulative polyimides are in great demand for the fabrication of microelectronic devices. In this investigation dielectric and optical properties have been studied for several series of aromatic polyimides. The effect of polarizability, fluorine content, and free volume on dielectric constant was examined. In general, minimizing polarizability, maximizing free volume and fluorination all lowered dielectric constants in the polyimides studied.

Comparison of large-insert, small-insert and pyrosequencing libraries for metagenomic analysis.

PubMed

Danhorn, Thomas; Young, Curtis R; DeLong, Edward F

2012-11-01

The development of DNA sequencing methods for characterizing microbial communities has evolved rapidly over the past decades. To evaluate more traditional, as well as newer methodologies for DNA library preparation and sequencing, we compared fosmid, short-insert shotgun and 454 pyrosequencing libraries prepared from the same metagenomic DNA samples. GC content was elevated in all fosmid libraries, compared with shotgun and 454 libraries. Taxonomic composition of the different libraries suggested that this was caused by a relative underrepresentation of dominant taxonomic groups with low GC content, notably Prochlorales and the SAR11 cluster, in fosmid libraries. While these abundant taxa had a large impact on library representation, we also observed a positive correlation between taxon GC content and fosmid library representation in other low-GC taxa, suggesting a general trend. Analysis of gene category representation in different libraries indicated that the functional composition of a library was largely a reflection of its taxonomic composition, and no additional systematic biases against particular functional categories were detected at the level of sequencing depth in our samples. Another important but less predictable factor influencing the apparent taxonomic and functional library composition was the read length afforded by the different sequencing technologies. Our comparisons and analyses provide a detailed perspective on the influence of library type on the recovery of microbial taxa in metagenomic libraries and underscore the different uses and utilities of more traditional, as well as contemporary 'next-generation' DNA library construction and sequencing technologies for exploring the genomics of the natural microbial world.

Photoinduced Electron Transfer between Psoralens and DNA: Influence of DNA Sequence and Substitution.

PubMed

Fröbel, Sascha; Levi, Lucilla; Ulamec, Sabine M; Gilch, Peter

2016-05-04

Psoralens are heterocyclic compounds which are, among other uses, used to treat skin deseases in the framework of PUVA therapy. In the dark, they intercalate into DNA and can form photoadducts with thymines upon UV-A excitation, which harms the affected cells. We have recently discovered that after excitation of intercalated psoralens, an efficient photoinduced electron transfer (PET) from DNA occurs. Here, the PET is studied in detail by means of femtosecond transient absorption spectroscopy. Using DNA samples that contain either only GC or AT base pairs, we show that only guanine donates the electrons. Additionally, the substituent effects on PET are studied relying on three different psoralen derivatives. The substitution alters spectroscopic and electrochemical properties of the psoralens, which are determined by cyclic voltammetry and steady state spectroscopy. These experiments allow us to estimate the PET energetics, which are in line with the measured kinetics. Implications for the applications of psoralens are discussed. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Benchmarking of the Oxford Nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations.

PubMed

Oikonomopoulos, Spyros; Wang, Yu Chang; Djambazian, Haig; Badescu, Dunarel; Ragoussis, Jiannis

2016-08-24

To assess the performance of the Oxford Nanopore Technologies MinION sequencing platform, cDNAs from the External RNA Controls Consortium (ERCC) RNA Spike-In mix were sequenced. This mix mimics mammalian mRNA species and consists of 92 polyadenylated transcripts with known concentration. cDNA libraries were generated using a template switching protocol to facilitate the direct comparison between different sequencing platforms. The MinION performance was assessed for its ability to sequence the cDNAs directly with good accuracy in terms of abundance and full length. The abundance of the ERCC cDNA molecules sequenced by MinION agreed with their expected concentration. No length or GC content bias was observed. The majority of cDNAs were sequenced as full length. Additionally, a complex cDNA population derived from a human HEK-293 cell line was sequenced on an Illumina HiSeq 2500, PacBio RS II and ONT MinION platforms. We observed that there was a good agreement in the measured cDNA abundance between PacBio RS II and ONT MinION (rpearson = 0.82, isoforms with length more than 700bp) and between Illumina HiSeq 2500 and ONT MinION (rpearson = 0.75). This indicates that the ONT MinION can sequence quantitatively both long and short full length cDNA molecules.

The formation of diploid and triploid hybrids of female grass carp × male blunt snout bream and their 5S rDNA analysis.

PubMed

He, Weiguo; Xie, Lihua; Li, Tangluo; Liu, Shaojun; Xiao, Jun; Hu, Jie; Wang, Jing; Qin, Qinbo; Liu, Yun

2013-11-23

Hybridization is a useful strategy to alter the genotypes and phenotypes of the offspring. It could transfer the genome of one species to another through combing the different genome of parents in the hybrid offspring. And the offspring may exhibit advantages in growth rate, disease resistance, survival rate and appearance, which resulting from the combination of the beneficial traits from both parents. Diploid and triploid hybrids of female grass carp (Ctenopharyngodon idellus, GC, Cyprininae, 2n = 48) × male blunt snout bream (Megalobrama amblycephala, BSB, Cultrinae, 2n = 48) were successfully obtained by distant hybridization. Diploid hybrids had 48 chromosomes, with one set from GC and one set from BSB. Triploid hybrids possessed 72 chromosomes, with two sets from GC and one set from BSB.The morphological traits, growth rates, and feeding ecology of the parents and hybrid offspring were compared and analyzed. The two kinds of hybrid offspring exhibited significantly phenotypic divergence from GC and BSB. 2nGB hybrids showed similar growth rate compared to that of GC, and 3nGB hybrids significantly higher results. Furthermore, the feeding ecology of hybrid progeny was omnivorous.The 5S rDNA of GC, BSB and their hybrid offspring were also cloned and sequenced. There was only one type of 5S rDNA (designated type I: 180 bp) in GC and one type of 5S rDNA (designated type II: 188 bp) in BSB. However, in the hybrid progeny, diploid and triploid hybrids both inherited type I and type II from their parents, respectively. In addition, a chimera of type I and type II was observed in the genome of diploid and triploid hybrids, excepting a 10 bp of polyA insertion in type II sequence of the chimera of the diploid hybrids. This is the first report of diploid and triploid hybrids being produced by crossing GC and BSB, which have the same chromosome number. The obtainment of two new hybrid offspring has significance in fish genetic breeding. The results illustrate the effect

A novel automatic quantification method for high-content screening analysis of DNA double strand-break response.

PubMed

Feng, Jingwen; Lin, Jie; Zhang, Pengquan; Yang, Songnan; Sa, Yu; Feng, Yuanming

2017-08-29

High-content screening is commonly used in studies of the DNA damage response. The double-strand break (DSB) is one of the most harmful types of DNA damage lesions. The conventional method used to quantify DSBs is γH2AX foci counting, which requires manual adjustment and preset parameters and is usually regarded as imprecise, time-consuming, poorly reproducible, and inaccurate. Therefore, a robust automatic alternative method is highly desired. In this manuscript, we present a new method for quantifying DSBs which involves automatic image cropping, automatic foci-segmentation and fluorescent intensity measurement. Furthermore, an additional function was added for standardizing the measurement of DSB response inhibition based on co-localization analysis. We tested the method with a well-known inhibitor of DSB response. The new method requires only one preset parameter, which effectively minimizes operator-dependent variations. Compared with conventional methods, the new method detected a higher percentage difference of foci formation between different cells, which can improve measurement accuracy. The effects of the inhibitor on DSB response were successfully quantified with the new method (p = 0.000). The advantages of this method in terms of reliability, automation and simplicity show its potential in quantitative fluorescence imaging studies and high-content screening for compounds and factors involved in DSB response.

Comparison of DNA-Reactive Metabolites from Nitrosamine and Styrene Using Voltammetric DNA/Microsomes Sensors

PubMed Central

Krishnan, Sadagopan; Bajrami, Besnik; Mani, Vigneshwaran; Pan, Shenmin; Rusling, James F.

2012-01-01

Voltammetric sensors made with films of polyions, double-stranded DNA and liver microsomes adsorbed layer-by-layer onto pyrolytic graphite electrodes were evaluated for reactive metabolite screening. This approach features simple, inexpensive screening without enzyme purification for applications in drug or environmental chemical development. Cytochrome P450 enzymes (CYPs) in the liver microsomes were activated by an NADPH regenerating system or by electrolysis to metabolize model carcinogenic compounds nitrosamine and styrene. Reactive metabolites formed in the films were trapped as adducts with nucleobases on DNA. The DNA damage was detected by square-wave voltammetry (SWV) using Ru(bpy)32+ as a DNA-oxidation catalyst. These sensors showed a larger rate of increase in signal vs. reaction time for a highly toxic nitrosamine than for the moderately toxic styrene due to more rapid reactive metabolite-DNA adduct formation. Results were consistent with reported in vivo TD50 data for the formation of liver tumors in rats. Analogous polyion/ liver microsome films prepared on 500 nm silica nanoparticles (nanoreactors) and reacted with nitrosamine or styrene, provided LC-MS or GC analyses of metabolite formation rates that correlated well with sensor response. PMID:23100998

ADAP-GC 3.0: Improved Peak Detection and Deconvolution of Co-eluting Metabolites from GC/TOF-MS Data for Metabolomics Studies

PubMed Central

Ni, Yan; Su, Mingming; Qiu, Yunping; Jia, Wei

2017-01-01

ADAP-GC is an automated computational pipeline for untargeted, GC-MS-based metabolomics studies. It takes raw mass spectrometry data as input and carries out a sequence of data processing steps including construction of extracted ion chromatograms, detection of chromatographic peak features, deconvolution of co-eluting compounds, and alignment of compounds across samples. Despite the increased accuracy from the original version to version 2.0 in terms of extracting metabolite information for identification and quantitation, ADAP-GC 2.0 requires appropriate specification of a number of parameters and has difficulty in extracting information of compounds that are in low concentration. To overcome these two limitations, ADAP-GC 3.0 was developed to improve both the robustness and sensitivity of compound detection. In this paper, we report how these goals were achieved and compare ADAP-GC 3.0 against three other software tools including ChromaTOF, AnalyzerPro, and AMDIS that are widely used in the metabolomics community. PMID:27461032

ADAP-GC 3.0: Improved Peak Detection and Deconvolution of Co-eluting Metabolites from GC/TOF-MS Data for Metabolomics Studies.

PubMed

Ni, Yan; Su, Mingming; Qiu, Yunping; Jia, Wei; Du, Xiuxia

2016-09-06

ADAP-GC is an automated computational pipeline for untargeted, GC/MS-based metabolomics studies. It takes raw mass spectrometry data as input and carries out a sequence of data processing steps including construction of extracted ion chromatograms, detection of chromatographic peak features, deconvolution of coeluting compounds, and alignment of compounds across samples. Despite the increased accuracy from the original version to version 2.0 in terms of extracting metabolite information for identification and quantitation, ADAP-GC 2.0 requires appropriate specification of a number of parameters and has difficulty in extracting information on compounds that are in low concentration. To overcome these two limitations, ADAP-GC 3.0 was developed to improve both the robustness and sensitivity of compound detection. In this paper, we report how these goals were achieved and compare ADAP-GC 3.0 against three other software tools including ChromaTOF, AnalyzerPro, and AMDIS that are widely used in the metabolomics community.

DNAzymes in DNA Nanomachines and DNA Analysis

NASA Astrophysics Data System (ADS)

He, Yu; Tian, Ye; Chen, Yi; Mao, Chengde

This chapter discusses our efforts in using DNAzymes in DNA nano-machines and DNA analysis systems. 10-23 DNAzymes can cleave specific phos-phodiester bonds in RNA. We use them to construct an autonomous DNA-RNA chimera nanomotor, which constantly extracts chemical energy from RNA substrates and transduces the energy into a mechanical motion: cycles of contraction and extension. The motor's motion can be reversibly turned on and off by a DNA analogue (brake) of the RNA substrate. Addition and removal of the brake stops and restarts, respectively, the motor's motion. Furthermore, when the RNA substrates are preorganized into a one-dimensional track, a DNAzyme can continuously move along the track so long as there are substrates available ahead. Based on a similar mechanism, a novel DNA detection system has been developed. A target DNA activates a DNAzyme to cleave RNA-containing molecular beacons (MB), which generates an enhanced fluorescence signal. A following work integrates two steps of signal amplifications: a rolling-circle amplification (RCA) to synthesize multiple copies of DNAzymes, and the DNAzymes catalyze a chemical reaction to generate a colorimetric signal. This method allows detection of DNA analytes whose concentration is as low as 1 pM.

Gc protein-derived macrophage-activating factor (GcMAF) stimulates cAMP formation in human mononuclear cells and inhibits angiogenesis in chick embryo chorionallantoic membrane assay.

PubMed

Pacini, Stefania; Morucci, Gabriele; Punzi, Tiziana; Gulisano, Massimo; Ruggiero, Marco

2011-04-01

The effects of Gc protein-derived macrophage-activating factor (GcMAF) have been studied in cancer and other conditions where angiogenesis is deregulated. In this study, we demonstrate for the first time that the mitogenic response of human peripheral blood mononuclear cells (PBMCs) to GcMAF was associated with 3'-5'-cyclic adenosine monophosphate (cAMP) formation. The effect was dose dependent, and maximal stimulation was achieved using 0.1 ng/ml. Heparin inhibited the stimulatory effect of GcMAF on PBMCs. In addition, we demonstrate that GcMAF (1 ng/ml) inhibited prostaglandin E(1)- and human breast cancer cell-stimulated angiogenesis in chick embryo chorionallantoic membrane (CAM) assay. Finally, we tested different GcMAF preparations on CAM, and the assay proved to be a reliable, reproducible and inexpensive method to determine the relative potencies of different preparations and their stability; we observed that storage at room temperature for 15 days decreased GcMAF potency by about 50%. These data could prove useful for upcoming clinical trials on GcMAF.

Evaluation of simultaneous binding of Chromomycin A3 to the multiple sites of DNA by the new restriction enzyme assay.

PubMed

Murase, Hirotaka; Noguchi, Tomoharu; Sasaki, Shigeki

2018-06-01

Chromomycin A3 (CMA3) is an aureolic acid-type antitumor antibiotic. CMA3 forms dimeric complexes with divalent cations, such as Mg 2+ , which strongly binds to the GC rich sequence of DNA to inhibit DNA replication and transcription. In this study, the binding property of CMA3 to the DNA sequence containing multiple GC-rich binding sites was investigated by measuring the protection from hydrolysis by the restriction enzymes, AccII and Fnu4HI, for the center of the CGCG site and the 5'-GC↓GGC site, respectively. In contrast to the standard DNase I footprinting method, the DNA substrates are fully hydrolyzed by the restriction enzymes, therefore, the full protection of DNA at all the cleavable sites indicates that CMA3 simultaneously binds to all the binding sites. The restriction enzyme assay has suggested that CMA3 has a high tendency to bind the successive CGCG sites and the CGG repeat. Copyright © 2018 Elsevier Ltd. All rights reserved.

Detection biomarkers of lung cancer using mini-GC-PID system integrated with micro GC column and micro pre-concentrator

PubMed Central

2014-01-01

The survival rate of lung cancer can be significantly improved by monitoring biomarkers in exhaled air that indicate diseases in early stage, so it is very important to develop micro analytical systems which can offer a fast, on-site, real-time detecting biomarkers in exhaled air. In this paper, a mini-gas chromatography (GC)-photo-ionization detector (PID) system integrated with a micro GC column and a micro pre-concentrator was developed for forming an inexpensive, fast, and non-invasive diagnostic tool for lung cancer. This system has very strong concentrate ability owing to its integrated micro pre-concentrator, which make the detection of trace components in exhaled air very easy. In addition, the integrated micro GC column can separate complex mixtures, which overcome low resolution and poor anti-interference ability of other instruments. The results indicated that the mini-GC-PID system can effectively separate and detect the biomarkers at parts-per-billion (ppb) level. PMID:25339856

Efficient quantification of water content in edible oils by headspace gas chromatography with vapour phase calibration.

PubMed

Xie, Wei-Qi; Gong, Yi-Xian; Yu, Kong-Xian

2018-06-01

An automated and accurate headspace gas chromatographic (HS-GC) technique was investigated for rapidly quantifying water content in edible oils. In this method, multiple headspace extraction (MHE) procedures were used to analyse the integrated water content from the edible oil sample. A simple vapour phase calibration technique with an external vapour standard was used to calibrate both the water content in the gas phase and the total weight of water in edible oil sample. After that the water in edible oils can be quantified. The data showed that the relative standard deviation of the present HS-GC method in the precision test was less than 1.13%, the relative differences between the new method and a reference method (i.e. the oven-drying method) were no more than 1.62%. The present HS-GC method is automated, accurate, efficient, and can be a reliable tool for quantifying water content in edible oil related products and research. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Characterization of volatile profile from ten different varieties of Chinese jujubes by HS-SPME/GC-MS coupled with E-nose.

PubMed

Chen, Qinqin; Song, Jianxin; Bi, Jinfeng; Meng, Xianjun; Wu, Xinye

2018-03-01

Volatile profile of ten different varieties of fresh jujubes was characterized by HS-SPME/GC-MS (headspace solid phase micro-extraction combined with gas chromatography-mass spectrometry) and E-nose (electronic nose). GC-MS results showed that a total of 51 aroma compounds were identified in jujubes, hexanoic acid, hexanal, (E)-2-hexenal, (Z)-2-heptenal, benzaldehyde and (E)-2-nonenal were the main aroma components with contributions that over 70%. Differentiation of jujube varieties was conducted by cluster analysis of GC-MS data and principal component analysis & linear discriminant analysis of E-nose data. Both results showed that jujubes could be mainly divided into two groups: group A (JZ, PDDZ, JSXZ and LWZZ) and group B (BZ, YZ, MZ, XZ and DZ). There were significant differences in contents of alcohols, acids and aromatic compounds between group A and B. GC-MS coupled with E-nose could be a fast and accurate method to identify the general flavor difference in different varieties of jujubes. Copyright © 2017 Elsevier Ltd. All rights reserved.

DNA nanostructure-based drug delivery nanosystems in cancer therapy.

PubMed

Wu, Dandan; Wang, Lei; Li, Wei; Xu, Xiaowen; Jiang, Wei

2017-11-25

DNA as a novel biomaterial can be used to fabricate different kinds of DNA nanostructures based on its principle of GC/AT complementary base pairing. Studies have shown that DNA nanostructure is a nice drug carrier to overcome big obstacles existing in cancer therapy such as systemic toxicity and unsatisfied drug efficacy. Thus, different types of DNA nanostructure-based drug delivery nanosystems have been designed in cancer therapy. To improve treating efficacy, they are also developed into more functional drug delivery nanosystems. In recent years, some important progresses have been made. The objective of this review is to make a retrospect and summary about these different kinds of DNA nanostructure-based drug delivery nanosystems and their latest progresses: (1) active targeting; (2) mutidrug co-delivery; (3) construction of stimuli-responsive/intelligent nanosystems. Copyright © 2017 Elsevier B.V. All rights reserved.

[The study of complex-formation of DNA with the antimicrobial drug decamethoxine].

PubMed

Sorokin, V A; Blagoĭ, Iu P; Valeev, V A; Gladchenko, G O; Sukhodub, L F; Volianskiĭ, Iu L

1990-01-01

The interaction of effective antibacterial drug decametoxyn with natural DNA was studied by UV-spectroscopy. Decametoxyn shows a specificity to nucleotides: it decreases the cooperativity of melting and the thermal stability of DNA parts enriched by AT pairs. The characteristics of the helix-coil transition on the DNA parts enriched by GC-pairs are invariable. Interaction with AT-pairs results in their partial or complete melting at room temperature, followed by intermolecule aggregation. Interacting with phosphates decametoxyn manifests itself not as a dication but as two single-charged ions.

Investigation of Imposter Perfumes Using GC-MS

ERIC Educational Resources Information Center

Mowery, Kelly A.; Blanchard, Daniel E.; Smith, Stephanie; Betts, Thomas A.

2004-01-01

An experiment to initiate students into several features of gas chromatograph-mass spectrometers (GC-MS) through the study of branded and counterfeit perfumes is described. The experiment enables the students to appreciate the power of GC-MS in distinguishing between original and imitation perfumes, and in analyzing data with its application…

[Studies on GC fingerprint of volatile oil of Houttuynia cordata].

PubMed

Yang, Bin; Wang, Ruo-Jing; Wang, Qian-Peng; Lu, Dong-Mei; Li, Hua; Xiao, Yong-Qing; Wang, Yong-Yan

2006-12-01

To establish a GC fingerprint of the volatile oil of Houttuynia cordata. The volatile oil was extracted from H. cordata by water stream distillation method, and analyzed by GC coupled with FID. 12 bathes of samples collected from different regions were analyzed; the GC fingerprint of the volatile oil of H. cordata was subsequently established. The established GC fingerprint can be used for the identification of H. cordata.

Gastric lymphomas in Turkey. Analysis of prognostic factors with special emphasis on flow cytometric DNA content.

PubMed

Aydin, Z D; Barista, I; Canpinar, H; Sungur, A; Tekuzman, G

2000-07-01

In contrast to DNA ploidy, to the authors' knowledge the prognostic significance of S-phase fraction (SPF) in gastric lymphomas has not been determined. In the current study, the prognostic significance of various parameters including SPF and DNA aneuploidy were analyzed and some distinct epidemiologic and biologic features of gastric lymphomas in Turkey were found. A series of 78 gastric lymphoma patients followed at Hacettepe University is reported. DNA flow cytometry was performed for 34 patients. The influence of various parameters on survival was investigated with the log rank test. The Cox proportional hazards model was fitted to identify independent prognostic factors. The median age of the patients was 50 years. There was no correlation between patient age and tumor grade. DNA content analysis revealed 4 of the 34 cases to be aneuploid with DNA index values < 1.0. The mean SPF was 33.5%. In the univariate analysis, surgical resection of the tumor, modified Ann Arbor stage, performance status, response to first-line chemotherapy, lactate dehydrogenase (LDH) level, and SPF were important prognostic factors for disease free survival (DFS). The same parameters, excluding LDH level, were important for determining overall survival (OS). In the multivariate analysis, surgical resection of the tumor, disease stage, performance status, and age were found to be important prognostic factors for OS. To the authors' knowledge the current study is the first to demonstrate the prognostic significance of SPF in gastric lymphomas. The distinguishing features of Turkish gastric lymphoma patients are 1) DNA indices of aneuploid cases that all are < 1.0, which is a unique feature; 2) a lower percentage of aneuploid cases; 3) a higher SPF; 4) a younger age distribution; and 5) lack of an age-grade correlation. The authors conclude that gastric lymphomas in Turkey have distinct biologic and epidemiologic characteristics. Copyright 2000 American Cancer Society.

One-pot synthesis of K-doped g-C3N4 nanosheets with enhanced photocatalytic hydrogen production under visible-light irradiation

NASA Astrophysics Data System (ADS)

Wang, Yanyun; Zhao, Shuo; Zhang, Yiwei; Fang, Jiasheng; Zhou, Yuming; Yuan, Shenhao; Zhang, Chao; Chen, Wenxia

2018-05-01

Graphite carbon nitride (g-C3N4), as a promising low cost, visible light driven conjugated polymer semiconductor photocatalyst, has attracted wide attentions from researchers. However, low light absorption efficiency and inadequate charge separation limit the potential applications of g-C3N4. This paper exhibits K-doped g-C3N4 prepared by a facile thermal polymerization with KBr as the K source. The experiments of photocatalytic hydrogen evolution demonstrate that KBr content strongly affects the activity of the catalyst. XRD, FT-IR, XPS, SEM, TEM, UV-vis diffuse reflectance spectra, photoluminescence (PL) characterization methods are used to study the effects of potassium on the catalyst performance. The results find that K-modified g-C3N4 has a narrower band gap and enhanced light harvesting properties. Moreover, the photocatalytic hydrogen evolution rate (HER) of the optimized K-doped g-C3N4 nanosheets (10 wt % KBr) reaches 1337.2 μmol g-1h-1, which is about 5.6 times in comparison with that of pure g-C3N4 (239.8 μmol g-1h-1). The doping of the potassium may increase the π-conjugated systems and accelerate the electron transport rate, then improve the photocatalytic properties. Based on the results of the analysis, a possible mechanism is proposed.

Calcination Method Synthesis of SnO2/g-C3N4 Composites for a High-Performance Ethanol Gas Sensing Application

PubMed Central

Cao, Jianliang; Qin, Cong; Wang, Yan; Zhang, Bo; Gong, Yuxiao; Zhang, Huoli; Sun, Guang; Bala, Hari; Zhang, Zhanying

2017-01-01

The SnO2/g-C3N4 composites were synthesized via a facile calcination method by using SnCl4·5H2O and urea as the precursor. The structure and morphology of the as-synthesized composites were characterized by the techniques of X-ray diffraction (XRD), the field-emission scanning electron microscopy and transmission electron microscopy (SEM and TEM), energy dispersive spectrometry (EDS), thermal gravity and differential thermal analysis (TG-DTA), and N2-sorption. The analysis results indicated that the as-synthesized samples possess the two dimensional structure. Additionally, the SnO2 nanoparticles were highly dispersed on the surface of the g-C3N4nanosheets. The gas-sensing performance of the as-synthesized composites for different gases was tested. Moreover, the composite with 7 wt % g-C3N4 content (SnO2/g-C3N4-7) SnO2/g-C3N4-7 exhibits an admirable gas-sensing property to ethanol, which possesses a higher response and better selectivity than that of the pure SnO2-based sensor. The high surface area of the SnO2/g-C3N4 composite and the good electronic characteristics of the two dimensional graphitic carbon nitride are in favor of the elevated gas-sensing property. PMID:28468245

The quest for a unified view of bacterial land colonization

PubMed Central

Wu, Hao; Fang, Yongjun; Yu, Jun; Zhang, Zhang

2014-01-01

Exploring molecular mechanisms underlying bacterial water-to-land transition represents a critical start toward a better understanding of the functioning and stability of the terrestrial ecosystems. Here, we perform comprehensive analyses based on a large variety of bacteria by integrating taxonomic, phylogenetic and metagenomic data, in the quest for a unified view that elucidates genomic, evolutionary and ecological dynamics of the marine progenitors in adapting to nonaquatic environments. We hypothesize that bacterial land colonization is dominated by a single-gene sweep, that is, the emergence of dnaE2 derived from an early duplication event of the primordial dnaE, followed by a series of niche-specific genomic adaptations, including GC content increase, intensive horizontal gene transfer and constant genome expansion. In addition, early bacterial radiation may be stimulated by an explosion of land-borne hosts (for example, plants and animals) after initial land colonization events. PMID:24451209

Special antitumor immune effects of laser immunotherapy with SWNT-GC

NASA Astrophysics Data System (ADS)

Zhou, Feifan; Song, Sheng; Chen, Wei R.

2014-02-01

In our previous work, we constructed a multifunction nano system SWNT-GC, which can synergize photothermal and immunological effects. To further improve the application of this system, we study the cytotoxicity of SWNT-GC and investigate the effects on malignant tumor therapy. Here, we selected the optimal concentration of GC and SWNTs for the stable SWNT-GC construction. No cytotoxicity was observed under the dose used in the experiments. Using mouse melanoma tumor model, Laser+SWNT-GC treatment resulted in a significant mice survival rate, there were no long-term survivors under other treatment. It is providing a promising treatment modality for the malignancy.

Migration of mitochondrial DNA in the nuclear genome of colorectal adenocarcinoma.

PubMed

Srinivasainagendra, Vinodh; Sandel, Michael W; Singh, Bhupendra; Sundaresan, Aishwarya; Mooga, Ved P; Bajpai, Prachi; Tiwari, Hemant K; Singh, Keshav K

2017-03-29

Colorectal adenocarcinomas are characterized by abnormal mitochondrial DNA (mtDNA) copy number and genomic instability, but a molecular interaction between mitochondrial and nuclear genome remains unknown. Here we report the discovery of increased copies of nuclear mtDNA (NUMT) in colorectal adenocarcinomas, which supports link between mtDNA and genomic instability in the nucleus. We name this phenomenon of nuclear occurrence of mitochondrial component as numtogenesis. We provide a description of NUMT abundance and distribution in tumor versus matched blood-derived normal genomes. Whole-genome sequence data were obtained for colon adenocarcinoma and rectum adenocarcinoma patients participating in The Cancer Genome Atlas, via the Cancer Genomics Hub, using the GeneTorrent file acquisition tool. Data were analyzed to determine NUMT proportion and distribution on a genome-wide scale. A NUMT suppressor gene was identified by comparing numtogenesis in other organisms. Our study reveals that colorectal adenocarcinoma genomes, on average, contains up to 4.2-fold more somatic NUMTs than matched normal genomes. Women colorectal tumors contained more NUMT than men. NUMT abundance in tumor predicted parallel abundance in blood. NUMT abundance positively correlated with GC content and gene density. Increased numtogenesis was observed with higher mortality. We identified YME1L1, a human homolog of yeast YME1 (yeast mitochondrial DNA escape 1) to be frequently mutated in colorectal tumors. YME1L1 was also mutated in tumors derived from other tissues. We show that inactivation of YME1L1 results in increased transfer of mtDNA in the nuclear genome. Our study demonstrates increased somatic transfer of mtDNA in colorectal tumors. Our study also reveals sex-based differences in frequency of NUMT occurrence and that NUMT in blood reflects NUMT in tumors, suggesting NUMT may be used as a biomarker for tumorigenesis. We identify YME1L1 as the first NUMT suppressor gene in human and

Effect of hGC-MSCs from human gastric cancer tissue on cell proliferation, invasion and epithelial-mesenchymal transition in tumor tissue of gastric cancer tumor-bearing mice.

PubMed

Song, Lin; Zhou, Xin; Jia, Hong-Jun; Du, Mei; Zhang, Jin-Ling; Li, Liang

2016-08-01

To study the effect of hGC-MSCs from human gastric cancer tissue on cell proliferation, invasion and epithelial-mesenchymal transition in tumor tissue of gastric cancer tumor-bearing mice. BABL/c nude mice were selected as experimental animals and gastric cancer tumor-bearing mice model were established by subcutaneous injection of gastric cancer cells, randomly divided into different intervention groups. hGC-MSCs group were given different amounts of gastric cancer cells for subcutaneous injection, PBS group was given equal volume of PBS for subcutaneous injection. Then tumor tissue volume were determined, tumor-bearing mice were killed and tumor tissues were collected, mRNA expression of proliferation, invasion, EMT-related molecules were determined. 4, 8, 12, 16, 20 d after intervention, tumor tissue volume of hGC-MSCs group were significantly higher than those of PBS group and the more the number of hGC-MSCs, the higher the tumor tissue volume; mRNA contents of Ki-67, PCNA, Bcl-2, MMP-2, MMP-7, MMP-9, MMP-14, N-cadherin, vimentin, Snail and Twist in tumor tissue of hGC-MSCs group were higher than those of PBS group, and mRNA contents of Bax, TIMP1, TIMP2 and E-cadherin were lower than those of PBS group. hGC-MSCs from human gastric cancer tissue can promote the tumor growth in gastric cancer tumor-bearing mice, and the molecular mechanism includes promoting cell proliferation, invasion and epithelial-mesenchymal transition. Copyright © 2016 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.

The −174G/C and −572G/C Interleukin 6 Promoter Gene Polymorphisms in Mexican Patients with Rheumatoid Arthritis: A Case-Control Study

PubMed Central

Zavaleta-Muñiz, S. A.; Martín-Márquez, B. T.; Gonzalez-Lopez, L.; Gonzalez-Montoya, N. G.; Díaz-Toscano, M. L.; Ponce-Guarneros, J. M.; Ruiz-Padilla, A. J.; Mercado, M. Vázquez-Del; Maldonado-González, M.; Fafutis-Morris, M.; Flores-Martínez, S. E.; Martínez-García, E. A.; Gamez-Nava, J. I.

2013-01-01

Objective. There is a lack of information about the genotype frequencies of IL-6 −174G/C and −572G/C polymorphisms in Mexicans with rheumatoid arthritis (RA). Therefore, the aim of this study was to evaluate the association of the IL-6 −174G/C and −572G/C polymorphisms in Mexican mestizo with RA. Methods. We included 137 patients with RA and 102 healthy controls. Patients were assessed for clinical characteristics. IL-6 −174G/C and −572G/C polymorphisms were genotyped using PCR-RFLP analysis. Allele and genotype frequencies and the Hardy-Weinberg equilibrium were computed. Odds ratios (ORs) were computed to identify the risk for RA associated with the presence of GG genotype in comparison with the GC or CC genotypes. Results. The genotype −174GG occurred at a higher frequency in cases and controls (77.4% versus 78.4%, P = 0.845). We found similar results for the genotype −572GG (54% in patients versus 60.8% in controls, P = 0.295). Conclusions. This is the first study to evaluate the association of −174G/C and −572G/C polymorphisms of the IL-6 gene with RA in Mexican mestizo patients. These two polymorphisms were not associated with RA in the studied sample. Additional studies are required to evaluate if these IL-6 polymorphisms have relevance to the development of more severe disease. PMID:24223608

GC-Biased Gene Conversion in Yeast Is Specifically Associated with Crossovers: Molecular Mechanisms and Evolutionary Significance

PubMed Central

Lesecque, Yann; Mouchiroud, Dominique; Duret, Laurent

2013-01-01

GC-biased gene conversion (gBGC) is a process associated with recombination that favors the transmission of GC alleles over AT alleles during meiosis. gBGC plays a major role in genome evolution in many eukaryotes. However, the molecular mechanisms of gBGC are still unknown. Different steps of the recombination process could potentially cause gBGC: the formation of double-strand breaks (DSBs), the invasion of the homologous or sister chromatid, and the repair of mismatches in heteroduplexes. To investigate these models, we analyzed a genome-wide data set of crossovers (COs) and noncrossovers (NCOs) in Saccharomyces cerevisiae. We demonstrate that the overtransmission of GC alleles is specific to COs and that it occurs among conversion tracts in which all alleles are converted from the same donor haplotype. Thus, gBGC results from a process that leads to long-patch repair. We show that gBGC is associated with longer tracts and that it is driven by the nature (GC or AT) of the alleles located at the extremities of the tract. These observations invalidate the hypotheses that gBGC is due to the base excision repair machinery or to a bias in DSB formation and suggest that in S. cerevisiae, gBGC is caused by the mismatch repair (MMR) system. We propose that the presence of nicks on both DNA strands during CO resolution could be the cause of the bias in MMR activity. Our observations are consistent with the hypothesis that gBGC is a nonadaptive consequence of a selective pressure to limit the mutation rate in mitotic cells. PMID:23505044

GC and GC-MS determination of fluoroacetic acid and phenoxy acid herbicides via triphasal extractive pentafluorobenzylation using a polymer-bound phase-transfer catalyst.

PubMed

Miki, A; Tsuchihashi, H; Yamashita, M

1998-01-01

A simple and sensitive gas chromatography and gas chromatography-mass spectrometry (GC-MS) procedure has been developed for fluoroacetic acid (FA) and phenoxy acid herbicides (PAHs) via triphasal extractive pentafluorobenzylation. The triphasal system consisted of an aqueous sample, the extraction solvent toluene containing pentafluorobenzyl bromide as the derivatization reagent, and polymer-bound tri-n-butyl-methylphosphonium bromide as a phase-transfer catalyst, FA spiked in beverages, such as orange juice and milk, was extracted as its pentafluorobenzyl (PFB) derivative under moderate conditions (i.e., at a pH value of 6.5 at 60 degrees C). The detection limits were 0.10-0.20 microgram/mL by GC with electron-capture detection (GC-ECD), and 0.42-0.50 microgram/mL by full-scan GC-MS. PAHs were also detectable in the same manner within the detection limits of 0.05-0.10 microgram/mL by GC-ECD and 0.13-0.25 microgram/mL by full-scan GC-MS. Urine and serum which both contained 2,4-dichlorophenoxyacetic acid could also be analyzed by GC-MS after the triphasal pentafluorobenzylation. The detection limit was 0.20 microgram/mL in the full-scan mode and 10 ng/mL in the selected ion monitoring mode both for the urine and serum.

'Junk' DNA and long-term phenotypic evolution in Silene section Elisanthe (Caryophyllaceae).

PubMed Central

Meagher, Thomas R; Costich, Denise E

2004-01-01

Nuclear DNA content variation over orders of magnitude across species has been attributed to 'junk' repetitive DNA with limited adaptive significance. By contrast, our previous work on Silene latifolia showed that DNA content is negatively correlated with flower size, a character of clear adaptive relevance. The present paper explores this relationship in a broader phylogenetic context to investigate the long-term evolutionary impacts of DNA content variation. The relationship between nuclear DNA content and phenotype variation was determined for four closely related species of Silene section Elisanthe (Caryophyllaceae). In addition to a consistent sexual dimorphism in DNA content across all of the species, we found DNA content variation among populations within, as well as among, species. We also found a general trend towards a negative correlation between DNA content and flower and leaf size over all four species, within males and females as well as overall. These results indicate that repetitive DNA may play a role in long-term phenotypic evolution. PMID:15801614

Oxidatively Generated Guanine(C8)-Thymine(N3) Intrastrand Cross-links in Double-stranded DNA Are Repaired by Base Excision Repair Pathways*

PubMed Central

Talhaoui, Ibtissam; Shafirovich, Vladimir; Liu, Zhi; Saint-Pierre, Christine; Akishev, Zhiger; Matkarimov, Bakhyt T.; Gasparutto, Didier; Geacintov, Nicholas E.; Saparbaev, Murat

2015-01-01

Oxidatively generated guanine radical cations in DNA can undergo various nucleophilic reactions including the formation of C8-guanine cross-links with adjacent or nearby N3-thymines in DNA in the presence of O2. The G*[C8-N3]T* lesions have been identified in the DNA of human cells exposed to oxidative stress, and are most likely genotoxic if not removed by cellular defense mechanisms. It has been shown that the G*[C8-N3]T* lesions are substrates of nucleotide excision repair in human cell extracts. Cleavage at the sites of the lesions was also observed but not further investigated (Ding et al. (2012) Nucleic Acids Res. 40, 2506–2517). Using a panel of eukaryotic and prokaryotic bifunctional DNA glycosylases/lyases (NEIL1, Nei, Fpg, Nth, and NTH1) and apurinic/apyrimidinic (AP) endonucleases (Apn1, APE1, and Nfo), the analysis of cleavage fragments by PAGE and MALDI-TOF/MS show that the G*[C8-N3]T* lesions in 17-mer duplexes are incised on either side of G*, that none of the recovered cleavage fragments contain G*, and that T* is converted to a normal T in the 3′-fragment cleavage products. The abilities of the DNA glycosylases to incise the DNA strand adjacent to G*, while this base is initially cross-linked with T*, is a surprising observation and an indication of the versatility of these base excision repair proteins. PMID:25903131

H3.3 demarcates GC-rich coding and subtelomeric regions and serves as potential memory mark for virulence gene expression in Plasmodium falciparum

PubMed Central

Fraschka, Sabine Anne-Kristin; Henderson, Rob Wilhelmus Maria; Bártfai, Richárd

2016-01-01

Histones, by packaging and organizing the DNA into chromatin, serve as essential building blocks for eukaryotic life. The basic structure of the chromatin is established by four canonical histones (H2A, H2B, H3 and H4), while histone variants are more commonly utilized to alter the properties of specific chromatin domains. H3.3, a variant of histone H3, was found to have diverse localization patterns and functions across species but has been rather poorly studied in protists. Here we present the first genome-wide analysis of H3.3 in the malaria-causing, apicomplexan parasite, P. falciparum, which revealed a complex occupancy profile consisting of conserved and parasite-specific features. In contrast to other histone variants, PfH3.3 primarily demarcates euchromatic coding and subtelomeric repetitive sequences. Stable occupancy of PfH3.3 in these regions is largely uncoupled from the transcriptional activity and appears to be primarily dependent on the GC-content of the underlying DNA. Importantly, PfH3.3 specifically marks the promoter region of an active and poised, but not inactive antigenic variation (var) gene, thereby potentially contributing to immune evasion. Collectively, our data suggest that PfH3.3, together with other histone variants, indexes the P. falciparum genome to functionally distinct domains and contribute to a key survival strategy of this deadly pathogen. PMID:27555062

Getting it done at the ends: Pif1 family DNA helicases and telomeres

PubMed Central

Geronimo, Carly L.; Zakian, Virginia A.

2017-01-01

It is widely appreciated that the ends of linear DNA molecules cannot be fully replicated by the conventional replication apparatus. Less well known is that semi-conservative replication of telomeric DNA also presents problems for DNA replication. These problems likely arise from the atypical chromatin structure of telomeres, the GC-richness of telomeric DNA that makes it prone to forming DNA secondary structures, and from RNA-DNA hybrids, formed by transcripts of one or both DNA strands. Given the different aspects of telomeres that complicate their replication, it is not surprising that multiple DNA helicases promote replication of telomeric DNA. This review focuses on one such class of DNA helicases, the Pif1 family of 5′–3′ DNA helicases. In budding and fission yeasts, Pif1 family helicases impact both telomerase-mediated and semi-conservative replication of telomeric DNA as well as recombination-mediated telomere lengthening. PMID:27233114

Gaussian content as a laser beam quality parameter.

PubMed

Ruschin, Shlomo; Yaakobi, Elad; Shekel, Eyal

2011-08-01

We propose the Gaussian content (GC) as an optional quality parameter for the characterization of laser beams. It is defined as the overlap integral of a given field with an optimally defined Gaussian. The definition is especially suited for applications where coherence properties are targeted. Mathematical definitions and basic calculation procedures are given along with results for basic beam profiles. The coherent combination of an array of laser beams and the optimal coupling between a diode laser and a single-mode fiber are elaborated as application examples. The measurement of the GC and its conservation upon propagation are experimentally confirmed.

Comparative analysis of three sperm DNA damage assays and sperm nuclear protein content in couples undergoing assisted reproduction treatment.

PubMed

Simon, L; Liu, L; Murphy, K; Ge, S; Hotaling, J; Aston, K I; Emery, B; Carrell, D T

2014-05-01

Is there an association between sperm DNA damage, measured by three different assays, sperm nuclear protein content and clinical outcomes in assisted reproduction treatment (ART)? Sperm DNA damage measured by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) and the Comet assay were significantly associated with ART outcomes in our single institution study. Abnormal protamine expression is known to be associated with sperm DNA damage and male infertility. A number of studies have shown a significant relationship between sperm DNA damage and ART outcomes. To date, there are no large studies providing direct comparisons of DNA damage tests within the same study population. Thus, the prognostic value for each method remains unknown. Cross-sectional study of 238 men from infertile couples undergoing ART at the University Center for Reproductive Medicine, Utah, USA, between April 2011 and March 2013. Sperm from men undergoing ART were tested for DNA damage using the alkaline Comet assay, TUNEL and flow cytometric chromatin evaluation (FCCE) assays. Histone retention was analysed using the aniline blue staining method, whereas protamine content (proteins P1 and P2) and ratio were analysed using acid urea gel electrophoresis. The prognostic value of each sperm DNA test to predict clinical pregnancy was calculated. Histone retention was associated with sperm DNA damage (P < 0.001), reduced embryo quality (P = 0.005) and clinical pregnancies (P < 0.001). The mean percentage of sperm with DNA damage was significantly higher in sperm from non-pregnant couples compared with that from pregnant couples, as measured by TUNEL assay (15.04 ± 1.16% versus 8.79 ± 0.56%; P < 0.001) and alkaline Comet assay (72.79 ± 2.49% versus 55.86 ± 2.29%; P < 0.001). There was no association between clinical pregnancies and DNA fragmentation index measured by FCCE (12.97 ± 1.46 versus 14.93 ± 1.65; P = 0.379). Of the protamine parameters analysed, only the P1/P2

Assessment of Telomere Length, Phenotype, and DNA Content

PubMed Central

Kelesidis, Theodoros; Schmid, Ingrid

2017-01-01

Telomere sequences at the end of chromosomes control somatic cell division; therefore, telomere length in a given cell population provides information about its replication potential. This unit describes a method for flow cytometric measurement of telomere length in subpopulations using fluorescence in situ hybridization of fluorescently-labeled probes (Flow-FISH) without prior cell separation. After cells are stained for surface immunofluorescence, antigen-antibody complexes are covalently cross-linked onto cell membranes before FISH with a telomere-specific probe. Cells with long telomeres are included as internal standards. Addition of a DNA dye permits exclusion of proliferating cells during data analysis. DNA ploidy measurements of cells of interest and internal standard are performed on separate aliquots in parallel to Flow-FISH. Telomere fluorescence of G0/1 cells of subpopulations and internal standards obtained from Flow-FISH are normalized for DNA ploidy, and telomere length in subsets of interest is expressed as a fraction of the internal standard telomere length. PMID:28055113

GC-MS analyses of the volatiles of Houttuynia cordata Thunb.

PubMed

Yang, Zhan-Nan; Luo, Shi-Qiong; Ma, Jing; Wu, Dan; Hong, Liang; Yu, Zheng-Wen

2016-09-01

GC-MS is the basis of analysis of plant volatiles. Several protocols employed for the assay have resulted in inconsistent results in the literature. We developed a GC-MS method, which were applied to analyze 25 volatiles (α-pinene, camphene, β-pinene, 2-methyl-2-pentenal, myrcene, (+)-limonene, eucalyptol, trans-2-hexenal, γ-terpinene, cis-3-hexeneyl-acetate, 1-hexanol, α-pinene oxide, cis-3-hexen-1-ol, trans-2-hexen-1-ol, decanal, linalool, acetyl-borneol, β-caryophyllene, 2-undecanone, 4-terpineol, borneol, decanol, eugenol, isophytol and phytol) of Houttuynia cordata Thunb. Linear behaviors for all analytes were observed with a linear regression relationship (r2>0.9991) at the concentrations tested. Recoveries of the 25 analytes were 98.56-103.77% with RSDs <3.0%. Solution extraction (SE), which involved addition of an internal standard, could avoid errors for factors in sample preparation by steam distillation (SD) and solidphase micro extraction (SPME). Less sample material (≍0.05g fresh leaves of H. cordata) could be used to determine the contents of 25 analytes by our proposed method and, after collection, did not affect the normal physiological activity or growth of H. cordata. This method can be used to monitor the metabolic accumulation of H. cordata volatiles.

Arginine-rich cross-linking peptides with different SV40 nuclear localization signal content as vectors for intranuclear DNA delivery.

PubMed

Bogacheva, Mariia; Egorova, Anna; Slita, Anna; Maretina, Marianna; Baranov, Vladislav; Kiselev, Anton

2017-11-01

The major barriers for intracellular DNA transportation by cationic polymers are their toxicity, poor endosomal escape and inefficient nuclear uptake. Therefore, we designed novel modular peptide-based carriers modified with SV40 nuclear localization signal (NLS). Core peptide consists of arginine, histidine and cysteine residues for DNA condensation, endosomal escape promotion and interpeptide cross-linking, respectively. We investigated three polyplexes with different NLS content (10 mol%, 50 mol% and 90 mol% of SV40 NLS) as vectors for intranuclear DNA delivery. All carriers tested were able to condense DNA, to protect it from DNAase I and were not toxic to the cells. We observed that cell cycle arrest by hydroxyurea did not affect transfection efficacy of NLS-modified carriers which we confirmed using quantitative confocal microscopy analysis. Overall, peptide carrier modified with 90 mol% of SV40 NLS provided efficient transfection and nuclear uptake in non-dividing cells. Thus, incorporation of NLS into arginine-rich cross-linking peptides is an adequate approach to the development of efficient intranuclear gene delivery vehicles. Copyright © 2017 Elsevier Ltd. All rights reserved.

Metal organic framework g-C3N4/MIL-53(Fe) heterojunctions with enhanced photocatalytic activity for Cr(VI) reduction under visible light

NASA Astrophysics Data System (ADS)

Huang, Wenyuan; Liu, Ning; Zhang, Xiaodong; Wu, Minghong; Tang, Liang

2017-12-01

In this study, hybrid nanocomposites based on Fe-based MOF and graphitic carbon nitride (g-C3N4) were developed by a facile solvothermal method. The as-prepared materials were characterized by XRD, FESEM, TEM, XPS and PL analysis. It was showed that the introduction of a certain amount of g-C3N4 on the surface of MIL-53(Fe) would improve the separation and migration rate of photo-induced charges, consequently resulting in the boost of photocatalytic efficiency. Compared with g-C3N4 and MIL-53(Fe), the CMFe composites displayed more excellent visible light-resposive photocatalytic activity for the reduction of Cr(VI). The optimal doping content of g-C3N4 in g-C3N4/MIL-53(Fe) composite was determined to be 3.0 wt%, and it showed about 2.1 and 2.0 times as high photocatalytic efficiency for the reduction of Cr(VI) as that of pure g-C3N4 and MIL-53(Fe), respectively. Meanwhile, the composite exhibited good reusability and stability in the process of cyclic experiments. A possible photocatalytic reaction mechanism was also investigated in detail by the related electrochemical analysis.

GC and GC/MS Analysis of Essential Oil Composition of the Endemic Soqotraen Leucas virgata Balf.f. and Its Antimicrobial and Antioxidant Activities

PubMed Central

Mothana, Ramzi A.; Al-Said, Mansour S.; Al-Yahya, Mohammed A.; Al-Rehaily, Adnan J.; Khaled, Jamal M.

2013-01-01

Leucas virgata Balf.f. (Lamiaceae) was collected from the Island Soqotra (Yemen) and its essential oil was obtained by hydrodistillation. The chemical composition of the oil was investigated by GC and GC-MS. Moreover, the essential oil was evaluated for its antimicrobial activity against two Gram-positive bacteria, two Gram-negative bacteria, and one yeast species by using broth micro-dilution assay for minimum inhibitory concentrations (MIC) and antioxidant activity by measuring the scavenging activity of the DPPH radical. The investigation led to the identification of 43 constituents, representing 93.9% of the total oil. The essential oil of L. virgata was characterized by a high content of oxygenated monoterpenes (50.8%). Camphor (20.5%) exo-fenchol (3.4%), fenchon (5.4%), and borneol (3.1%) were identified as the main components. Oxygenated sesquiterpenes were found as the second major group of compounds (21.0%). β-Eudesmol (6.1%) and caryophyllene oxide (5.1%) were the major compounds among oxygenated sesquiterpenes. The results of the antimicrobial assay showed that the oil exhibited a great antibacterial activity against the tested S. aureus, B. subtilis, and E. coli. No activity was found against P. aeruginosa and C. albicans. Moreover, the DPPH-radical scavenging assay exhibited only a moderate antioxidant activity (31%) for the oil at the highest concentration tested (1 mg/mL). PMID:24284402

Outdoor and indoor benzene evaluation by GC-FID and GC-MS/MS.

PubMed

Sousa, José A; Domingues, Valentina F; Rosas, Mónica S; Ribeiro, Susana O; Alvim-Ferraz, Conceiçao M; Delerue-Matos, Cristina F

2011-01-01

The evaluation of benzene in different environments such as indoor (with and without tobacco smoke), a city area, countryside, gas stations and near exhaust pipes from cars running on different types of fuels was performed. The samples were analyzed using gas chromatography (GC) with flame ionization detection (FID) and tandem mass spectrometric detection (MS/MS) (to confirm the identification of benzene in the air samples). Operating conditions for the GC-MS analysis were optimized as well as the sampling and sample preparation. The results obtained in this work indicate that i) the type of fuel directly influences the benzene concentration in the air. Gasoline with additives provided the highest amount of benzene followed by unleaded gasoline and diesel; ii) the benzene concentration in the gas station was always higher than the advisable limit established by law (5 μg m⁻³) and during the unloading of gasoline the achieved concentration was 8371 μg m⁻³; iii) the data from the countryside (Taliscas) and the urban city (Matosinhos) were below 5 μg m⁻³ except 5 days after a fire on a petroleum refinery plant located near the city; iv) it was proven that in coffee shops where smoking is allowed the benzene concentration is higher (6 μg m⁻³) than in coffee shops where this is forbidden (4 μg m⁻³). This method may also be helpful for environmental analytical chemists who use GC-MS/MS for the confirmation or/and quantification of benzene.

Preservation and Significance of Extracellular DNA in Ferruginous Sediments from Lake Towuti, Indonesia

PubMed Central

Vuillemin, Aurèle; Horn, Fabian; Alawi, Mashal; Henny, Cynthia; Wagner, Dirk; Crowe, Sean A.; Kallmeyer, Jens

2017-01-01

GC contents. We conclude that extracellular DNA preserved in shallow lacustrine sediments reflects the initial environmental context, but is gradually modified and thereby shifts from its stratigraphic context. Discrimination of exogenous and endogenous sources of extracellular DNA allows simultaneously addressing in-lake and post-depositional processes. In deeper sediments, the accumulation of resting stages and sequences from cell lysis would require stringent extraction and specific primers if ancient DNA is targeted. PMID:28798742

Interaction of Sulforaphane with DNA and RNA

PubMed Central

Abassi Joozdani, Farzaneh; Yari, Faramarz; Abassi Joozdani, Parvaneh; Nafisi, Shohreh

2015-01-01

Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables with anti-inflammatory, anti-oxidant and anti-cancer activities. However, the antioxidant and anticancer mechanism of sulforaphane is not well understood. In the present research, we reported binding modes, binding constants and stability of SFN–DNA and -RNA complexes by Fourier transform infrared (FTIR) and UV–Visible spectroscopic methods. Spectroscopic evidence showed DNA intercalation with some degree of groove binding. SFN binds minor and major grooves of DNA and backbone phosphate (PO2), while RNA binding is through G, U, A bases with some degree of SFN–phosphate (PO2) interaction. Overall binding constants were estimated to be K(SFN–DNA)=3.01 (± 0.035)×104 M-1 and K(SFN–RNA)= 6.63 (±0.042)×103 M-1. At high SFN concentration (SFN/RNA = 1/1), DNA conformation changed from B to A occurred, while RNA remained in A-family structure. PMID:26030290

Quantification of Lignin and Its Structural Features in Plant Biomass Using 13C Lignin as Internal Standard for Pyrolysis-GC-SIM-MS.

PubMed

van Erven, Gijs; de Visser, Ries; Merkx, Donny W H; Strolenberg, Willem; de Gijsel, Peter; Gruppen, Harry; Kabel, Mirjam A

2017-10-17

Understanding the mechanisms underlying plant biomass recalcitrance at the molecular level can only be achieved by accurate analyses of both the content and structural features of the molecules involved. Current quantification of lignin is, however, majorly based on unspecific gravimetric analysis after sulfuric acid hydrolysis. Hence, our research aimed at specific lignin quantification with concurrent characterization of its structural features. Hereto, for the first time, a polymeric 13 C lignin was used as internal standard (IS) for lignin quantification via analytical pyrolysis coupled to gas chromatography with mass-spectrometric detection in selected ion monitoring mode (py-GC-SIM-MS). In addition, relative response factors (RRFs) for the various pyrolysis products obtained were determined and applied. First, 12 C and 13 C lignin were isolated from nonlabeled and uniformly 13 C labeled wheat straw, respectively, and characterized by heteronuclear single quantum coherence (HSQC), nuclear magnetic resonance (NMR), and py-GC/MS. The two lignin isolates were found to have identical structures. Second, 13 C-IS based lignin quantification by py-GC-SIM-MS was validated in reconstituted biomass model systems with known contents of the 12 C lignin analogue and was shown to be extremely accurate (>99.9%, R 2 > 0.999) and precise (RSD < 1.5%). Third, 13 C-IS based lignin quantification was applied to four common poaceous biomass sources (wheat straw, barley straw, corn stover, and sugar cane bagasse), and lignin contents were in good agreement with the total gravimetrically determined lignin contents. Our robust method proves to be a promising alternative for the high-throughput quantification of lignin in milled biomass samples directly and simultaneously provides a direct insight into the structural features of lignin.

Application of comprehensive multidimensional gas chromatography combined with time-of-flight mass spectrometry (GC x GC-TOFMS) for high resolution analysis of hop essential oil.

PubMed

Roberts, Mark T; Dufour, Jean-Pierre; Lewis, Alastair C

2004-04-01

The selection and quality of hops is a major determinant in beer flavour. Brewers acknowledge that distinctive characteristics of different hop varieties can be traced to the composition of their essential oils. The difficulty in characterising complex mixtures such as hop oil using 1-D chromatography is that many compounds co-elute. With the introduction of comprehensive multidimensional capillary gas chromatography (GC x GC), there is a tremendous improvement in the separation power or peak capacity. Recent work using GC x GC with flame ionisation detection has suggested that there may be over 1,000 compounds in hop oil. This work describes the use of GC x GC combined with TOFMS detection (Leco Pegasus 4D instrument) to analyse Target hop oil. The TOFMS spectral acquisition rate of 60 Hz provided sufficient spectra per peak (2-D peak base width of 0.1-0.2 s) for identification (119 components were identified with 45 previously unreported compounds). When analysing results, an advantage of GC x GC coupled to TOFMS is that 2-D chromatograms can be viewed for individual masses that are characteristic of particular functional groups. This allows the analyst to view the various homologous series of compounds although in certain cases coelution may still be present as shown by the esters with mass 75.

Novel ternary g-C3N4/Ag3VO4/AgBr nanocomposites with excellent visible-light-driven photocatalytic performance for environmental applications

NASA Astrophysics Data System (ADS)

Barzegar, Javid; Habibi-Yangjeh, Aziz; Akhundi, Anise; Vadivel, S.

2018-04-01

Novel visible-light-induced photocatalysts were fabricated by integration of Ag3VO4 and AgBr semiconductors with graphitic carbon nitride (g-C3N4) through a facile refluxing method. The fabricated photocatalysts were extensively characterized by XRD, EDX, SEM, TEM, FT-IR, UV-vis DRS, BET, TGA, and PL instruments. The photocatalytic performance of these samples was studied by degradations of three dye contaminants under visible-light exposure. Among the ternary photocatalysts, the g-C3N4/Ag3VO4/AgBr (10%) nanocomposite displayed the maximum activity for RhB degradation with rate constant of 1366.6 × 10-4 min-1, which is 116, 7.23, and 38.5 times as high as those of the g-C3N4, g-C3N4/AgBr (10%), and g-C3N4/Ag3VO4 (30%) photocatalysts, respectively. The effects of synthesis time and calcination temperature were also investigated and discussed. Furthermore, according to the trapping experiments, it was found that superoxide anion radicals were the predominant reactive species in this system. Finally, the ternary photocatalyst displayed superlative activity in removal of the contaminants under visible-light exposure, displaying great potential of this ternary photocatalyst for environmental remediation, because of a facile synthesis route and outstanding photocatalytic performance.

Fabrication of novel ternary Au/CeO2@g-C3N4 nanocomposite: kinetics and mechanism investigation of 4-nitrophenol reduction, and benzyl alcohol oxidation

NASA Astrophysics Data System (ADS)

Kohantorabi, Mona; Gholami, Mohammad Reza

2018-06-01

Au nanoparticles supported on cerium oxide/graphitic carbon nitride (CeO2@g-C3N4) was synthesized and used as heterogeneous catalyst in redox reaction. The catalyst was characterized by different techniques such as FT-IR, XRD, FE-SEM, EDS, TEM, BET, TGA, and ICP. The as-prepared ternary nanocomposite was used as an effective catalyst for the reduction of toxic 4-nitrophenol to useful 4-aminophenol by NaBH4. The rate constant value of reduction reaction reached up to 0.106 s-1 by Au/CeO2@g-C3N4, which was 3.8, and 8.8 times higher than that of Au@CeO2 (0.028 s-1), and Au@g-C3N4 (0.012 s-1) nanocomposites, respectively. The superior catalytic performance of as-prepared catalyst in 4-NP reduction can be attributed to synergistic effect between Au nanoparticles and CeO2@g-C3N4 support, and efficient electron transfer. The reduction reaction was carried out at different temperatures, and the energy of activation ({Ea}), and thermodynamic parameters including, activation of entropy (Δ S^ ≠), enthalpy (Δ H^ ≠), and Gibbs free energy (Δ G^ ≠) were determined. Additionally, the mechanism of reaction was studied in details, and equilibrium constants of 4-NP ( K 4-NP), and {BH}4^{ - } ({K_{{BH}4^{{ - }} }}) were calculated using Langmuir-Hinshelwood model. Furthermore, this nanocomposite exhibited excellent catalytic activity in oxidation of benzyl alcohol by molecular oxygen as a green oxidant. This study revealed that the ternary Au/CeO2@g-C3N4 nanocomposite is an attractive candidate for catalytic applications.

Greenhouse Gas Analysis by GC/MS

NASA Astrophysics Data System (ADS)

Bock, E. M.; Easton, Z. M.; Macek, P.

2015-12-01

Current methods to analyze greenhouse gases rely on designated complex, multiple-column, multiple-detector gas chromatographs. A novel method was developed in partnership with Shimadzu for simultaneous quantification of carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O) in environmental gas samples. Gas bulbs were used to make custom standard mixtures by injecting small volumes of pure analyte into the nitrogen-filled bulb. Resulting calibration curves were validated using a certified gas standard. The use of GC/MS systems to perform this analysis has the potential to move the analysis of greenhouse gasses from expensive, custom GC systems to standard single-quadrupole GC/MS systems that are available in most laboratories, which wide variety of applications beyond greenhouse gas analysis. Additionally, use of mass spectrometry can provide confirmation of identity of target analytes, and will assist in the identification of unknown peaks should they be present in the chromatogram.

The total alkaloid and anagyrine contents of some bitter and sweet selections of lupin species used as food.

PubMed

Keeler, R F; Gross, R

1980-01-01

The total alkaloid and anagyrine contents of bitter and sweet Lupinus luteus, Lupinus angustifolius, Lupinus albus, Lupinus mutabilis, Lupinus polyphyllus, and Lupinus perennis were analyzed by gas chromatography (GC) and mass spectrometry. No anagyrine was detected in any of the samples. The GC peak in some of the samples which corresponded to anagyrine in retention of time seems to be identical to 17-oxolupanine. The alkaloid content of samples ranged from 3.17 to 0.003 percent.

DNA Binding Mode Transitions of Escherichia coli HUαβ: Evidence for Formation of a Bent DNA – Protein Complex on Intact, Linear Duplex DNA

PubMed Central

Koh, Junseock; Saecker, Ruth M.; Record, M. Thomas

2008-01-01

Escherichia coli HUαβ, a major nucleoid associated protein (NAP), organizes the DNA chromosome and facilitates numerous DNA transactions. Using isothermal titration calorimetry (ITC), fluorescence resonance energy transfer (FRET) and a series of DNA lengths (8, 15, 34, 38 and 160 base pairs) we establish that HUαβ interacts with duplex DNA using three different nonspecific binding modes. Both the HU to DNA mole ratio ([HU]/[DNA]) and DNA length dictate the dominant HU binding mode. On sufficiently long DNA (≥ 34 base pairs), at low [HU]/[DNA], HU populates a noncooperative 34 bp binding mode with a binding constant of 2.1 (± 0.4) × 106 M−1, and a binding enthalpy of +7.7 (± 0.6) kcal/mol at 15 °C and 0.15 M Na+. With increasing [HU]/[DNA], HU bound in the noncooperative 34 bp mode progressively converts to two cooperative (ω ~ 20) modes with site sizes of 10 bp and 6 bp. These latter modes exhibit smaller binding constants (1.1 (± 0.2) × 105 M−1 for the 10 bp mode, 3.5 (± 1.4) × 104 M−1 for the 6 bp mode) and binding enthalpies (4.2 (± 0.3) kcal/mol for the 10 bp mode, −1.6 (±0.3) kcal/mol for the 6 bp mode). As DNA length increases to 34 bp or more at low [HU]/[DNA], the small modes are replaced by the 34 bp binding mode. FRET data demonstrate that the 34 bp mode bends DNA by 143 ± 6° whereas the 6 and 10 bp modes do not. The model proposed in this study provides a novel quantitative and comprehensive framework for reconciling previous structural and solution studies of HU, including single molecule (force extension measurement, AFM), fluorescence, and electrophoretic gel mobility shift assays. In particular, it explains how HU condenses or extends DNA depending on the relative concentrations of HU and DNA. PMID:18657548

The role of ethylene in the development of constant-light injury of potato and tomato

NASA Technical Reports Server (NTRS)

Cushman, K. E.; Tibbitts, T. W.

1998-01-01

The role of ethylene in the development of constant-light injury of potato (Solanum tuberosum L.) and tomato (Lycopersicon esculentum Mill.) was investigated. In one study, silver thiosulfate (STS) was applied to the foliage of four potato cultivars growing under constant light. Leaf area and shoot dry mass of 'Kennebec' and 'Superior', cultivars normally injured by constant light, were greater (P < 0.05) than those of control plants given foliar applications of distilled water. Examination of STS-treated 'Kennebec' leaflets revealed significantly less injury (necrotic spotting and reduced starch content) than the water-treated controls. 'Norland' and 'Denali', cultivars tolerant of constant light, exhibited no differences in growth between treatments. In a second study, injury (necrotic spotting and reduced starch content) was induced in leaflets of 'Denali' when exposed to spray applications of 0.5 mmol L-1 ethephon or air containing 0.5 to 0.8 microL L-1 ethylene. In a third study, three genotypes of 'Ailsa Craig' tomato were grown under constant light. Leaves of the normal 'Ailsa Craig' exhibited epinasty, reduced chlorophyll concentration, and reduced starch content. Leaves of a mutant 'Ailsa Craig', containing the Never ripe mutation, did not exhibit epinasty but exhibited the same amount of reduced chlorophyll concentration and starch content as normal plants. Leaves of a transgenic 'Ailsa Craig', containing an antisense gene of 1-aminocyclopropane 1-carboxylate (ACC) oxidase, were epinastic, but chlorophyll concentration and starch content were greater than in leaves of normal and mutant plants. These results suggest that transgenic plants were more tolerant of constant light than the other genotypes. Evidence from these studies indicates that ethylene, combined with constant light, has an important role in the development of constant-light injury.

A Specific Mutational Signature Associated with DNA 8-Oxoguanine Persistence in MUTYH-defective Colorectal Cancer

DOE PAGES

Viel, Alessandra; Bruselles, Alessandro; Meccia, Ettore; ...

2017-04-13

8-Oxoguanine, a common mutagenic DNA lesion, generates G:C > T:A transversions via mispairing with adenine during DNA replication. When operating normally, the MUTYH DNA glycosylase prevents 8-oxoguanine-related mutagenesis by excising the incorporated adenine. Biallelic MUTYH mutations impair this enzymatic function and are associated with colorectal cancer (CRC) in MUTYH-Associated Polyposis (MAP) syndrome. Here in this paper, we perform whole-exome sequencing that reveals a modest mutator phenotype in MAP CRCs compared to sporadic CRC stem cell lines or bulk tumours. The excess G:C > T:A transversion mutations in MAP CRCs exhibits a novel mutational signature, termed Signature 36, with a strongmore » sequence dependence. The MUTYH mutational signature reflecting persistent 8-oxoG:A mismatches occurs frequently in the APC, KRAS, PIK3CA, FAT4, TP53, FAT1, AMER1, KDM6A, SMAD4 and SMAD2 genes that are associated with CRC. In conclusion, the occurrence of Signature 36 in other types of human cancer indicates that DNA 8-oxoguanine-related mutations might contribute to the development of cancer in other organs.« less

A Specific Mutational Signature Associated with DNA 8-Oxoguanine Persistence in MUTYH-defective Colorectal Cancer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Viel, Alessandra; Bruselles, Alessandro; Meccia, Ettore

8-Oxoguanine, a common mutagenic DNA lesion, generates G:C > T:A transversions via mispairing with adenine during DNA replication. When operating normally, the MUTYH DNA glycosylase prevents 8-oxoguanine-related mutagenesis by excising the incorporated adenine. Biallelic MUTYH mutations impair this enzymatic function and are associated with colorectal cancer (CRC) in MUTYH-Associated Polyposis (MAP) syndrome. Here in this paper, we perform whole-exome sequencing that reveals a modest mutator phenotype in MAP CRCs compared to sporadic CRC stem cell lines or bulk tumours. The excess G:C > T:A transversion mutations in MAP CRCs exhibits a novel mutational signature, termed Signature 36, with a strongmore » sequence dependence. The MUTYH mutational signature reflecting persistent 8-oxoG:A mismatches occurs frequently in the APC, KRAS, PIK3CA, FAT4, TP53, FAT1, AMER1, KDM6A, SMAD4 and SMAD2 genes that are associated with CRC. In conclusion, the occurrence of Signature 36 in other types of human cancer indicates that DNA 8-oxoguanine-related mutations might contribute to the development of cancer in other organs.« less

Case Report: GcMAF Treatment in a Patient with Multiple Sclerosis.

PubMed

Inui, Toshio; Katsuura, Goro; Kubo, Kentaro; Kuchiike, Daisuke; Chenery, Leslye; Uto, Yoshihiro; Nishikata, Takahito; Mette, Martin

2016-07-01

Gc protein-derived macrophage-activating factor (GcMAF) has various functions as an immune modulator, such as macrophage activation, anti-angiogenic activity and anti-tumor activity. Clinical trials of second-generation GcMAF demonstrated remarkable clinical effects in several types of cancers. Thus, GcMAF-based immunotherapy has a wide application for use in the treatment of many diseases via macrophage activation that can be used as a supportive therapy. Multiple sclerosis (MS) is considered to be an autoimmune disorder that affects the myelinated axons in the central nervous system (CNS). This study was undertaken to examine the effects of second-generation GcMAF in a patient with MS. This case study demonstrated that treatments of GcMAF in a patient with MS have potent therapeutic actions with early beneficial responses, especially improvement of motor dysfunction. GcMAF shows therapeutic potency in the treatment of MS. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Guanine Plus Cytosine Contents of the Deoxyribonucleic Acids of Some Sulfate-Reducing Bacteria: a Reassessment

PubMed Central

Skyring, G. W.; Jones, H. E.

1972-01-01

Guanine plus cytosine (GC) contents of the deoxyribonucleic acids of Desulfovibrio and Desulfotomaculum have been used as a basis for classification. Some of these data have been incorrectly calculated, resulting in errors of as much as 5% GC. This situation has been corrected by a reanalysis of existing data and by the contribution of new data. PMID:5011245

Ambient VOC-Measurements by GC-PTR-TOF

NASA Astrophysics Data System (ADS)

Langebner, S.; Schnitzhofer, R.; Hasler, C.; Jocher, M.; Hansel, A.; Brilli, F.

2011-12-01

Authors: Stephan LANGEBNER, Federico BRILLI, Ralf SCHNITZHOFER, Christoph HASLER, Markus JOCHER, Armin HANSEL; During the past 16 years PTR MS (Proton Transfer Reaction Mass Spectrometry) became a well established technique for real time measurements of environmentally important volatile organic compounds (VOCs) [HANSEL 1995]. The recent development of PTR ToF [GRAUS 2010] increased the VOC separation capability by strongly improving the mass separation capability and the duty cycle. Now isobaric compounds can be separated and whole mass spectra are recorded within a fraction of a second. Isomeric VOCs, however, remain undistinguishable with this technique. Therefore a Thermo-Desorption-System-Gas-Chromatograph (TDS GC) with isomeric separation capabilities was coupled with a PTR ToF. The performance of this new GC PTR TOF instrument was evaluated analysing ambient air for several days. The measurement cycle started with simultaneous GC-sampling and direct PTR ToF measurements of ambient air. After the fifteen minute TDS cycle, the output of the GC column was directed to the PTR ToF and the timely separated VOC peaks were recorded for 40 minutes. We will present first results which look very promising e.g. different monoterpene isomers can be clearly distinguished at ambient levels.

Processing of N-linked oligosaccharides from precursor- to mature-form herpes simplex virus type 1 glycoprotein gC.

PubMed Central

Serafini-Cessi, F; Dall'Olio, F; Pereira, L; Campadelli-Fiume, G

1984-01-01

Immature and mature forms of glycoprotein gC were purified by immunoadsorbent from herpes simplex virus type 1-infected BHK cells labeled with [3H]mannose for a 20-min pulse or for 11 h followed by a 3-h chase. The nature of N-asparagine-linked oligosaccharides carried by the immature form, pgC (molecular weight = 92,000), and the mature gC (molecular weight = 120,000) has been investigated. All pronase-digested glycopeptides of pgC were susceptible to endo-beta-N-acetylglucosaminidase H treatment; thus they have a high-mannose structure. Using thin-layer chromatography to separate endo-beta-N-acetylglucosaminidase H-cleaved oligosaccharides, polymannosyl chains of different sizes, ranging from Man9GlcNAc to Man5GlcNAc, were separated. The major components were Man8GlcNAc and Man7GlcNAc, suggesting that pgC labeled in a 20-min pulse represents the form of glycoprotein already routed to the Golgi apparatus. Analysis of glycopeptides of mature gC showed that the majority (95%) of N-linked glycans were converted to complex-type glycans. Ion-exchange chromatography and affinity chromatography on concanavalin A-Sepharose and leucoagglutinin-agarose revealed that diantennary and triantennary glycans predominated, whereas tetrantennary chains were not present. Parts of the di- and triantennary chains were not fully sialylated. The high heterogeneity of complex-type chains found in mature gC may be related to the high number of N-glycosylation sites of the glycoprotein as predicted by DNA sequencing studies (Frink et al., J. Virol. 45:634-647, 1983). Images PMID:6088806

Processing of N-linked oligosaccharides from precursor- to mature-form herpes simplex virus type 1 glycoprotein gC.

PubMed

Serafini-Cessi, F; Dall'Olio, F; Pereira, L; Campadelli-Fiume, G

1984-09-01

Immature and mature forms of glycoprotein gC were purified by immunoadsorbent from herpes simplex virus type 1-infected BHK cells labeled with [3H]mannose for a 20-min pulse or for 11 h followed by a 3-h chase. The nature of N-asparagine-linked oligosaccharides carried by the immature form, pgC (molecular weight = 92,000), and the mature gC (molecular weight = 120,000) has been investigated. All pronase-digested glycopeptides of pgC were susceptible to endo-beta-N-acetylglucosaminidase H treatment; thus they have a high-mannose structure. Using thin-layer chromatography to separate endo-beta-N-acetylglucosaminidase H-cleaved oligosaccharides, polymannosyl chains of different sizes, ranging from Man9GlcNAc to Man5GlcNAc, were separated. The major components were Man8GlcNAc and Man7GlcNAc, suggesting that pgC labeled in a 20-min pulse represents the form of glycoprotein already routed to the Golgi apparatus. Analysis of glycopeptides of mature gC showed that the majority (95%) of N-linked glycans were converted to complex-type glycans. Ion-exchange chromatography and affinity chromatography on concanavalin A-Sepharose and leucoagglutinin-agarose revealed that diantennary and triantennary glycans predominated, whereas tetrantennary chains were not present. Parts of the di- and triantennary chains were not fully sialylated. The high heterogeneity of complex-type chains found in mature gC may be related to the high number of N-glycosylation sites of the glycoprotein as predicted by DNA sequencing studies (Frink et al., J. Virol. 45:634-647, 1983).

Assessment of Telomere Length, Phenotype, and DNA Content.