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Sample records for human dna quantification

  1. Quantification of human mitochondrial DNA using synthesized DNA standards.

    Science.gov (United States)

    Kavlick, Mark F; Lawrence, Helen S; Merritt, R Travis; Fisher, Constance; Isenberg, Alice; Robertson, James M; Budowle, Bruce

    2011-11-01

    Successful mitochondrial DNA (mtDNA) forensic analysis depends on sufficient quantity and quality of mtDNA. A real-time quantitative PCR assay was developed to assess such characteristics in a DNA sample, which utilizes a duplex, synthetic DNA to ensure optimal quality assurance and quality control. The assay's 105-base pair target sequence facilitates amplification of degraded DNA and is minimally homologous to nonhuman mtDNA. The primers and probe hybridize to a region that has relatively few sequence polymorphisms. The assay can also identify the presence of PCR inhibitors and thus indicate the need for sample repurification. The results show that the assay provides information down to 10 copies and provides a dynamic range spanning seven orders of magnitude. Additional experiments demonstrated that as few as 300 mtDNA copies resulted in successful hypervariable region amplification, information that permits sample conservation and optimized downstream PCR testing. The assay described is rapid, reliable, and robust.

  2. Human DNA quantification and sample quality assessment: Developmental validation of the PowerQuant(®) system.

    Science.gov (United States)

    Ewing, Margaret M; Thompson, Jonelle M; McLaren, Robert S; Purpero, Vincent M; Thomas, Kelli J; Dobrowski, Patricia A; DeGroot, Gretchen A; Romsos, Erica L; Storts, Douglas R

    2016-07-01

    Quantification of the total amount of human DNA isolated from a forensic evidence item is crucial for DNA normalization prior to short tandem repeat (STR) DNA analysis and a federal quality assurance standard requirement. Previous commercial quantification methods determine the total human DNA and total human male DNA concentrations, but provide limited information about the condition of the DNA sample. The PowerQuant(®) System includes targets for quantification of total human and total human male DNA as well as targets for evaluating whether the human DNA is degraded and/or PCR inhibitors are present in the sample. A developmental validation of the PowerQuant(®) System was completed, following SWGDAM Validation Guidelines, to evaluate the assay's specificity, sensitivity, precision and accuracy, as well as the ability to detect degraded DNA or PCR inhibitors. In addition to the total human DNA and total human male DNA concentrations in a sample, data from the degradation target and internal PCR control (IPC) provide a forensic DNA analyst meaningful information about the quality of the isolated human DNA and the presence of PCR inhibitors in the sample that can be used to determine the most effective workflow and assist downstream interpretation.

  3. HUMAN DNA QUANTIFICATION IN THE STOOLS OF PATIENTS WITH COLORECTAL CANCER

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    Yolanda TEIXEIRA

    2015-12-01

    Full Text Available Background - Colorectal cancer is one of the main cause of cancer in the world. Colonoscopy is the best screen method, however the compliance is less than 50%. Quantification of human DNA (hDNA in the feces may be a possible screen non-invasive method that is a consequence of the high proliferation and exfoliation of cancer cells. Objective - To quantify the human DNA in the stools of patients with colorectal cancer or polyps. Methods - Fifty patients with CRC, 26 polyps and 53 with normal colonoscopy were included. Total and human DNA were analyzed from the frozen stools. Results - An increased concentration of hDNA in the stools was observed in colorectal cancer patients compared to controls and polyps. Tumors localized in the left side of the colon had higher concentrations of hDNA. There were no difference between polyps and controls. A cut off of 0.87 ng/mL of human DNA was determined for colorectal cancer patients by the ROC curve, with a sensitivity of 66% and a specificity of 86.8%. For polyps the cut off was 0.41, the sensitivity was 41% and the specificity 77.4%. Conclusion - A higher concentration of hDNA had been found in colorectal cancer patients The quantification of hDNA from the stools can be a trial method for the diagnosis of colorectal cancer.

  4. Absolute quantification of somatic DNA alterations in human cancer.

    Science.gov (United States)

    Carter, Scott L; Cibulskis, Kristian; Helman, Elena; McKenna, Aaron; Shen, Hui; Zack, Travis; Laird, Peter W; Onofrio, Robert C; Winckler, Wendy; Weir, Barbara A; Beroukhim, Rameen; Pellman, David; Levine, Douglas A; Lander, Eric S; Meyerson, Matthew; Getz, Gad

    2012-05-01

    We describe a computational method that infers tumor purity and malignant cell ploidy directly from analysis of somatic DNA alterations. The method, named ABSOLUTE, can detect subclonal heterogeneity and somatic homozygosity, and it can calculate statistical sensitivity for detection of specific aberrations. We used ABSOLUTE to analyze exome sequencing data from 214 ovarian carcinoma tumor-normal pairs. This analysis identified both pervasive subclonal somatic point-mutations and a small subset of predominantly clonal and homozygous mutations, which were overrepresented in the tumor suppressor genes TP53 and NF1 and in a candidate tumor suppressor gene CDK12. We also used ABSOLUTE to infer absolute allelic copy-number profiles from 3,155 diverse cancer specimens, revealing that genome-doubling events are common in human cancer, likely occur in cells that are already aneuploid, and influence pathways of tumor progression (for example, with recessive inactivation of NF1 being less common after genome doubling). ABSOLUTE will facilitate the design of clinical sequencing studies and studies of cancer genome evolution and intra-tumor heterogeneity.

  5. Absolute quantification of somatic DNA alterations in human cancer

    OpenAIRE

    Carter, Scott L.; Cibulskis, Kristian; Helman, Elena; McKenna, Aaron; Shen, Hui; Zack, Travis; Laird, Peter W.; Onofrio, Robert C.; Winckler, Wendy; Weir, Barbara A; Beroukhim, Rameen; Pellman, David; Levine, Douglas A.; Lander, Eric S.; Meyerson, Matthew

    2012-01-01

    We developed a computational method (ABSOLUTE) that infers tumor purity and malignant cell ploidy directly from analysis of somatic DNA alterations. ABSOLUTE can detect subclonal heterogeneity, somatic homozygosity, and calculate statistical sensitivity to detect specific aberrations. We used ABSOLUTE to analyze ovarian cancer data and identified pervasive subclonal somatic point mutations. In contrast, mutations occurring in key tumor suppressor genes, TP53 and NF1 were predominantly clonal ...

  6. Absolute quantification of somatic DNA alterations in human cancer

    OpenAIRE

    Carter, Scott L.; Cibulskis, Kristian; Helman, Elena; McKenna, Aaron; Shen, Hui; Zack, Travis; Laird, Peter W.; Onofrio, Robert C.; Winckler, Wendy; Weir, Barbara A; Beroukhim, Rameen; Pellman, David; Levine, Douglas A.; Lander, Eric S.; Meyerson, Matthew

    2015-01-01

    We developed a computational method (ABSOLUTE) that infers tumor purity and malignant cell ploidy directly from analysis of somatic DNA alterations. ABSOLUTE can detect subclonal heterogeneity, somatic homozygosity, and calculate statistical sensitivity to detect specific aberrations. We used ABSOLUTE to analyze ovarian cancer data and identified pervasive subclonal somatic point mutations. In contrast, mutations occurring in key tumor suppressor genes, TP53 and NF1 were predominantly clonal ...

  7. Comparison of five DNA quantification methods

    DEFF Research Database (Denmark)

    Nielsen, Karsten; Mogensen, Helle Smidt; Hedman, Johannes;

    2008-01-01

    Six commercial preparations of human genomic DNA were quantified using five quantification methods: UV spectrometry, SYBR-Green dye staining, slot blot hybridization with the probe D17Z1, Quantifiler Human DNA Quantification kit and RB1 rt-PCR. All methods measured higher DNA concentrations than ...

  8. Comparison of methods for quantification of global DNA methylation in human cells and tissues.

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    Sofia Lisanti

    Full Text Available DNA methylation is a key epigenetic modification which, in mammals, occurs mainly at CpG dinucleotides. Most of the CpG methylation in the genome is found in repetitive regions, rich in dormant transposons and endogenous retroviruses. Global DNA hypomethylation, which is a common feature of several conditions such as ageing and cancer, can cause the undesirable activation of dormant repeat elements and lead to altered expression of associated genes. DNA hypomethylation can cause genomic instability and may contribute to mutations and chromosomal recombinations. Various approaches for quantification of global DNA methylation are widely used. Several of these approaches measure a surrogate for total genomic methyl cytosine and there is uncertainty about the comparability of these methods. Here we have applied 3 different approaches (luminometric methylation assay, pyrosequencing of the methylation status of the Alu repeat element and of the LINE1 repeat element for estimating global DNA methylation in the same human cell and tissue samples and have compared these estimates with the "gold standard" of methyl cytosine quantification by HPLC. Next to HPLC, the LINE1 approach shows the smallest variation between samples, followed by Alu. Pearson correlations and Bland-Altman analyses confirmed that global DNA methylation estimates obtained via the LINE1 approach corresponded best with HPLC-based measurements. Although, we did not find compelling evidence that the gold standard measurement by HPLC could be substituted with confidence by any of the surrogate assays for detecting global DNA methylation investigated here, the LINE1 assay seems likely to be an acceptable surrogate in many cases.

  9. The effects of metal ion PCR inhibitors on results obtained with the Quantifiler(®) Human DNA Quantification Kit.

    Science.gov (United States)

    Combs, Laura Gaydosh; Warren, Joseph E; Huynh, Vivian; Castaneda, Joanna; Golden, Teresa D; Roby, Rhonda K

    2015-11-01

    Forensic DNA samples may include the presence of PCR inhibitors, even after extraction and purification. Studies have demonstrated that metal ions, co-purified at specific concentrations, inhibit DNA amplifications. Metal ions are endogenous to sample types, such as bone, and can be introduced from environmental sources. In order to examine the effect of metal ions as PCR inhibitors during quantitative real-time PCR, 2800 M DNA was treated with 0.0025-18.750 mM concentrations of aluminum, calcium, copper, iron, nickel, and lead. DNA samples, both untreated and metal-treated, were quantified using the Quantifiler(®) Human DNA Quantification Kit. Quantification cycle (Cq) values for the Quantifiler(®) Human DNA and internal PCR control (IPC) assays were measured and the estimated concentrations of human DNA were obtained. Comparisons were conducted between metal-treated and control DNA samples to determine the accuracy of the quantification estimates and to test the efficacy of the IPC inhibition detection. This kit is most resistant to the presence of calcium as compared to all metals tested; the maximum concentration tested does not affect the amplification of the IPC or quantification of the sample. This kit is most sensitive to the presence of aluminum; concentrations greater than 0.0750 mM negatively affected the quantification, although the IPC assay accurately assessed the presence of PCR inhibition. The Quantifiler(®) Human DNA Quantification Kit accurately quantifies human DNA in the presence of 0.5000 mM copper, iron, nickel, and lead; however, the IPC does not indicate the presence of PCR inhibition at this concentration of these metals. Unexpectedly, estimates of DNA quantity in samples treated with 18.750 mM copper yielded values in excess of the actual concentration of DNA in the samples; fluorescence spectroscopy experiments indicated this increase was not a direct interaction between the copper metal and 6-FAM dye used to label the probe that

  10. Development and assessment of a multiplex real-time PCR assay for quantification of human immunodeficiency virus type 1 DNA.

    Science.gov (United States)

    Beloukas, A; Paraskevis, D; Haida, C; Sypsa, V; Hatzakis, A

    2009-07-01

    Previous studies showed that high levels of human immunodeficiency virus type 1 (HIV-1) DNA are associated with a faster progression to AIDS, an increased risk of death, and a higher risk of HIV RNA rebound in patients on highly active antiretroviral therapy. Our objective was to develop and assess a highly sensitive real-time multiplex PCR assay for the quantification of HIV-1 DNA (RTMP-HIV) based on molecular beacons. HIV-1 DNA quantification was carried out by RTMP in a LightCycler 2.0 apparatus. HIV-1 DNA was quantified in parallel with CCR5 as a reference gene, and reported values are numbers of HIV-1 DNA copies/10(6) peripheral blood mononuclear cells (PBMCs). The clinical sensitivity of the assay was assessed for 115 newly diagnosed HIV-1-infected individuals. The analytical sensitivity was estimated to be 12.5 copies of HIV-1 DNA per 10(6) PBMCs, while the clinical sensitivity was 100%, with levels ranging from 1.23 to 4.25 log(10) HIV-1 DNA copies/10(6) PBMCs. In conclusion, we developed and assessed a new RTMP-HIV assay based on molecular beacons, using a LightCycler 2.0 instrument. This multiplex assay has comparable sensitivity, reproducibility, and accuracy to single real-time PCR assays.

  11. Identification of person and quantification of human DNA recovered from mosquitoes (Culicidae).

    Science.gov (United States)

    Curic, Goran; Hercog, Rajna; Vrselja, Zvonimir; Wagner, Jasenka

    2014-01-01

    Mosquitoes (Culicidae) are widespread insects and can be important in forensic context as a source of human DNA. In order to establish the quantity of human DNA in mosquitoes' gut after different post-feeding interval and for how long after taking a bloodmeal the human donor could be identified, 174 blood-engorged mosquitoes (subfamily Anophelinae and Culicinae) were captured, kept alive and sacrificed at 8h intervals. Human DNA was amplified using forensic PCR kits (Identifiler, MiniFiler, and Quantifiler). A full DNA profiles were obtained from all Culicinae mosquitoes (74/74) up to 48 h and profiling was successful up to 88 h after a bloodmeal. Duration of post-feeding interval had a significant negative effect on the possibility of obtaining a full profile (pmosquitoes are a suitable source of human DNA for forensic STR kits more than three days after a bloodmeal. Human DNA recovered from mosquito can be used for matching purposes and could be useful in revealing spatial and temporal relation of events that took place at the crime scene. Therefore, mosquitoes at the crime scene, dead or alive, could be a valuable piece of forensic evidence.

  12. Quantification of mitochondrial DNA in human blood cells using an automated detection system.

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    Meissner, C; Mohamed, S A; Klueter, H; Hamann, K; von Wurmb, N; Oehmichen, M

    2000-09-11

    The 4977 bp deletion of mitochondrial DNA (mtDNA) accumulates in postmitotic tissues with advancing age. The purpose of our study was to detect and quantify these deletion even in blood cells with a high turnover activity. Whole venous blood, isolated human platelets and peripheral blood mononuclear cells (PBMCs) were collected from 10 unrelated donors aged 20-71 years and total DNA was extracted. PCR was performed for total and mutated mtDNA using two different primer pairs and two fluorogenic probes labeled with the fluorescent dyes FAM and VIC. Specific PCR products were generated, detected and quantified in a real-time PCR. The amplification products of total and deleted mtDNA could be detected in each sample and did not exhibit any differences in the amount of the deleted mtDNA in whole blood, human platelets or PBMCs. Our data did not show any accumulation of the 4977 bp deletion with increasing age as it was observed for several other tissues.

  13. Human Cytomegalovirus DNA Quantification and Gene Expression in Gliomas of Different Grades

    Science.gov (United States)

    Medeiros, Raphael Salles Scortegagna; Guerra, Juliana Mariotti; Kimura, Lidia Midori; Shirata, Neuza Kazumi; Nonogaki, Suely; dos Santos, Claudia Januário; Carlan Silva, Maria Cristina

    2016-01-01

    Gliomas are the most common type of primary brain tumors. The most aggressive type, Glioblastoma multiforme (GBM), is one of the deadliest human diseases, with an average survival at diagnosis of about 1 year. Previous evidence suggests a link between human cytomegalovirus (HCMV) and gliomas. HCMV has been shown to be present in these tumors and several viral proteins can have oncogenic properties in glioma cells. Here we have investigated the presence of HCMV DNA, RNA and proteins in fifty-two gliomas of different grades of malignancy. The UL83 viral region, the early beta 2.7 RNA and viral protein were detected in 73%, 36% and 57% by qPCR, ISH and IHC, respectively. Positivity of the viral targets and viral load was independent of tumor type or grade suggesting no correlation between viral presence and tumor progression. Our results demonstrate high prevalence of the virus in gliomas from Brazilian patients, contributing to a better understanding of the association between HCMV infection and gliomas worldwide and supporting further investigations of the virus oncomodulatory properties. PMID:27458810

  14. Micro-RNA quantification using DNA polymerase and pyrophosphate quantification.

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    Yu, Hsiang-Ping; Hsiao, Yi-Ling; Pan, Hung-Yin; Huang, Chih-Hung; Hou, Shao-Yi

    2011-12-15

    A rapid quantification method for micro-RNA based on DNA polymerase activity and pyrophosphate quantification has been developed. The tested micro-RNA serves as the primer, unlike the DNA primer in all DNA sequencing methods, and the DNA probe serves as the template for DNA replication. After the DNA synthesis, the pyrophosphate detection and quantification indicate the existence and quantity of the tested miRNA. Five femtomoles of the synthetic RNA could be detected. In 20-100 μg RNA samples purified from SiHa cells, the measurement was done using the proposed assay in which hsa-miR-16 and hsa-miR-21 are 0.34 fmol/μg RNA and 0.71 fmol/μg RNA, respectively. This simple and inexpensive assay takes less than 5 min after total RNA purification and preparation. The quantification is not affected by the pre-miRNA which cannot serve as the primer for the DNA synthesis in this assay. This assay is general for the detection of the target RNA or DNA with a known matched DNA template probe, which could be widely used for detection of small RNA, messenger RNA, RNA viruses, and DNA. Therefore, the method could be widely used in RNA and DNA assays. Copyright © 2011 Elsevier Inc. All rights reserved.

  15. Quantitative real-time PCR (qPCR) assay for human-dog-cat species identification and nuclear DNA quantification.

    Science.gov (United States)

    Kanthaswamy, S; Premasuthan, A; Ng, J; Satkoski, J; Goyal, V

    2012-03-01

    In the United States, human forensic evidence collected from crime scenes is usually comingled with biomaterial of canine and feline origins. Knowledge of the concentration of nuclear DNA extracted from a crime scene biological sample and the species from which the sample originated is essential for DNA profiling. The ability to accurately detect and quantify target DNA in mixed-species samples is crucial when target DNA may be overwhelmed by non-target DNA. We have designed and evaluated a species-specific (human, dog and cat) nuclear DNA identification assay based on the TaqMan(®) quantitative real-time PCR (qPCR) technology that can simultaneously detect and measure minute quantities of DNA specific to either humans, dogs and/or cats. The fluorogenic triplex assay employs primers and hydrolysis probes that target the human TH01 locus as well as the dog and cat Melanocortin 1 Receptor (MC1R) sequences in a species-specific manner. We also demonstrate that the assay is a highly sensitive, reliable and robust method for identifying and quantifying mixed-species templates of human-dog-cat origin with as little as 0.4 pg of human and cat nuclear DNA, respectively, and 4.0 pg of dog nuclear DNA.

  16. Automated Template Quantification for DNA Sequencing Facilities

    Science.gov (United States)

    Ivanetich, Kathryn M.; Yan, Wilson; Wunderlich, Kathleen M.; Weston, Jennifer; Walkup, Ward G.; Simeon, Christian

    2005-01-01

    The quantification of plasmid DNA by the PicoGreen dye binding assay has been automated, and the effect of quantification of user-submitted templates on DNA sequence quality in a core laboratory has been assessed. The protocol pipets, mixes and reads standards, blanks and up to 88 unknowns, generates a standard curve, and calculates template concentrations. For pUC19 replicates at five concentrations, coefficients of variance were 0.1, and percent errors were from 1% to 7% (n = 198). Standard curves with pUC19 DNA were nonlinear over the 1 to 1733 ng/μL concentration range required to assay the majority (98.7%) of user-submitted templates. Over 35,000 templates have been quantified using the protocol. For 1350 user-submitted plasmids, 87% deviated by ≥ 20% from the requested concentration (500 ng/μL). Based on data from 418 sequencing reactions, quantification of user-submitted templates was shown to significantly improve DNA sequence quality. The protocol is applicable to all types of double-stranded DNA, is unaffected by primer (1 pmol/μL), and is user modifiable. The protocol takes 30 min, saves 1 h of technical time, and costs approximately $0.20 per unknown. PMID:16461949

  17. Chrysotile effects on human lung cell carcinoma in culture: 3-D reconstruction and DNA quantification by image analysis

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    Machado-Santelli Glaucia M

    2008-06-01

    Full Text Available Abstract Background Chrysotile is considered less harmful to human health than other types of asbestos fibers. Its clearance from the lung is faster and, in comparison to amphibole forms of asbestos, chrysotile asbestos fail to accumulate in the lung tissue due to a mechanism involving fibers fragmentation in short pieces. Short exposure to chrysotile has not been associated with any histopathological alteration of lung tissue. Methods The present work focuses on the association of small chrysotile fibers with interphasic and mitotic human lung cancer cells in culture, using for analyses confocal laser scanning microscopy and 3D reconstructions. The main goal was to perform the analysis of abnormalities in mitosis of fibers-containing cells as well as to quantify nuclear DNA content of treated cells during their recovery in fiber-free culture medium. Results HK2 cells treated with chrysotile for 48 h and recovered in additional periods of 24, 48 and 72 h in normal medium showed increased frequency of multinucleated and apoptotic cells. DNA ploidy of the cells submitted to the same chrysotile treatment schedules showed enhanced aneuploidy values. The results were consistent with the high frequency of multipolar spindles observed and with the presence of fibers in the intercellular bridge during cytokinesis. Conclusion The present data show that 48 h chrysotile exposure can cause centrosome amplification, apoptosis and aneuploid cell formation even when long periods of recovery were provided. Internalized fibers seem to interact with the chromatin during mitosis, and they could also interfere in cytokinesis, leading to cytokinesis failure which forms aneuploid or multinucleated cells with centrosome amplification.

  18. Optimizing a Method for the Quantification by Quantitative Real-Time Polymerase Chain Reaction of Host Cell DNA in Plasmid Vector Batches Used in Human Gene Therapy.

    Science.gov (United States)

    Ferro, Serge; Fabre, Isabelle; Chenivesse, Xavier

    2016-08-01

    Gene therapy products are very complex advanced therapy medicinal products produced using different processes that require many chemical and biological reagents and production intermediates, such as producing cells. The quantification of residual impurities in gene therapy vectors is a major quality control step when these vectors are used for therapeutic purposes, whether or not they are derived from viruses. Indeed, in nonviral gene therapy products, particularly plasmid vectors used to transfer genetic material, the presence of host-cell DNA (HCDNA) from the bacterial cells used for the vector production is an important concern because of the risk of immunogenicity and insertional mutagenesis. Several methods have been developed to quantify residual HCDNA, but real-time quantitative polymerase chain reaction (qPCR) seems to be most suitable because it allows detecting traces of "contaminating" DNA. The French National Agency for Medicines and Health Products Safety (ANSM) ensures the quality and safety of gene transfer medicinal products and must be able to quantify, in its own laboratories, the amount of HCDNA present in plasmid vector batches. Therefore, we developed and validated a qPCR method to quantify at the femtogram level the presence of Escherichia coli residual DNA in plasmid vectors. This approach uses the capillary-based LightCycler 1.5 System (Roche) with SYBR Green I, a primer pair against the E. coli 23S ribosomal RNA gene and different concentrations of a linearized plasmid that contains the 23S target sequence, as standard. This qPCR method is linear on an 8-decade logarithmic scale, accurate, reproducible, and sensitive (quantification of up to 10 copies of 23S target sequence per reaction, or 1.4 E. coli genome, or 7 fg of bacterial DNA). This technique allows ensuring that batches of plasmid vectors to be used in clinical trials comply with the specifications on HCDNA content.

  19. Mutant DNA quantification by digital PCR can be confounded by heating during DNA fragmentation.

    Science.gov (United States)

    Kang, Qing; Parkin, Brian; Giraldez, Maria D; Tewari, Muneesh

    2016-04-01

    Digital PCR (dPCR) is gaining popularity as a DNA mutation quantification method for clinical specimens. Fragmentation prior to dPCR is required for non-fragmented genomic DNA samples; however, the effect of fragmentation on DNA analysis has not been well-studied. Here we evaluated three fragmentation methods for their effects on dPCR point mutation assay performance. Wild-type (WT) human genomic DNA was fragmented by heating, restriction digestion, or acoustic shearing using a Covaris focused-ultrasonicator. dPCR was then used to determine the limit of blank (LoB) by quantifying observed WT and mutant allele counts of the proto-oncogenes KRAS and BRAF in the WT DNA sample. DNA fragmentation by heating to 95°C, while the simplest and least expensive method, produced a high background mutation frequency for certain KRAS mutations relative to the other methods. This was due to heat-induced mutations, specifically affecting dPCR assays designed to interrogate guanine to adenine (G>A) mutations. Moreover, heat-induced fragmentation overestimated gene copy number, potentially due to denaturation and partition of single-stranded DNA into different droplets. Covaris acoustic shearing and restriction enzyme digestion showed similar LoBs and gene copy number estimates to one another. It should be noted that moderate heating, commonly used in genomic DNA extraction protocols, did not significantly increase observed KRAS mutation counts.

  20. Development of a Broad-Range 23S rDNA Real-Time PCR Assay for the Detection and Quantification of Pathogenic Bacteria in Human Whole Blood and Plasma Specimens

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    Paolo Gaibani

    2013-01-01

    Full Text Available Molecular methods are important tools in the diagnosis of bloodstream bacterial infections, in particular in patients treated with antimicrobial therapy, due to their quick turn-around time. Here we describe a new broad-range real-time PCR targeting the 23S rDNA gene and capable to detect as low as 10 plasmid copies per reaction of targeted bacterial 23S rDNA gene. Two commercially available DNA extraction kits were evaluated to assess their efficiency for the extraction of plasma and whole blood samples spiked with different amount of either Staphylococcus aureus or Escherichia coli, in order to find the optimal extraction method to be used. Manual QIAmp extraction method with enzyme pre-treatment resulted the most sensitive for detection of bacterial load. Sensitivity of this novel assay ranged between 10 and 103 CFU per PCR reaction for E. coli and S. aureus in human whole blood samples depending on the extraction methods used. Analysis of plasma samples showed a 10- to 100-fold reduction of bacterial 23S rDNA in comparison to the corresponding whole blood specimens, thus indicating that whole blood is the preferential sample type to be used in this real-time PCR protocol. Our results thus show that the 23S rDNA gene represents an optimal target for bacteria quantification in human whole blood.

  1. Real-Time PCR Quantification of Chloroplast DNA Supports DNA Barcoding of Plant Species.

    Science.gov (United States)

    Kikkawa, Hitomi S; Tsuge, Kouichiro; Sugita, Ritsuko

    2016-03-01

    Species identification from extracted DNA is sometimes needed for botanical samples. DNA quantification is required for an accurate and effective examination. If a quantitative assay provides unreliable estimates, a higher quantity of DNA than the estimated amount may be used in additional analyses to avoid failure to analyze samples from which extracting DNA is difficult. Compared with conventional methods, real-time quantitative PCR (qPCR) requires a low amount of DNA and enables quantification of dilute DNA solutions accurately. The aim of this study was to develop a qPCR assay for quantification of chloroplast DNA from taxonomically diverse plant species. An absolute quantification method was developed using primers targeting the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene using SYBR Green I-based qPCR. The calibration curve was generated using the PCR amplicon as the template. DNA extracts from representatives of 13 plant families common in Japan. This demonstrates that qPCR analysis is an effective method for quantification of DNA from plant samples. The results of qPCR assist in the decision-making will determine the success or failure of DNA analysis, indicating the possibility of optimization of the procedure for downstream reactions.

  2. Pitfalls of DNA Quantification Using DNA-Binding Fluorescent Dyes and Suggested Solutions.

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    Yuki Nakayama

    Full Text Available The Qubit fluorometer is a DNA quantification device based on the fluorescence intensity of fluorescent dye binding to double-stranded DNA (dsDNA. Qubit is generally considered useful for checking DNA quality before next-generation sequencing because it measures intact dsDNA. To examine the most accurate and suitable methods for quantifying DNA for quality assessment, we compared three quantification methods: NanoDrop, which measures UV absorbance; Qubit; and quantitative PCR (qPCR, which measures the abundance of a target gene. For the comparison, we used three types of DNA: 1 DNA extracted from fresh frozen liver tissues (Frozen-DNA; 2 DNA extracted from formalin-fixed, paraffin-embedded liver tissues comparable to those used for Frozen-DNA (FFPE-DNA; and 3 DNA extracted from the remaining fractions after RNA extraction with Trizol reagent (Trizol-DNA. These DNAs were serially diluted with distilled water and measured using three quantification methods. For Frozen-DNA, the Qubit values were not proportional to the dilution ratio, in contrast with the NanoDrop and qPCR values. This non-proportional decrease in Qubit values was dependent on a lower salt concentration, and over 1 mM NaCl in the DNA solution was required for the Qubit measurement. For FFPE-DNA, the Qubit values were proportional to the dilution ratio and were lower than the NanoDrop values. However, electrophoresis revealed that qPCR reflected the degree of DNA fragmentation more accurately than Qubit. Thus, qPCR is superior to Qubit for checking the quality of FFPE-DNA. For Trizol-DNA, the Qubit values were proportional to the dilution ratio and were consistently lower than the NanoDrop values, similar to FFPE-DNA. However, the qPCR values were higher than the NanoDrop values. Electrophoresis with SYBR Green I and single-stranded DNA (ssDNA quantification demonstrated that Trizol-DNA consisted mostly of non-fragmented ssDNA. Therefore, Qubit is not always the most accurate method

  3. Effects of humic substances on fluorometric DNA quantification and DNA hybridization

    NARCIS (Netherlands)

    Bachoon, DS; Otero, E; Hodson, RE

    2001-01-01

    DNA extracts from sediment and water samples are often contaminated with coextracted humic-like impurities, Estuarine humic substances and vascular plant extract were used to evaluate the effect of the presence of such impurities on DNA hybridization and quantification. The presence of humic

  4. Construction and Evaluation of Cytomegalovirus DNA Quantification System with Real-Time Detection Polymerase Chain Reaction

    OpenAIRE

    Hatayama, Yuki; Hashimoto, Yuki; Hara, Ayako; Motokura, Toru

    2016-01-01

    Background For patients with reactivation of human cytomegalovirus (CMV), a highly sensitive and accurate CMV quantification system is essential to monitor viral load. Methods We constructed a real-time detection PCR (RTD-PCR) system for CMV DNA and evaluated its linearity, lower detection limit, dynamic range and accuracy using two CMV standards. We used 219 clinical samples derived from 101 patients to compare the system with the pp65 antigen test. Results The 95% detection limit was determ...

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

    Energy Technology Data Exchange (ETDEWEB)

    Jackson, Christopher B., E-mail: Christopher.jackson@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland); Gallati, Sabina, E-mail: sabina.gallati@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland); Schaller, Andre, E-mail: andre.schaller@insel.ch [Division of Human Genetics, Departements of Pediatrics and Clinical Research, Inselspital, University of Berne, Freiburgstrasse, CH-3010 Berne (Switzerland)

    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 serial 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 measurements in

  6. Identification and quantification of Lactobacillus casei strain Shirota in human feces with strain-specific primers derived from randomly amplified polymorphic DNA.

    Science.gov (United States)

    Fujimoto, Junji; Matsuki, Takahiro; Sasamoto, Masae; Tomii, Yasuaki; Watanabe, Koichi

    2008-08-15

    Lactobacillus casei strain Shirota (LcS) has been used in the production of fermented milk products for many years and is one of the most intensively studied probiotics. To evaluate the ability of LcS to proliferate in human intestines after it has been ingested, we developed a PCR-based method to identify and quantify LcS using an LcS-specific primer set (pLcS) derived from a randomly amplified polymorphic DNA (RAPD) analysis. We confirmed the high specificity of the pLcS primer set in 167 bacterial strains (57 strains of L. casei and 110 other strains of bacteria commonly isolated from human feces). The method's ability to identify LcS matched that of an ELISA using a monoclonal antibody and a RAPD analysis in a representative sample of colonies cultured from human feces. The detection limit of quantitative PCR (qPCR) using pLcS was 10(4.6) per gram of feces. The number of LcS in feces detected with qPCR was highly and significantly correlated with the number of LcS added to fecal samples within the range of 10(4.6) to 10(9.6) per gram feces (r(2)=0.999, P<0.001). After 14 healthy subjects ingested 10(11.0) CFU of LcS daily for 7 days, 10(9.1+/-0.5) LcS g(-1) (mean+/-S.D.) was detected in the fecal samples of all subjects by qPCR, and 10(8.0+/-0.9) CFU g(-1) was detected by culture; these values were significantly different (P<0.001, paired t-test). After the subjects stopped ingesting LcS, fecal LcS counts obtained with both methods decreased daily. The values produced by the 2 methods might have differed because of an overestimation in the PCR analysis due to the presence of dead LcS cells or an underestimation in the culture system due to the use of selective culture media; however, dead LcS cells can also be beneficial as immunomodulators. We confirmed that qPCR with an LcS-specific primer set was a rapid and accurate method for determining the total amount of LcS in feces including dead or less active cells which could not be detected by culture method.

  7. Optimal purification and sensitive quantification of DNA from fecal samples

    DEFF Research Database (Denmark)

    Jensen, Annette Nygaard; Hoorfar, Jeffrey

    2002-01-01

    Application of reliable, rapid and sensitive methods to laboratory diagnosis of zoonotic infections continues to challenge microbiological laboratories. The recovery of DNA from a swine fecal sample and a bacterial culture extracted by a conventional phenol-chloroform extraction method was compared......, the detection range of X-DNA of a spectrophotometric and a fluorometric (PicoGreen) method was compared. The PicoGreen showed a quantification limit of 1 ng/mL, consistent triplicate measurements, and finally a linear relationship between the concentrations of DNA standards and the fluorescence readings (R-2...... = 0.99 and R-2 = 1.00). In conclusion, silica-membrane, columns can provide a more convenient and less hazardous alternative to the conventional phenol-based method. The results have implication for further improvement of sensitive amplification methods for laboratory diagnosis....

  8. Measurement of DNA single-strand breaks by alkaline elution and fluorometric DNA quantification.

    Science.gov (United States)

    Goumenou, Marina; Machera, Kyriaki

    2004-03-15

    The method presented is based on the alkaline elution procedure for the determination of DNA single-stand (ss) breaks developed by Kohn and on the principles of DNA quantification after binding with the dye Hoechst 33258. In the present study, modification of the alkaline elution procedure with regard to the elution solution volume was performed. The influences of the DNA strandedness, the ethylenediaminetetraacetate/tetraethylammonium hydroxide denaturation and elution solution presence, the DNA solution pH, the dye amount, and the incubation time for the formation of the dye-ssDNA complex on the DNA fluorometric quantification were also studied. The modified DNA alkaline elution procedure followed by the optimized fluorometric determination of the ssDNA was applied on liver tissue from both untreated and treated (N-nitroso-N-methylurea- administered) Wistar rats. The criteria for the selection of the appropriate estimator and statistical analysis of the obtained results are also presented. The method of the DNA alkaline elution followed by fluorometric determination of ssDNA as modified and evaluated is an accurate and reliable approach for the determination of in vivo induced ssDNA strand breaks.

  9. Preanalytical Conditions and DNA Isolation Methods Affect Telomere Length Quantification in Whole Blood.

    Directory of Open Access Journals (Sweden)

    Alexander Tolios

    Full Text Available Telomeres are located at chromosome ends and their length (TL has been associated with aging and human diseases such as cancer. Whole blood DNA is frequently used for TL measurements but the influence of preanalytical conditions and DNA isolation methods on TL quantification has not been thoroughly investigated. To evaluate potential preanalytical as well as methodological bias on TL, anonymized leftover EDTA-whole blood samples were pooled according to leukocyte counts and were incubated with and without actinomycin D to induce apoptosis as a prototype of sample degradation. DNA was isolated from fresh blood pools and after freezing at -80°C. Commercially available kits using beads (Invitrogen, spin columns (Qiagen, Macherey-Nagel and 5prime or precipitation (Stratec/Invisorb and a published isopropanol precipitation protocol (IPP were used for DNA isolation. TL was assessed by qPCR, and normalized to the single copy reference gene 36B4 using two established single-plex and a new multiplex protocol. We show that the method of DNA isolation significantly affected TL (e.g. 1.86-fold longer TL when comparing IPP vs. Invitrogen. Sample degradation led to an average TL decrease of 22% when using all except for one DNA isolation method (5prime. Preanalytical storage conditions did not affect TL with exception of samples that were isolated with the 5prime kit, where a 27% increase in TL was observed after freezing. Finally, performance of the multiplex qPCR protocol was comparable to the single-plex assays, but showed superior time- and cost-effectiveness and required > 80% less DNA. Findings of the current study highlight the need for standardization of whole blood processing and DNA isolation in clinical study settings to avoid preanalytical bias of TL quantification and show that multiplex assays may improve TL/SCG measurements.

  10. DNA isolation and sample preparation for quantification of adduct levels by accelerator mass spectrometry.

    Science.gov (United States)

    Dingley, Karen H; Ubick, Esther A; Vogel, John S; Ognibene, Ted J; Malfatti, Michael A; Kulp, Kristen; Haack, Kurt W

    2014-01-01

    Accelerator mass spectrometry (AMS) is a highly sensitive technique used for the quantification of adducts following exposure to carbon-14- or tritium-labeled chemicals, with detection limits in the range of one adduct per 10(11)-10(12) nucleotides. The protocol described in this chapter provides an optimal method for isolating and preparing DNA samples to measure isotope-labeled DNA adducts by AMS. When preparing samples, special precautions must be taken to avoid cross-contamination of isotope among samples and produce a sample that is compatible with AMS. The DNA isolation method described is based upon digestion of tissue with proteinase K, followed by extraction of DNA using Qiagen isolation columns. The extracted DNA is precipitated with isopropanol, washed repeatedly with 70 % ethanol to remove salt, and then dissolved in water. DNA samples are then converted to graphite or titanium hydride and the isotope content measured by AMS to quantify adduct levels. This method has been used to reliably generate good yields of uncontaminated, pure DNA from animal and human tissues for analysis of adduct levels.

  11. Comparison of DNA quantification methodology used in the DNA extraction protocol for the UK Biobank cohort.

    Science.gov (United States)

    Welsh, Samantha; Peakman, Tim; Sheard, Simon; Almond, Rachael

    2017-01-05

    UK Biobank is a large prospective cohort study in the UK established by the Medical Research Council (MRC) and the Wellcome Trust to enable approved researchers to investigate the role of genetic factors, environmental exposures and lifestyle in the causes of major diseases of late and middle age. A wide range of phenotypic data has been collected at recruitment and has recently been enhanced by the UK Biobank Genotyping Project. All UK Biobank participants (500,000) have been genotyped on either the UK Biobank Axiom® Array or the Affymetrix UK BiLEVE Axiom® Array and the workflow for preparing samples for genotyping is described. The genetic data is hoped to provide further insight into the genetics of disease. All data, including the genetic data, is available for access to approved researchers. Data for two methods of DNA quantification (ultraviolet-visible spectroscopy [UV/Vis]) measured on the Trinean DropSense™ 96 and PicoGreen®) were compared by two laboratories (UK Biobank and Affymetrix). The sample processing workflow established at UK Biobank, for genotyping on the custom Affymetrix Axiom® array, resulted in high quality DNA (average DNA concentration 38.13 ng/μL, average 260/280 absorbance 1.91). The DNA generated high quality genotype data (average call rate 99.48% and pass rate 99.45%). The DNA concentration measured on the Trinean DropSense™ 96 at UK Biobank correlated well with DNA concentration measured by PicoGreen® at Affymetrix (r = 0.85). The UK Biobank Genotyping Project demonstrated that the high throughput DNA extraction protocol described generates high quality DNA suitable for genotyping on the Affymetrix Axiom array. The correlation between DNA concentration derived from UV/Vis and PicoGreen® quantification methods suggests, in large-scale genetic studies involving two laboratories, it may be possible to remove the DNA quantification step in one laboratory without affecting downstream analyses. This would result in

  12. Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms

    Directory of Open Access Journals (Sweden)

    Žel Jana

    2006-08-01

    Full Text Available Abstract Background Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Results Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was

  13. Critical points of DNA quantification by real-time PCR – effects of DNA extraction method and sample matrix on quantification of genetically modified organisms

    Science.gov (United States)

    Cankar, Katarina; Štebih, Dejan; Dreo, Tanja; Žel, Jana; Gruden, Kristina

    2006-01-01

    Background Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Results Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary

  14. Critical points of DNA quantification by real-time PCR--effects of DNA extraction method and sample matrix on quantification of genetically modified organisms.

    Science.gov (United States)

    Cankar, Katarina; Stebih, Dejan; Dreo, Tanja; Zel, Jana; Gruden, Kristina

    2006-08-14

    Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary criterion by which to

  15. A direct droplet digital PCR method for quantification of residual DNA in protein drugs produced in yeast cells.

    Science.gov (United States)

    Hussain, Musaddeq; Fantuzzo, Rebecca; Mercorelli, Suzanne; Cullen, Constance

    2016-05-10

    Yeast cells, in particular Pichia pastoris, are the host cell of choice for manufacturing several protein therapeutic agents in the biopharmaceutical industry. Host cell DNA is an impurity of such manufacturing process and the residual DNA after the purification process of the drug must be monitored to ensure drug purity and safety. Currently, real-time PCR (qPCR) based methods are widely employed for quantification of host residual DNA. At the same time the digital PCR technology is coming into prominence with promise of higher sensitivity. Here we report a method where the protein drug is directly added to the droplet digital PCR (ddPCR) reaction including yeast-specific primers and fluorescent-tagged probe and nanoliter-sized droplets are generated. The droplets are then subjected to PCR followed by analysis for fluorescence. This Pichia residual DNA direct ddPCR method for yeast can be used to test higher amount of drug compared to the corresponding qPCR method thereby increasing sensitivity, retaining high precision and accuracy and has a wide linear range of determination. The method has been successfully tested with three batches of a recombinant human IgG1-Fc-based drug (RP-1) and with commercially available human insulin, both manufactured in yeast cells. This method simplifies the residual DNA quantification protocol by eliminating DNA extraction or protease digestion and eliminates use of DNA standards in day-to-day running of the method.

  16. Stereological quantification of mast cells in human synovium

    DEFF Research Database (Denmark)

    Damsgaard, T E; Sørensen, Flemming Brandt; Herlin, T;

    1999-01-01

    Mast cells participate in both the acute allergic reaction as well as in chronic inflammatory diseases. Earlier studies have revealed divergent results regarding the quantification of mast cells in the human synovium. The aim of the present study was therefore to quantify these cells in the human...... synovium, using stereological techniques. Different methods of staining and quantification have previously been used for mast cell quantification in human synovium. Stereological techniques provide precise and unbiased information on the number of cell profiles in two-dimensional tissue sections of......, in this case, human synovium. In 10 patients suffering from osteoarthritis a median of 3.6 mast cells/mm2 synovial membrane was found. The total number of cells (synoviocytes, fibroblasts, lymphocytes, leukocytes) present was 395.9 cells/mm2 (median). The mast cells constituted 0.8% of all the cell profiles...

  17. Detection and quantification of 4-ABP adducts in DNA from bladder cancer patients.

    Science.gov (United States)

    Zayas, Beatriz; Stillwell, Sara W; Wishnok, John S; Trudel, Laura J; Skipper, Paul; Yu, Mimi C; Tannenbaum, Steven R; Wogan, Gerald N

    2007-02-01

    We analyzed bladder DNA from 27 cancer patients for dG-C8-4-aminobiphenyl (dG-C8-ABP) adducts using the liquid chromatography tandem mass spectrometry method with a 700 attomol (1 adduct in 10(9) bases) detection limit. Hemoglobin (Hb) 4-aminobiphenyl (4-ABP) adduct levels were measured by gas chromatography-mass spectrometry. After isolation of dG-C8-ABP by immunoaffinity chromatography and further purification, deuterated (d9) dG-C8-ABP (MW=443 Da) was added to each sample. Structural evidence and adduct quantification were determined by selected reaction monitoring, based on the expected adduct ion [M+H+]+1, at m/z 435 with fragmentation to the product ion at m/z 319, and monitoring of the transition for the internal standard, m/z 444-->328. The method was validated by analysis of DNA (100 microg each) from calf thymus; livers from ABP-treated and untreated rats; human placentas; and TK6 lymphoblastoid cells. Adduct was detected at femtomol levels in DNA from livers of ABP-treated rats and calf thymus, but not in other controls. The method was applied to 41 DNA samples (200 microg each) from 27 human bladders; 28 from tumor and 14 from surrounding non-tumor tissue. Of 27 tissues analyzed, 44% (12) contained 5-80 dG-C8-ABP adducts per 10(9) bases; only 1 out of 27 (4%) contained adduct in both tumor and surrounding tissues. The Hb adduct was detected in samples from all patients, at levels of 12-1960 pg per gram Hb. There was no correlation between levels of DNA and Hb adducts. The presence of DNA adducts in 44% of the subjects and high levels of Hb adducts in these non-smokers indicate environmental sources of exposure to 4-ABP.

  18. Performance of the Real-Q EBV Quantification Kit for Epstein-Barr Virus DNA Quantification in Whole Blood.

    Science.gov (United States)

    Huh, Hee Jae; Park, Jong Eun; Kim, Ji Youn; Yun, Sun Ae; Lee, Myoung Keun; Lee, Nam Yong; Kim, Jong Won; Ki, Chang Seok

    2017-03-01

    There has been increasing interest in standardized and quantitative Epstein-Barr virus (EBV) DNA testing for the management of EBV disease. We evaluated the performance of the Real-Q EBV Quantification Kit (BioSewoom, Korea) in whole blood (WB). Nucleic acid extraction and real-time PCR were performed by using the MagNA Pure 96 (Roche Diagnostics, Germany) and 7500 Fast real-time PCR system (Applied Biosystems, USA), respectively. Assay sensitivity, linearity, and conversion factor were determined by using the World Health Organization international standard diluted in EBV-negative WB. We used 81 WB clinical specimens to compare performance of the Real-Q EBV Quantification Kit and artus EBV RG PCR Kit (Qiagen, Germany). The limit of detection (LOD) and limit of quantification (LOQ) for the Real-Q kit were 453 and 750 IU/mL, respectively. The conversion factor from EBV genomic copies to IU was 0.62. The linear range of the assay was from 750 to 10⁶ IU/mL. Viral load values measured with the Real-Q assay were on average 0.54 log₁₀ copies/mL higher than those measured with the artus assay. The Real-Q assay offered good analytical performance for EBV DNA quantification in WB.

  19. Relationship between DAPI-fluorescence fading and nuclear DNA content: An alternative method to DNA quantification?

    Science.gov (United States)

    Gallardo-Escárate, Cristian; Alvarez-Borrego, Josué; Von Brand, Elisabeth; Dupré, Enrique; Del Río-Portilla, Miguel Angel

    2007-01-01

    In observations by confocal or conventional fluorescence microscopy, important factors should be considered in order to obtain accurate images. One of them, such as the fluorescence bleaching from highest intensity to lowest signal of fluorescence is a common problem with several DNA fluorochromes and especially for DAPI stain. The fluorescence of DAPI fades rapidly when it is exposed to UV light, under optimal conditions of observation. Although the fading process can be retarded using a mounting medium with antifading reagents, the photochemical process underlying the fluorescence decay has not yet been fully explained. In addition, no relationship between fluorescence fading and nuclear DNA content has been tested. In order to test this relationship, we measured by means of image analysis the DAPI-fluorescence intensity in several cellular types (spermatozoa, erythrocytes and haemocytes) during their fluorescence bleaching. An algorithm specifically built in MATLAB software was used for this approach. The correlation coefficient between nuclear DNA content and DAPI-fluorescence fading was found equal to 99%. This study demonstrates the feasibility to measure nuclear DNA content by fluorescence fading quantification, as an alternative method concurrently with image analysis procedures.

  20. Accurate quantification of microRNA via single strand displacement reaction on DNA origami motif.

    Directory of Open Access Journals (Sweden)

    Jie Zhu

    Full Text Available DNA origami is an emerging technology that assembles hundreds of staple strands and one single-strand DNA into certain nanopattern. It has been widely used in various fields including detection of biological molecules such as DNA, RNA and proteins. MicroRNAs (miRNAs play important roles in post-transcriptional gene repression as well as many other biological processes such as cell growth and differentiation. Alterations of miRNAs' expression contribute to many human diseases. However, it is still a challenge to quantitatively detect miRNAs by origami technology. In this study, we developed a novel approach based on streptavidin and quantum dots binding complex (STV-QDs labeled single strand displacement reaction on DNA origami to quantitatively detect the concentration of miRNAs. We illustrated a linear relationship between the concentration of an exemplary miRNA as miRNA-133 and the STV-QDs hybridization efficiency; the results demonstrated that it is an accurate nano-scale miRNA quantifier motif. In addition, both symmetrical rectangular motif and asymmetrical China-map motif were tested. With significant linearity in both motifs, our experiments suggested that DNA Origami motif with arbitrary shape can be utilized in this method. Since this DNA origami-based method we developed owns the unique advantages of simple, time-and-material-saving, potentially multi-targets testing in one motif and relatively accurate for certain impurity samples as counted directly by atomic force microscopy rather than fluorescence signal detection, it may be widely used in quantification of miRNAs.

  1. Accurate Quantification of microRNA via Single Strand Displacement Reaction on DNA Origami Motif

    Science.gov (United States)

    Lou, Jingyu; Li, Weidong; Li, Sheng; Zhu, Hongxin; Yang, Lun; Zhang, Aiping; He, Lin; Li, Can

    2013-01-01

    DNA origami is an emerging technology that assembles hundreds of staple strands and one single-strand DNA into certain nanopattern. It has been widely used in various fields including detection of biological molecules such as DNA, RNA and proteins. MicroRNAs (miRNAs) play important roles in post-transcriptional gene repression as well as many other biological processes such as cell growth and differentiation. Alterations of miRNAs' expression contribute to many human diseases. However, it is still a challenge to quantitatively detect miRNAs by origami technology. In this study, we developed a novel approach based on streptavidin and quantum dots binding complex (STV-QDs) labeled single strand displacement reaction on DNA origami to quantitatively detect the concentration of miRNAs. We illustrated a linear relationship between the concentration of an exemplary miRNA as miRNA-133 and the STV-QDs hybridization efficiency; the results demonstrated that it is an accurate nano-scale miRNA quantifier motif. In addition, both symmetrical rectangular motif and asymmetrical China-map motif were tested. With significant linearity in both motifs, our experiments suggested that DNA Origami motif with arbitrary shape can be utilized in this method. Since this DNA origami-based method we developed owns the unique advantages of simple, time-and-material-saving, potentially multi-targets testing in one motif and relatively accurate for certain impurity samples as counted directly by atomic force microscopy rather than fluorescence signal detection, it may be widely used in quantification of miRNAs. PMID:23990889

  2. A simple identification method for vaginal secretions using relative quantification of Lactobacillus DNA.

    Science.gov (United States)

    Doi, Masanori; Gamo, Shinsuke; Okiura, Tatsuyuki; Nishimukai, Hiroaki; Asano, Migiwa

    2014-09-01

    In criminal investigations there are some cases in which identifying the presence of vaginal secretions provides crucial evidence in proving sexual assault. However, there are no methods for definitively identifying vaginal secretions. In the present study, we focused on Lactobacillus levels in vaginal secretions and developed a novel identification method for vaginal secretions by relative quantification based on real time PCR. We designed a Lactobacillus conserved region primer pair (LCP) by aligning 16S rRNA gene sequences from major vaginal Lactobacillus species (Lactobacillus crispatus, Lactobacillus gasseri, Lactobacillus iners and Lactobacillus jensenii), and selected the human specific primer pair (HSP) as an endogenous control for relative quantification. As a result, the ΔCt (ΔCt=Ct[LCP]-Ct[HSP]) values of vaginal secretions (11 out of 12 samples) were significantly lower than those of saliva, semen and skin surface samples, and it was possible to discriminate between vaginal secretions and other body fluids. For the one remaining sample, it was confirmed that the predominant species in the microflora was not of the Lactobacillus genus. The ΔCt values in this study were calculated when the total DNA input used from the vaginal secretions was 10pg or more. Additionally, the ΔCt values of samples up to 6-months-old, which were kept at room temperature, remained unchanged. Thus, we concluded in this study that the simple ΔCt method by real time PCR is a useful tool for detecting the presence of vaginal secretions.

  3. Simultaneous quantification of sialyloligosaccharides from human milk by capillary electrophoresis

    OpenAIRE

    2007-01-01

    The acidic oligosaccharides of human milk are predominantly sialyloligosaccharides. Pathogens that bind sialic acid-containing glycans on their host mucosal surfaces may be inhibited by human milk sialyloligosaccharides, but testing this hypothesis requires their reliable quantification in milk. Sialyloligosaccharides have been quantified by anion exchange HPLC, reverse or normal phase HPLC, and capillary electrophoresis (CE) of fluorescent derivatives; in milk, these oligosaccharides have be...

  4. Quantification of cellular uptake of DNA nanostructures by qPCR.

    Science.gov (United States)

    Okholm, Anders Hauge; Nielsen, Jesper Sejrup; Vinther, Mathias; Sørensen, Rasmus Schøler; Schaffert, David; Kjems, Jørgen

    2014-05-15

    DNA nanostructures facilitating drug delivery are likely soon to be realized. In the past few decades programmed self-assembly of DNA building blocks have successfully been employed to construct sophisticated nanoscale objects. By conjugating functionalities to DNA, other molecules such as peptides, proteins and polymers can be precisely positioned on DNA nanostructures. This exceptional ability to produce modular nanoscale devices with tunable and controlled behavior has initiated an interest in employing DNA nanostructures for drug delivery. However, to obtain this the relationship between cellular interactions and structural and functional features of the DNA delivery device must be thoroughly investigated. Here, we present a rapid and robust method for the precise quantification of the component materials of DNA origami structures capable of entering cells in vitro. The quantification is performed by quantitative polymerase chain reaction, allowing a linear dynamic range of detection of five orders of magnitude. We demonstrate the use of this method for high-throughput screening, which could prove efficient to identify key features of DNA nanostructures enabling cell penetration. The method described here is suitable for quantification of in vitro uptake studies but should easily be extended to quantify DNA nanostructures in blood or tissue samples.

  5. Controls to validate plasma samples for cell free DNA quantification

    DEFF Research Database (Denmark)

    Pallisgaard, Niels; Spindler, Karen-Lise Garm; Andersen, Rikke Fredslund;

    2015-01-01

    Recent research has focused on the utility of cell free DNA (cfDNA) in serum and plasma for clinical application, especially in oncology. The literature holds promise of cfDNA as a valuable tumour marker to be used for treatment selection, monitoring and follow-up. The results, however, are diver...

  6. Quantification of human-structure interaction

    Directory of Open Access Journals (Sweden)

    Caprani Colin

    2015-01-01

    Full Text Available In lightweight structural systems there is increasing evidence that the presence of humans influences the dynamics characteristics of the system. In the past, most effort on determining the footfall-induced vertical force to the walking surface has been conducted using rigid or non-flexible surfaces such as treadmills. However, should the walking surface be vibrating, the characteristics of human walking could change to maximize comfort. This interaction between the structure and human may account for the discrepancy between the levels of vibration predicted by theory and those observed in practice. Indeed, many design rules can be seen to be conservative, perhaps partly because knowledge of this human-structure interaction is limited. This work aims to address this problem by quantifying the magnitude of human-structure interaction through a comprehensive experimental programme. Novel experimental techniques are used to measure the human-imparted force on the walking surface. Both rigid and flexible (vibrating surfaces are used, and we measure the imparted vibration response on a lively footbridge (the Warwick Bridge which acts as the flexible surface. A range of test subjects is considered, walking at a range of pacing frequencies. Comparison is made between a notional vibration response from the footfall force imparted to the rigid surface and the actual vibration response caused by the footfall force imparted to the flexible surface. Key aspects of the experimental regime are also explained. Finally, some comparisons are made using footfall force models from the literature. It is concluded that human-structure interaction is a key phenomenon that should be taken into account in the design and assessment of vibration-sensitive structures.

  7. Accurate episomal HIV 2-LTR circles quantification using optimized DNA isolation and droplet digital PCR

    Directory of Open Access Journals (Sweden)

    Eva Malatinkova

    2014-11-01

    Full Text Available Introduction: In HIV-infected patients on combination antiretroviral therapy (cART, the detection of episomal HIV 2-LTR circles is a potential marker for ongoing viral replication. Quantification of 2-LTR circles is based on quantitative PCR or more recently on digital PCR assessment, but is hampered due to its low abundance. Sample pre-PCR processing is a critical step for 2-LTR circles quantification, which has not yet been sufficiently evaluated in patient derived samples. Materials and Methods: We compared two sample processing procedures to more accurately quantify 2-LTR circles using droplet digital PCR (ddPCR. Episomal HIV 2-LTR circles were either isolated by genomic DNA isolation or by a modified plasmid DNA isolation, to separate the small episomal circular DNA from chromosomal DNA. This was performed in a dilution series of HIV-infected cells and HIV-1 infected patient derived samples (n=59. Samples for the plasmid DNA isolation method were spiked with an internal control plasmid. Results: Genomic DNA isolation enables robust 2-LTR circles quantification. However, in the lower ranges of detection, PCR inhibition caused by high genomic DNA load substantially limits the amount of sample input and this impacts sensitivity and accuracy. Moreover, total genomic DNA isolation resulted in a lower recovery of 2-LTR templates per isolate, further reducing its sensitivity. The modified plasmid DNA isolation with a spiked reference for normalization was more accurate in these low ranges compared to genomic DNA isolation. A linear correlation of both methods was observed in the dilution series (R2=0.974 and in the patient derived samples with 2-LTR numbers above 10 copies per million peripheral blood mononuclear cells (PBMCs, (R2=0.671. Furthermore, Bland–Altman analysis revealed an average agreement between the methods within the 27 samples in which 2-LTR circles were detectable with both methods (bias: 0.3875±1.2657 log10. Conclusions: 2-LTR

  8. One-step assay for the quantification of T4 DNA ligase.

    Science.gov (United States)

    Franke, Steffi; Kreisig, Thomas; Buettner, Karin; Zuchner, Thole

    2015-02-01

    As one of the most commonly used enzyme in molecular biology, the T4 DNA ligase presents an important tool for the manipulation of DNA. T4 DNA ligase activity measurements are based on the use of radioactivity or rather labor-intense procedures including gel-based analysis. We therefore established a homogeneous T4 DNA ligase assay utilizing a specifically designed fluorescein- and dark quencher-labeled DNA molecule. Upon ligation of both DNA molecules, a quenching occurs and the fluorescence intensity decreases with increasing ligase concentrations. The assay allows a sensitive and precise quantification (CV, 4.6-5.5 %) of T4 DNA ligase activities and showed a high specificity when tested against other ligases of related and different species. Most importantly, this T4 DNA ligase assay requires only one working and incubation step before measurement can take place at room temperature and may therefore offer an interesting alternative to existing, more laborious ligase assays.

  9. Quantification of phospholipids classes in human milk.

    Science.gov (United States)

    Giuffrida, Francesca; Cruz-Hernandez, Cristina; Flück, Brigitte; Tavazzi, Isabelle; Thakkar, Sagar K; Destaillats, Frédéric; Braun, Marcel

    2013-10-01

    Phospholipids are integral constituents of the milk fat globule membranes and they play a central role in infants' immune and inflammatory responses. A methodology employing liquid chromatography coupled with evaporative light scattering detector has been optimized and validated to quantify the major phospholipids classes in human milk. Phospholipids were extracted using chloroform and methanol and separated on C18 column. Repeatability, intermediate reproducibility, and recovery values were calculated and a large sample set of human milk analyzed. In human milk, phospholipid classes were quantified at concentrations of 0.6 mg/100 g for phosphatidylinositol; 4.2 mg/100 g for phosphatidylethanolamine, 0.4 mg/100 g for phosphatidylserine, 2.8 mg/100 g for phosphatidylcholine, and 4.6 mg/100 g for sphingomyelin. Their relative standard deviation of repeatability and intermediate reproducibility values ranging between 0.8 and 13.4 % and between 2.4 and 25.7 %, respectively. The recovery values ranged between 67 and 112 %. Finally, the validated method was used to quantify phospholipid classes in human milk collected from 50 volunteers 4 weeks postpartum providing absolute content of these lipids in a relatively large cohort. The average content of total phospholipids was 23.8 mg/100 g that corresponds to an estimated mean intake of 140 mg phospholipids/day in a 4-week old infant when exclusively breast-fed.

  10. A Novel Immunological Assay for Hepcidin Quantification in Human Serum

    OpenAIRE

    Vasiliki Koliaraki; Martha Marinou; Vassilakopoulos, Theodoros P.; Eustathios Vavourakis; Emmanuel Tsochatzis; Pangalis, Gerassimos A.; George Papatheodoridis; Alexandra Stamoulakatou; Dorine W Swinkels; George Papanikolaou; Avgi Mamalaki

    2009-01-01

    BACKGROUND: Hepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Increased hepcidin concentrations lead to iron sequestration in macrophages, contributing to the pathogenesis of anaemia of chronic disease whereas decreased hepcidin is observed in iron deficiency and primary iron overload diseases such as hereditary hemochromatosis. Hepcidin quantification in human blood or urine may provide further insights for the pathogenesis of disorders of iron homeostasis and might prove a v...

  11. Association of Global DNA Methylation and Global DNA Hydroxymethylation with Metals and Other Exposures in Human Blood DNA Samples

    Science.gov (United States)

    Tang, Wan-yee; Shang, Yan; Umans, Jason G.; Francesconi, Kevin A.; Goessler, Walter; Ledesma, Marta; Leon, Montserrat; Laclaustra, Martin; Pollak, Jonathan; Guallar, Eliseo; Cole, Shelley A.; Fallin, M. Dani; Navas-Acien, Ana

    2014-01-01

    Background: The association between human blood DNA global methylation and global hydroxymethylation has not been evaluated in population-based studies. No studies have evaluated environmental determinants of global DNA hydroxymethylation, including exposure to metals. Objective: We evaluated the association between global DNA methylation and global DNA hydroxymethylation in 48 Strong Heart Study participants for which selected metals had been measured in urine at baseline and DNA was available from 1989–1991 (visit 1) and 1998–1999 (visit 3). Methods: We measured the percentage of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in samples using capture and detection antibodies followed by colorimetric quantification. We explored the association of participant characteristics (i.e., age, adiposity, smoking, and metal exposure) with both global DNA methylation and global DNA hydroxymethylation. Results: The Spearman’s correlation coefficient for 5-mC and 5-hmC levels was 0.32 (p = 0.03) at visit 1 and 0.54 (p Ledesma M, Leon M, Laclaustra M, Pollak J, Guallar E, Cole SA, Fallin MD, Navas-Acien A. 2014. Association of global DNA methylation and global DNA hydroxymethylation with metals and other exposures in human blood DNA samples. Environ Health Perspect 122:946–954; http://dx.doi.org/10.1289/ehp.1306674 PMID:24769358

  12. DNA quantification of basidiomycetous fungi during storage of logging residues

    Directory of Open Access Journals (Sweden)

    Isabella Børja

    2015-04-01

    Full Text Available The demand for bioenergy caused an increased use of logging residues, branches and treetops that were previously left on the ground after harvesting. Residues are stored outdoors in piles and it is unclear to what extent fungi transform this material. Our objective was to quantify the amount of wood degrading fungi during storage using quantitative real-time PCR (qPCR to detect basidiomycetous DNA in logging residues, a novel approach in this field. We found that the qPCR method was accurate in quantifying the fungal DNA during storage. As the moisture content of the piled logging residues decreased during the storage period, the fungal DNA content also decreased. Scots pine residues contained more fungal DNA than residues from Norway spruce. Loose piles had generally more fungal DNA than bundled ones.

  13. Simultaneous quantification of sialyloligosaccharides from human milk by capillary electrophoresis.

    Science.gov (United States)

    Bao, Yuanwu; Zhu, Libin; Newburg, David S

    2007-11-15

    The acidic oligosaccharides of human milk are predominantly sialyloligosaccharides. Pathogens that bind sialic acid-containing glycans on their host mucosal surfaces may be inhibited by human milk sialyloligosaccharides, but testing this hypothesis requires their reliable quantification in milk. Sialyloligosaccharides have been quantified by anion exchange high-performance liquid chromatography (HPLC), reverse- or normal-phase HPLC, and capillary electrophoresis (CE) of fluorescent derivatives; in milk, these oligosaccharides have been analyzed by high pH anion exchange chromatography with pulsed amperometric detection and, in our laboratory, by CE with detection at 205nm. The novel method described here uses a running buffer of aqueous 200mM NaH2PO4 (pH 7.05) containing 100mM sodium dodecyl sulfate (SDS) mixed with 45% (v/v) methanol to baseline resolve 5 oligosaccharides and separate all 12. This allows automated simultaneous quantification of the 12 major sialyloligosaccharides of human milk in a single 35-min run. This method revealed differences in sialyloligosaccharide concentrations between less and more mature milk from the same donors. Individual donors also varied in expression of sialyloligosaccharides in their milk. Thus, the facile quantification of sialyloligosaccharides by this method is suitable for measuring variation in expression of specific sialyloligosaccharides in milk and their relationship to decreased risk of specific diseases in infants.

  14. A multistage volumetric bar chart chip for visualized quantification of DNA

    OpenAIRE

    Song, Yujun; Wang, Yuanchen; Qin, Lidong

    2013-01-01

    Nucleic acid detection is critical in disease diagnosis as well as in the environmental assays of harmful bacteria or viruses and forensic applications. Current methods for visualized quantification of DNA require costly and sophisticated instruments. Here, we report a multistage propelled volumetric bar chart chip (MV-Chip) for multiplexing and quantitative detection of DNA. Owing to its ‘rocket-like’ propelling reaction, the pre-deposited platinum films could perform cascade amplification a...

  15. How Severely Is DNA Quantification Hampered by RNA Co-extraction?

    Science.gov (United States)

    Sanchez, Ignacio; Remm, Matthieu; Frasquilho, Sonia; Betsou, Fay; Mathieson, William

    2015-10-01

    The optional RNase digest that is part of many DNA extraction protocols is often omitted, either because RNase is not provided in the kit or because users do not want to risk contaminating their laboratory. Consequently, co-eluting RNA can become a "contaminant" of unknown magnitude in a DNA extraction. We extracted DNA from liver, lung, kidney, and heart tissues and established that 28-52% of the "DNA" as assessed by spectrophotometry is actually RNA (depending on tissue type). Including an RNase digest in the extraction protocol reduced 260:280 purity ratios. Co-eluting RNA drives an overestimation of DNA yield when quantification is carried out using OD 260 nm spectrophotometry, or becomes an unquantified contaminant when spectrofluorometry is used for DNA quantification. This situation is potentially incompatible with the best practice guidelines for biobanks issued by organizations such as the International Society for Biological and Environmental Repositories, which state that biospecimens should be accurately characterized in terms of their identity, purity, concentration, and integrity. Consequently, we conclude that an RNase digest must be included in DNA extractions if pure DNA is required. We also discuss the implications of unquantified RNA contamination in DNA samples in the context of laboratory accreditation schemes.

  16. Label-free quantification of microRNAs using ligase-assisted sandwich hybridization on a DNA microarray.

    Directory of Open Access Journals (Sweden)

    Taro Ueno

    Full Text Available MicroRNAs (miRNAs can be used as biomarkers for cancer and other human diseases; therefore, high-throughput and reliable miRNA-quantification methods are required to exploit these markers for diagnostic testing. In this report, we describe the construction of a platform for miRNA-quantification using ligase-assisted sandwich hybridization (LASH without miRNA-labeling. T4 DNA ligase was used to compensate for the low affinity between miRNAs and two short complementary DNA probes, and it improved the hybridization yield ∼50,000 times. The LASH assay enabled synthesized miR-143 to be quantified at concentrations ranging from 30 fM to 30 pM. The LASH assay could also quantify endogenous miR-143 released from cultured cells as well as some miRNAs in total RNAs derived from blood. Furthermore, multi-color detection enabled us to distinguish between the highly homologous miR-141 and miR-200a. This simple label-free quantification technique is an easy-to-use approach that can be applied to disease diagnosis.

  17. Recurrence plots and recurrence quantification analysis of human motion data

    Science.gov (United States)

    Josiński, Henryk; Michalczuk, Agnieszka; Świtoński, Adam; Szczesna, Agnieszka; Wojciechowski, Konrad

    2016-06-01

    The authors present exemplary application of recurrence plots, cross recurrence plots and recurrence quantification analysis for the purpose of exploration of experimental time series describing selected aspects of human motion. Time series were extracted from treadmill gait sequences which were recorded in the Human Motion Laboratory (HML) of the Polish-Japanese Academy of Information Technology in Bytom, Poland by means of the Vicon system. Analysis was focused on the time series representing movements of hip, knee, ankle and wrist joints in the sagittal plane.

  18. [DNA quantification in nuclei of cultivated mushroom with DAPI staining].

    Science.gov (United States)

    Pancheva, E V; Volkova, V N; Kamzolkina, O V

    2004-01-01

    Agaricus bisporus (Lange) Imbach is actively cultivated amphithallic basidiomycete, in which various strains are primary homothallic, heterothallic or secondary homothallic. Countings of relative nuclear DNA content by means of DAPI stain and its comparison in different strains can help to understand the mushroom's life cycle features. The authors for the first time observed change of nuclear phases in basidia of A. bisporus strains with different types of life cycle and revealed that DNA content in diploid nuclei is about 1.3 times higher than in haploid ones. The method is highly sensitive and can be used for quantitative measurings of nuclear DNA even in objects with nuclei of about 1 mkm in diameter.

  19. Quantification of plasma DNA as a screening tool for lung cancer

    Institute of Scientific and Technical Information of China (English)

    谢广顺; 侯爱荣; 李龙芸; 高燕宁; 程书钧

    2004-01-01

    Background Recent studies suggest that circulating DNA may be a potential tumor marker for lung cancer, but most of these studies are conducted between healthy controls and lung cancer patients, with few or no benign lung disease patients included. The objective of this study was to evaluate the performance of plasma DNA quantification in discriminating lung cancer from the healthy and benign lung disease.Results Plasma DNA values were significantly increased in lung cancer patients, especially in those with metastases, and in benign lung disease patients compared with that in the healthy individuals (P<0.001, respectively). The values in lung cancer patients were significantly increased compared with that in the benign lung disease patients (P<0.001). The area under the curve was 0.96 [95% confidence interval (CI) 0.92-0.99] for the healthy versus lung cancer, 0.73 (95% CI 0.64-0.83) for lung cancer versus benign lung disease, and 0.86 (95% CI 0.80-0.91) for lung cancer versus the healthy and benign lung disease.Conclusions Plasma DNA quantification has a strong power to discriminate lung cancer from the healthy and from the healthy and benign lung disease, less power to discriminate lung cancer from benign lung disease. Plasma DNA quantification may be useful as a screening tool for lung cancer.

  20. Rapid quantification of semen hepatitis B virus DNA by real-time polymerase chain reaction

    Institute of Scientific and Technical Information of China (English)

    Wei-Ping Qian; Li-Ka Shing; Yue-Qiu Tan; Ying Chen; Ying Peng; Zhi Li; Guang-Xiu Lu; Marie C. Lin; Hsiang-Fu Kung; Ming-Ling He

    2005-01-01

    AIM: To examine the sensitivity and accuracy of real-time polymerase chain reaction (PCR) for the quantification of hepatitis B virus (HBV) DNA in semen.METHODS: Hepatitis B viral DNA was isolated from HBV carriers' semen and sera using phenol extraction method and QTAamp DNA blood mini kit (Qiagen, Germany). HBV DNA was detected by conventional PCR and quantified by TaqMan technology-based real-time PCR (quantitative polymerase chain reaction (qPCR)). The detection threshold was 200 copies of HBV DNA for conventional PCR and 10 copies of HBV DNA for real time PCR per reaction.RESULTS: Both methods of phenol extraction and QIAamp DNA blood mini kit were suitable for isolating HBV DNA from semen. The value of the detection thresholds was 500 copies of HBV DNA per mL in the semen. The viral loads were 7.5×107 and 1.67×107 copies of HBV DNA per mL in two HBV infected patients' sera, while 2.L4×105 and 3.02×105 copies of HBV DNA per mL in the semen.CONCLUSION: Real-time PCR is a more sensitive and accurate method to detect and quantify HBV DNA in the semen.

  1. DNA content in embryo and endosperm of maize kernel (Zea mays L.): impact on GMO quantification.

    Science.gov (United States)

    Trifa, Youssef; Zhang, David

    2004-03-10

    PCR-based techniques are the most widely used methods for the quantification of genetically modified organisms (GMOs) through the determination of the ratio of transgenic DNA to total DNA. It is shown that the DNA content per mass unit is significantly different among 10 maize cultivars. The DNA contents of endosperms, embryos, and teguments of individual kernels from 10 maize cultivars were determined. According to our results, the tegument's DNA ratio reaches at maximum 3.5% of the total kernel's DNA, whereas the endosperm's and the embryo's DNA ratios are nearly equal to 50%. The embryo cells are diploid and made of one paternal and one maternal haploid genome, whereas the endosperm is constituted of triploid cells made of two maternal haploid genomes and one paternal haploid genome. Therefore, it is shown, in this study, that the accuracy of the GMO quantification depends on the reference material used as well as on the category of the transgenic kernels present in the mixture.

  2. Quantification of DNA cleavage specificity in Hi-C experiments.

    Science.gov (United States)

    Meluzzi, Dario; Arya, Gaurav

    2016-01-01

    Hi-C experiments produce large numbers of DNA sequence read pairs that are typically analyzed to deduce genomewide interactions between arbitrary loci. A key step in these experiments is the cleavage of cross-linked chromatin with a restriction endonuclease. Although this cleavage should happen specifically at the enzyme's recognition sequence, an unknown proportion of cleavage events may involve other sequences, owing to the enzyme's star activity or to random DNA breakage. A quantitative estimation of these non-specific cleavages may enable simulating realistic Hi-C read pairs for validation of downstream analyses, monitoring the reproducibility of experimental conditions and investigating biophysical properties that correlate with DNA cleavage patterns. Here we describe a computational method for analyzing Hi-C read pairs to estimate the fractions of cleavages at different possible targets. The method relies on expressing an observed local target distribution downstream of aligned reads as a linear combination of known conditional local target distributions. We validated this method using Hi-C read pairs obtained by computer simulation. Application of the method to experimental Hi-C datasets from murine cells revealed interesting similarities and differences in patterns of cleavage across the various experiments considered.

  3. Quantification of the mutagenic potency and repair of glycidol-induced DNA lesions.

    Science.gov (United States)

    Aasa, Jenny; Vare, Daniel; Motwani, Hitesh V; Jenssen, Dag; Törnqvist, Margareta

    2016-07-01

    Glycidol (Gly) is an electrophilic low-molecular weight epoxide that is classified by IARC as probably carcinogenic to humans. Humans might be exposed to Gly from food, e.g. refined vegetable oils, where Gly has been found as a food process contaminant. It is therefore important to investigate and quantify the genotoxicity of Gly as a primary step towards cancer risk assessment of the human exposure. Here, quantification of the mutagenic potency expressed per dose (AUC: area under the concentration-time curve) of Gly has been performed in Chinese hamster ovary (CHO) cells, using the HPRT assay. The dose of Gly was estimated in the cell exposure medium by trapping Gly with a strong nucleophile, cob(I)alamin, to form stable cobalamin adducts for analysis by LC-MS/MS. Gly was stable in the exposure medium during the time for cell treatment, and thus the dose in vitro is the initial concentration×cell treatment time. Gly induced mutations in the hprt-gene at a rate of 0.08±0.01 mutations/10(5) cells/mMh. Through comparison with the effect of ionizing radiation in the same system a relative mutagenic potency of 9.5rad-eq./mMh was obtained, which could be used for comparison of genotoxicity of chemicals and between test systems and also in procedures for quantitative cancer risk assessment. Gly was shown to induce strand breaks, that were repaired by base excision repair. Furthermore, Gly-induced lesions, present during replication, were found to delay the replication fork elongation. From experiments with repair deficient cells, homologous recombination repair and the ERCC1-XPF complex were indicated to be recruited to support in the repair of the damage related to the stalled replication elongation. The type of DNA damage responsible for the mutagenic effect of Gly could not be concluded from the present study.

  4. DNA quantification based on FRET realized by combination with surfactant CPB.

    Science.gov (United States)

    Liu, Chunxia; Wang, Lei; Jiang, Wei

    2010-04-15

    In this work, we developed a novel DNA quantitative analysis based on fluorescence resonance energy transfer (FRET) realized by combination with a surfactant CPB. The approach was capable of detecting long-stranded DNA in a separation-free format. A sandwich-type FAM-c-DNA-t-DNA-r-DNA-TAMRA conjugate was first formed by the capture probe tagged with FAM, the reporter probe tagged with TAMRA and the target DNA through hybridization. The donor (FAM) and the acceptor (TAMRA) were bridged to afford a FRET system. Subsequently, an addition of the cationic surfactant CPB to the system resulted in a substantial change of the microenvironment and an effective condensation of DNA strands. Consequently, without altering the component of the double strands, an enhanced acceptor fluorescence signal from FRET was achieved and a quantification of the target DNA containing 30 bases was enabled. Under the optimal experimental conditions, an excellent linear relationship between the increase of acceptor fluorescent peak area and the target DNA concentration was obtained over the range from 1.0 x 10(-7) to 3.0 x 10(-9) mol L(-1). The proposed approach offered adequate sensitivity for the detection of the target DNA at 1.0 x 10(-9) mol L(-1).

  5. A probabilistic generative model for quantification of DNA modifications enables analysis of demethylation pathways.

    Science.gov (United States)

    Äijö, Tarmo; Huang, Yun; Mannerström, Henrik; Chavez, Lukas; Tsagaratou, Ageliki; Rao, Anjana; Lähdesmäki, Harri

    2016-03-14

    We present a generative model, Lux, to quantify DNA methylation modifications from any combination of bisulfite sequencing approaches, including reduced, oxidative, TET-assisted, chemical-modification assisted, and methylase-assisted bisulfite sequencing data. Lux models all cytosine modifications (C, 5mC, 5hmC, 5fC, and 5caC) simultaneously together with experimental parameters, including bisulfite conversion and oxidation efficiencies, as well as various chemical labeling and protection steps. We show that Lux improves the quantification and comparison of cytosine modification levels and that Lux can process any oxidized methylcytosine sequencing data sets to quantify all cytosine modifications. Analysis of targeted data from Tet2-knockdown embryonic stem cells and T cells during development demonstrates DNA modification quantification at unprecedented detail, quantifies active demethylation pathways and reveals 5hmC localization in putative regulatory regions.

  6. Quantification of human urinary exosomes by nanoparticle tracking analysis.

    Science.gov (United States)

    Oosthuyzen, Wilna; Sime, Nicole E L; Ivy, Jessica R; Turtle, Emma J; Street, Jonathan M; Pound, John; Bath, Louise E; Webb, David J; Gregory, Christopher D; Bailey, Matthew A; Dear, James W

    2013-12-01

    Exosomes are vesicles that are released from the kidney into urine. They contain protein and RNA from the glomerulus and all sections of the nephron and represent a reservoir for biomarker discovery. Current methods for the identification and quantification of urinary exosomes are time consuming and only semi-quantitative. Nanoparticle tracking analysis (NTA) counts and sizes particles by measuring their Brownian motion in solution. In this study, we applied NTA to human urine and identified particles with a range of sizes. Using antibodies against the exosomal proteins CD24 and aquaporin 2 (AQP2), conjugated to a fluorophore, we could identify a subpopulation of CD24- and AQP2-positive particles of characteristic exosomal size. Extensive pre-NTA processing of urine was not necessary. However, the intra-assay variability in the measurement of exosome concentration was significantly reduced when an ultracentrifugation step preceded NTA. Without any sample processing, NTA tracked exosomal AQP2 upregulation induced by desmopressin stimulation of kidney collecting duct cells. Nanoparticle tracking analysis was also able to track changes in exosomal AQP2 concentration that followed desmopressin treatment of mice and a patient with central diabetes insipidus. When urine was stored at room temperature, 4°C or frozen, nanoparticle concentration was reduced; freezing at -80°C with the addition of protease inhibitors produced the least reduction. In conclusion, with appropriate sample storage, NTA has potential as a tool for the characterization and quantification of extracellular vesicles in human urine.

  7. Quantification of Human Movement for Assessment in Automated Exercise Coaching

    CERN Document Server

    Hagler, Stuart; Bajczy, Ruzena; Pavel, Misha

    2016-01-01

    Quantification of human movement is a challenge in many areas, ranging from physical therapy to robotics. We quantify of human movement for the purpose of providing automated exercise coaching in the home. We developed a model-based assessment and inference process that combines biomechanical constraints with movement assessment based on the Microsoft Kinect camera. To illustrate the approach, we quantify the performance of a simple squatting exercise using two model-based metrics that are related to strength and endurance, and provide an estimate of the strength and energy-expenditure of each exercise session. We look at data for 5 subjects, and show that for some subjects the metrics indicate a trend consistent with improved exercise performance.

  8. Digital quantification of rolling circle amplified single DNA molecules in a resistive pulse sensing nanopore.

    Science.gov (United States)

    Kühnemund, M; Nilsson, M

    2015-05-15

    Novel portable, sensitive and selective DNA sensor methods for bio-sensing applications are required that can rival conventionally used non-portable and expensive fluorescence-based sensors. In this paper, rolling circle amplification (RCA) products are detected in solution and on magnetic particles using a resistive pulse sensing (RPS) nanopore. Low amounts of DNA molecules are detected by padlock probes which are circularized in a strictly target dependent ligation reaction. The DNA-padlock probe-complex is captured on magnetic particles by sequence specific capture oligonucleotides and amplified by a short RCA. Subsequent RPS analysis is used to identify individual particles with single attached RCA products from blank particles. This proof of concept opens up for a novel non-fluorescent digital DNA quantification method that can have many applications in bio-sensing and diagnostic approaches.

  9. Quantification of DNA synthesis in multicellular organisms by a combined DAPI and BrdU technique.

    Science.gov (United States)

    Knobloch, Jürgen; Kunz, Werner; Grevelding, Christoph G

    2002-12-01

    The development of a novel method to detect and quantify mitotic activity in multicellular organisms is reported. The method is based on the combinatorial use of 4',6-diamidino-2-phenylindole (DAPI) as a dye for the specific staining of DNA and the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) as a marker for DNA synthesis. It is shown that on nitrocellulose filters, the amount of DNA can be determined by DAPI as a prerequisite for the subsequent quantification of mitotic activity by BrdU. As a model system to prove the applicability of this technique, the blood fluke Schistosoma mansoni has been used. It is demonstrated that the DNA synthesis rate is higher in adult female schistosomes than in adult males. Furthermore, dimethyl sulfoxide, a widely used solvent for many mitogens and inhibitors of mitosis, has no influence on mitotic activity in adult schistosomes.

  10. A novel immunological assay for hepcidin quantification in human serum.

    Directory of Open Access Journals (Sweden)

    Vasiliki Koliaraki

    Full Text Available BACKGROUND: Hepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Increased hepcidin concentrations lead to iron sequestration in macrophages, contributing to the pathogenesis of anaemia of chronic disease whereas decreased hepcidin is observed in iron deficiency and primary iron overload diseases such as hereditary hemochromatosis. Hepcidin quantification in human blood or urine may provide further insights for the pathogenesis of disorders of iron homeostasis and might prove a valuable tool for clinicians for the differential diagnosis of anaemia. This study describes a specific and non-operator demanding immunoassay for hepcidin quantification in human sera. METHODS AND FINDINGS: An ELISA assay was developed for measuring hepcidin serum concentration using a recombinant hepcidin25-His peptide and a polyclonal antibody against this peptide, which was able to identify native hepcidin. The ELISA assay had a detection range of 10-1500 microg/L and a detection limit of 5.4 microg/L. The intra- and interassay coefficients of variance ranged from 8-15% and 5-16%, respectively. Mean linearity and recovery were 101% and 107%, respectively. Mean hepcidin levels were significantly lower in 7 patients with juvenile hemochromatosis (12.8 microg/L and 10 patients with iron deficiency anemia (15.7 microg/L and higher in 7 patients with Hodgkin lymphoma (116.7 microg/L compared to 32 age-matched healthy controls (42.7 microg/L. CONCLUSIONS: We describe a new simple ELISA assay for measuring hepcidin in human serum with sufficient accuracy and reproducibility.

  11. Assessment of morphological changes and DNA quantification: An in vitro study on acid-immersed teeth

    Directory of Open Access Journals (Sweden)

    K Sowmya

    2013-01-01

    Full Text Available Context: Acid immersion of victim′s body is one of the methods employed to subvert identification of the victim, and hence of the perpetrator. Being hardest and chemically the most stable tissue in the body, teeth can be an important forensic investigative medium in both living and nonliving populations. Teeth are also good reservoirs of both cellular and mitochondrial DNA; however, the quality and quantity of DNA obtained varies according to the environment the tooth has been subjected to. DNA extraction from acid-treated teeth has seldom been reported. Aims: The objectives of the present study were to assess the morphological changes along with DNA recovery from acid-immersed teeth. Materials and Methods: Concentrated hydrochloric acid, nitric acid, and sulfuric acid were employed for tooth decalcification. DNA was extracted on an hourly basis using phenol-chloroform method. Quantification of extracted DNA was done using a spectrophotometer. Results: Results showed that hydrochloric acid had more destructive capacity compared to other acids. Conclusion: Sufficient quantity of DNA was obtainable till the first 2 hours of acid immersion and there was an inverse proportional relation between mean absorbance ratio and quantity of obtained DNA on an hourly basis.

  12. Quantification of differential gene expression by multiplexed targeted resequencing of cDNA

    Science.gov (United States)

    Arts, Peer; van der Raadt, Jori; van Gestel, Sebastianus H.C.; Steehouwer, Marloes; Shendure, Jay; Hoischen, Alexander; Albers, Cornelis A.

    2017-01-01

    Whole-transcriptome or RNA sequencing (RNA-Seq) is a powerful and versatile tool for functional analysis of different types of RNA molecules, but sample reagent and sequencing cost can be prohibitive for hypothesis-driven studies where the aim is to quantify differential expression of a limited number of genes. Here we present an approach for quantification of differential mRNA expression by targeted resequencing of complementary DNA using single-molecule molecular inversion probes (cDNA-smMIPs) that enable highly multiplexed resequencing of cDNA target regions of ∼100 nucleotides and counting of individual molecules. We show that accurate estimates of differential expression can be obtained from molecule counts for hundreds of smMIPs per reaction and that smMIPs are also suitable for quantification of relative gene expression and allele-specific expression. Compared with low-coverage RNA-Seq and a hybridization-based targeted RNA-Seq method, cDNA-smMIPs are a cost-effective high-throughput tool for hypothesis-driven expression analysis in large numbers of genes (10 to 500) and samples (hundreds to thousands). PMID:28474677

  13. Evaluation of plasmid and genomic DNA calibrants used for the quantification of genetically modified organisms.

    Science.gov (United States)

    Caprioara-Buda, M; Meyer, W; Jeynov, B; Corbisier, P; Trapmann, S; Emons, H

    2012-07-01

    The reliable quantification of genetically modified organisms (GMOs) by real-time PCR requires, besides thoroughly validated quantitative detection methods, sustainable calibration systems. The latter establishes the anchor points for the measured value and the measurement unit, respectively. In this paper, the suitability of two types of DNA calibrants, i.e. plasmid DNA and genomic DNA extracted from plant leaves, for the certification of the GMO content in reference materials as copy number ratio between two targeted DNA sequences was investigated. The PCR efficiencies and coefficients of determination of the calibration curves as well as the measured copy number ratios for three powder certified reference materials (CRMs), namely ERM-BF415e (NK603 maize), ERM-BF425c (356043 soya), and ERM-BF427c (98140 maize), originally certified for their mass fraction of GMO, were compared for both types of calibrants. In all three systems investigated, the PCR efficiencies of plasmid DNA were slightly closer to the PCR efficiencies observed for the genomic DNA extracted from seed powders rather than those of the genomic DNA extracted from leaves. Although the mean DNA copy number ratios for each CRM overlapped within their uncertainties, the DNA copy number ratios were significantly different using the two types of calibrants. Based on these observations, both plasmid and leaf genomic DNA calibrants would be technically suitable as anchor points for the calibration of the real-time PCR methods applied in this study. However, the most suitable approach to establish a sustainable traceability chain is to fix a reference system based on plasmid DNA.

  14. The future of human DNA vaccines.

    Science.gov (United States)

    Li, Lei; Saade, Fadi; Petrovsky, Nikolai

    2012-12-31

    DNA vaccines have evolved greatly over the last 20 years since their invention, but have yet to become a competitive alternative to conventional protein or carbohydrate based human vaccines. Whilst safety concerns were an initial barrier, the Achilles heel of DNA vaccines remains their poor immunogenicity when compared to protein vaccines. A wide variety of strategies have been developed to optimize DNA vaccine immunogenicity, including codon optimization, genetic adjuvants, electroporation and sophisticated prime-boost regimens, with each of these methods having its advantages and limitations. Whilst each of these methods has contributed to incremental improvements in DNA vaccine efficacy, more is still needed if human DNA vaccines are to succeed commercially. This review foresees a final breakthrough in human DNA vaccines will come from application of the latest cutting-edge technologies, including "epigenetics" and "omics" approaches, alongside traditional techniques to improve immunogenicity such as adjuvants and electroporation, thereby overcoming the current limitations of DNA vaccines in humans.

  15. Quantification of Crypt and Stem Cell Evolution in the Normal and Neoplastic Human Colon

    Directory of Open Access Journals (Sweden)

    Ann-Marie Baker

    2014-08-01

    Full Text Available Human intestinal stem cell and crypt dynamics remain poorly characterized because transgenic lineage-tracing methods are impractical in humans. Here, we have circumvented this problem by quantitatively using somatic mtDNA mutations to trace clonal lineages. By analyzing clonal imprints on the walls of colonic crypts, we show that human intestinal stem cells conform to one-dimensional neutral drift dynamics with a “functional” stem cell number of five to six in both normal patients and individuals with familial adenomatous polyposis (germline APC−/+. Furthermore, we show that, in adenomatous crypts (APC−/−, there is a proportionate increase in both functional stem cell number and the loss/replacement rate. Finally, by analyzing fields of mtDNA mutant crypts, we show that a normal colon crypt divides around once every 30–40 years, and the division rate is increased in adenomas by at least an order of magnitude. These data provide in vivo quantification of human intestinal stem cell and crypt dynamics.

  16. DNA methylation-based variation between human populations.

    Science.gov (United States)

    Kader, Farzeen; Ghai, Meenu

    2017-02-01

    Several studies have proved that DNA methylation affects regulation of gene expression and development. Epigenome-wide studies have reported variation in methylation patterns between populations, including Caucasians, non-Caucasians (Blacks), Hispanics, Arabs, and numerous populations of the African continent. Not only has DNA methylation differences shown to impact externally visible characteristics, but is also a potential biomarker for underlying racial health disparities between human populations. Ethnicity-related methylation differences set their mark during early embryonic development. Genetic variations, such as single-nucleotide polymorphisms and environmental factors, such as age, dietary folate, socioeconomic status, and smoking, impacts DNA methylation levels, which reciprocally impacts expression of phenotypes. Studies show that it is necessary to address these external influences when attempting to differentiate between populations since the relative impacts of these factors on the human methylome remain uncertain. The present review summarises several reported attempts to establish the contribution of differential DNA methylation to natural human variation, and shows that DNA methylation could represent new opportunities for risk stratification and prevention of several diseases amongst populations world-wide. Variation of methylation patterns between human populations is an exciting prospect which inspires further valuable research to apply the concept in routine medical and forensic casework. However, trans-generational inheritance needs to be quantified to decipher the proportion of variation contributed by DNA methylation. The future holds thorough evaluation of the epigenome to understand quantification, heritability, and the effect of DNA methylation on phenotypes. In addition, methylation profiling of the same ethnic groups across geographical locations will shed light on conserved methylation differences in populations.

  17. Mitochondrial DNA as a non-invasive biomarker: accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias.

    Science.gov (United States)

    Malik, Afshan N; Shahni, Rojeen; Rodriguez-de-Ledesma, Ana; Laftah, Abas; Cunningham, Phil

    2011-08-19

    Circulating mitochondrial DNA (MtDNA) is a potential non-invasive biomarker of cellular mitochondrial dysfunction, the latter known to be central to a wide range of human diseases. Changes in MtDNA are usually determined by quantification of MtDNA relative to nuclear DNA (Mt/N) using real time quantitative PCR. We propose that the methodology for measuring Mt/N needs to be improved and we have identified that current methods have at least one of the following three problems: (1) As much of the mitochondrial genome is duplicated in the nuclear genome, many commonly used MtDNA primers co-amplify homologous pseudogenes found in the nuclear genome; (2) use of regions from genes such as β-actin and 18S rRNA which are repetitive and/or highly variable for qPCR of the nuclear genome leads to errors; and (3) the size difference of mitochondrial and nuclear genomes cause a "dilution bias" when template DNA is diluted. We describe a PCR-based method using unique regions in the human mitochondrial genome not duplicated in the nuclear genome; unique single copy region in the nuclear genome and template treatment to remove dilution bias, to accurately quantify MtDNA from human samples.

  18. Single DNA imaging and length quantification through a mobile phone microscope

    Science.gov (United States)

    Wei, Qingshan; Luo, Wei; Chiang, Samuel; Kappel, Tara; Mejia, Crystal; Tseng, Derek; Chan, Raymond Yan L.; Yan, Eddie; Qi, Hangfei; Shabbir, Faizan; Ozkan, Haydar; Feng, Steve; Ozcan, Aydogan

    2016-03-01

    The development of sensitive optical microscopy methods for the detection of single DNA molecules has become an active research area which cultivates various promising applications including point-of-care (POC) genetic testing and diagnostics. Direct visualization of individual DNA molecules usually relies on sophisticated optical microscopes that are mostly available in well-equipped laboratories. For POC DNA testing/detection, there is an increasing need for the development of new single DNA imaging and sensing methods that are field-portable, cost-effective, and accessible for diagnostic applications in resource-limited or field-settings. For this aim, we developed a mobile-phone integrated fluorescence microscopy platform that allows imaging and sizing of single DNA molecules that are stretched on a chip. This handheld device contains an opto-mechanical attachment integrated onto a smartphone camera module, which creates a high signal-to-noise ratio dark-field imaging condition by using an oblique illumination/excitation configuration. Using this device, we demonstrated imaging of individual linearly stretched λ DNA molecules (48 kilobase-pair, kbp) over 2 mm2 field-of-view. We further developed a robust computational algorithm and a smartphone app that allowed the users to quickly quantify the length of each DNA fragment imaged using this mobile interface. The cellphone based device was tested by five different DNA samples (5, 10, 20, 40, and 48 kbp), and a sizing accuracy of mobile DNA imaging and sizing platform can be very useful for various diagnostic applications including the detection of disease-specific genes and quantification of copy-number-variations at POC settings.

  19. Direct detection and quantification of abasic sites for in vivo studies of DNA damage and repair

    Energy Technology Data Exchange (ETDEWEB)

    Wang Yanming [Division of Radiopharmaceutical Science, Case Center for Imaging Research, Department of Radiology, Case Western Reserve University, Cleveland, OH 44122 (United States)], E-mail: yanming.wang@case.edu; Liu Lili [Department of Hematology and Oncology, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44122 (United States); Wu Chunying [Division of Radiopharmaceutical Science, Case Center for Imaging Research, Department of Radiology, Case Western Reserve University, Cleveland, OH 44122 (United States); Bulgar, Alina [Department of Hematology and Oncology, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44122 (United States); Somoza, Eduardo; Zhu Wenxia [Division of Radiopharmaceutical Science, Case Center for Imaging Research, Department of Radiology, Case Western Reserve University, Cleveland, OH 44122 (United States); Gerson, Stanton L. [Department of Hematology and Oncology, Case Comprehensive Cancer Center, Case Western Reserve University, Cleveland, OH 44122 (United States)

    2009-11-15

    Use of chemotherapeutic agents to induce cytotoxic DNA damage and programmed cell death is a key strategy in cancer treatments. However, the efficacy of DNA-targeted agents such as temozolomide is often compromised by intrinsic cellular responses such as DNA base excision repair (BER). Previous studies have shown that BER pathway resulted in formation of abasic or apurinic/apyrimidinic (AP) sites, and blockage of AP sites led to a significant enhancement of drug sensitivity due to reduction of DNA base excision repair. Since a number of chemotherapeutic agents also induce formation of AP sites, monitoring of these sites as a clinical correlate of drug effect will provide a useful tool in the development of DNA-targeted chemotherapies aimed at blocking abasic sites from repair. Here we report an imaging technique based on positron emission tomography (PET) that allows for direct quantification of AP sites in vivo. For this purpose, positron-emitting carbon-11 has been incorporated into methoxyamine ([{sup 11}C]MX) that binds covalently to AP sites with high specificity. The binding specificity of [{sup 11}C]MX for AP sites was demonstrated by in vivo blocking experiments. Using [{sup 11}C]MX as a radiotracer, animal PET studies have been conducted in melanoma and glioma xenografts for quantification of AP sites. Following induction of AP sites by temozolomide, both tumor models showed significant increase of [{sup 11}C]MX uptake in tumor regions in terms of radioactivity concentration as a function of time, which correlates well with conventional aldehyde reactive probe (ARP)-based bioassays for AP sites.

  20. Performance of Different Analytical Software Packages in Quantification of DNA Methylation by Pyrosequencing

    Science.gov (United States)

    Grasso, Chiara; Trevisan, Morena; Fiano, Valentina; Tarallo, Valentina; De Marco, Laura; Sacerdote, Carlotta; Richiardi, Lorenzo; Merletti, Franco; Gillio-Tos, Anna

    2016-01-01

    Background Pyrosequencing has emerged as an alternative method of nucleic acid sequencing, well suited for many applications which aim to characterize single nucleotide polymorphisms, mutations, microbial types and CpG methylation in the target DNA. The commercially available pyrosequencing systems can harbor two different types of software which allow analysis in AQ or CpG mode, respectively, both widely employed for DNA methylation analysis. Objective Aim of the study was to assess the performance for DNA methylation analysis at CpG sites of the two pyrosequencing software which allow analysis in AQ or CpG mode, respectively. Despite CpG mode having been specifically generated for CpG methylation quantification, many investigations on this topic have been carried out with AQ mode. As proof of equivalent performance of the two software for this type of analysis is not available, the focus of this paper was to evaluate if the two modes currently used for CpG methylation assessment by pyrosequencing may give overlapping results. Methods We compared the performance of the two software in quantifying DNA methylation in the promoter of selected genes (GSTP1, MGMT, LINE-1) by testing two case series which include DNA from paraffin embedded prostate cancer tissues (PC study, N = 36) and DNA from blood fractions of healthy people (DD study, N = 28), respectively. Results We found discrepancy in the two pyrosequencing software-based quality assignment of DNA methylation assays. Compared to the software for analysis in the AQ mode, less permissive criteria are supported by the Pyro Q-CpG software, which enables analysis in CpG mode. CpG mode warns the operators about potential unsatisfactory performance of the assay and ensures a more accurate quantitative evaluation of DNA methylation at CpG sites. Conclusion The implementation of CpG mode is strongly advisable in order to improve the reliability of the methylation analysis results achievable by pyrosequencing. PMID

  1. Performance of Different Analytical Software Packages in Quantification of DNA Methylation by Pyrosequencing.

    Directory of Open Access Journals (Sweden)

    Chiara Grasso

    Full Text Available Pyrosequencing has emerged as an alternative method of nucleic acid sequencing, well suited for many applications which aim to characterize single nucleotide polymorphisms, mutations, microbial types and CpG methylation in the target DNA. The commercially available pyrosequencing systems can harbor two different types of software which allow analysis in AQ or CpG mode, respectively, both widely employed for DNA methylation analysis.Aim of the study was to assess the performance for DNA methylation analysis at CpG sites of the two pyrosequencing software which allow analysis in AQ or CpG mode, respectively. Despite CpG mode having been specifically generated for CpG methylation quantification, many investigations on this topic have been carried out with AQ mode. As proof of equivalent performance of the two software for this type of analysis is not available, the focus of this paper was to evaluate if the two modes currently used for CpG methylation assessment by pyrosequencing may give overlapping results.We compared the performance of the two software in quantifying DNA methylation in the promoter of selected genes (GSTP1, MGMT, LINE-1 by testing two case series which include DNA from paraffin embedded prostate cancer tissues (PC study, N = 36 and DNA from blood fractions of healthy people (DD study, N = 28, respectively.We found discrepancy in the two pyrosequencing software-based quality assignment of DNA methylation assays. Compared to the software for analysis in the AQ mode, less permissive criteria are supported by the Pyro Q-CpG software, which enables analysis in CpG mode. CpG mode warns the operators about potential unsatisfactory performance of the assay and ensures a more accurate quantitative evaluation of DNA methylation at CpG sites.The implementation of CpG mode is strongly advisable in order to improve the reliability of the methylation analysis results achievable by pyrosequencing.

  2. Multiplex cDNA quantification method that facilitates the standardization of gene expression data

    Science.gov (United States)

    Gotoh, Osamu; Murakami, Yasufumi; Suyama, Akira

    2011-01-01

    Microarray-based gene expression measurement is one of the major methods for transcriptome analysis. However, current microarray data are substantially affected by microarray platforms and RNA references because of the microarray method can provide merely the relative amounts of gene expression levels. Therefore, valid comparisons of the microarray data require standardized platforms, internal and/or external controls and complicated normalizations. These requirements impose limitations on the extensive comparison of gene expression data. Here, we report an effective approach to removing the unfavorable limitations by measuring the absolute amounts of gene expression levels on common DNA microarrays. We have developed a multiplex cDNA quantification method called GEP-DEAN (Gene expression profiling by DCN-encoding-based analysis). The method was validated by using chemically synthesized DNA strands of known quantities and cDNA samples prepared from mouse liver, demonstrating that the absolute amounts of cDNA strands were successfully measured with a sensitivity of 18 zmol in a highly multiplexed manner in 7 h. PMID:21415008

  3. Total soil DNA quantification as an alternative microbial biomass determination approach

    Science.gov (United States)

    Semenov, Mikhail

    2015-04-01

    Many studies on geographically widespread soils from arctic permafrost to arid and tropical soils, as well as those studies on extreme events, such as freezing-thawing and drying-rewetting of soils, require immediate freezing of soil after sampling. The two common basic approaches, such as chloroform fumigation-extraction (CFE) and substrate-induced respiration (SIR), however, are not applicable in frozen or dry soil samples due to a partial destruction of microbial cells during freezing-thawing and drying-rewetting. This calls for approaches enabling correct estimation of microbial biomass in frozen or dried soil samples. This study was aimed to compare commonly used SIR and CFE techniques with total soil DNA quantification and demonstrate the applicability of DNA-based determination of microbial biomass in carbonate-containing, slightly (Chernozem) and strongly alkaline (Calcisol) soils of semi-arid climates. The samples of natural and agricultural ecosystems were taken throughout the soil profile from long-term static field experiments in the European part of Russia. The linear regression between SIR-Cmic and total soil dsDNA for the Chernozem showed very strong correlation. From the regression equation, the conversion factor of 5.10 with R2 = 0.96 was obtained. The effect of CO2 retention at alkaline pH (>8) and low microbial biomass-C resulted in an inability to obtain any SIR-CO2 release at deeper horizons of Calcisol, i.e. the CO2 retention potential was higher that the CO2 evolution. As a consequence, the values of SIR-Cmic of Calcisol at the horizons with pH > 8.0 were strongly underestimated (by a factor of 2-3). This smoothed the differences in Cmic between soil horizons. Nevertheless, reliable dsDNA values obtained for these soils demonstrated well-pronounced changes in microbial biomass within soil profile. The CFE and DNA-based approaches showed a good correspondence, with R2 = 0.96 for both soil types. The CFE-Cmic to DNA-Cmic factor of 0

  4. Quantification of applied dose in irradiated citrus fruits by DNA Comet Assay together with image analysis.

    Science.gov (United States)

    Cetinkaya, Nurcan; Ercin, Demet; Özvatan, Sümer; Erel, Yakup

    2016-02-01

    The experiments were conducted for quantification of applied dose for quarantine control in irradiated citrus fruits. Citrus fruits exposed to doses of 0.1 to 1.5 kGy and analyzed by DNA Comet Assay. Observed comets were evaluated by image analysis. The tail length, tail moment and tail DNA% of comets were used for the interpretation of comets. Irradiated citrus fruits showed the separated tails from the head of the comet by increasing applied doses from 0.1 to 1.5 kGy. The mean tail length and mean tail moment% levels of irradiated citrus fruits at all doses are significantly different (p Comet Assay may be a practical quarantine control method for irradiated citrus fruits since it has been possible to estimate the applied low doses as small as 0.1 kGy when it is combined with image analysis.

  5. Radiation dose determines the method for quantification of DNA double strand breaks

    Energy Technology Data Exchange (ETDEWEB)

    Bulat, Tanja; Keta, Olitija; Korićanac, Lela; Žakula, Jelena; Petrović, Ivan; Ristić-Fira, Aleksandra [University of Belgrade, Vinča Institute of Nuclear Sciences, Belgrade (Serbia); Todorović, Danijela, E-mail: dtodorovic@medf.kg.ac.rs [University of Kragujevac, Faculty of Medical Sciences, Kragujevac (Serbia)

    2016-03-15

    Ionizing radiation induces DNA double strand breaks (DSBs) that trigger phosphorylation of the histone protein H2AX (γH2AX). Immunofluorescent staining visualizes formation of γH2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of γH2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to γ-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany) microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany). Obtained results show that the level of γH2AX is time and dose dependent. Immunofluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of γH2AX foci. (author)

  6. Radiation dose determines the method for quantification of DNA double strand breaks

    Directory of Open Access Journals (Sweden)

    TANJA BULAT

    2016-03-01

    Full Text Available ABSTRACT Ionizing radiation induces DNA double strand breaks (DSBs that trigger phosphorylation of the histone protein H2AX (γH2AX. Immunofluorescent staining visualizes formation of γH2AX foci, allowing their quantification. This method, as opposed to Western blot assay and Flow cytometry, provides more accurate analysis, by showing exact position and intensity of fluorescent signal in each single cell. In practice there are problems in quantification of γH2AX. This paper is based on two issues: the determination of which technique should be applied concerning the radiation dose, and how to analyze fluorescent microscopy images obtained by different microscopes. HTB140 melanoma cells were exposed to γ-rays, in the dose range from 1 to 16 Gy. Radiation effects on the DNA level were analyzed at different time intervals after irradiation by Western blot analysis and immunofluorescence microscopy. Immunochemically stained cells were visualized with two types of microscopes: AxioVision (Zeiss, Germany microscope, comprising an ApoTome software, and AxioImagerA1 microscope (Zeiss, Germany. Obtained results show that the level of γH2AX is time and dose dependent. Immunofluorescence microscopy provided better detection of DSBs for lower irradiation doses, while Western blot analysis was more reliable for higher irradiation doses. AxioVision microscope containing ApoTome software was more suitable for the detection of γH2AX foci.

  7. Quantification of human upper extremity nerves and fascicular anatomy.

    Science.gov (United States)

    Brill, Natalie A; Tyler, Dustin J

    2017-09-01

    In this study we provide detailed quantification of upper extremity nerve and fascicular anatomy. The purpose is to provide values and trends in neural features useful for clinical applications and neural interface device design. Nerve cross-sections were taken from 4 ulnar, 4 median, and 3 radial nerves from 5 arms of 3 human cadavers. Quantified nerve features included cross-sectional area, minor diameter, and major diameter. Fascicular features analyzed included count, perimeter, area, and position. Mean fascicular diameters were 0.57 ± 0.39, 0.6 ± 0.3, 0.5 ± 0.26 mm in the upper arm and 0.38 ± 0.18, 0.47 ± 0.18, 0.4 ± 0.27 mm in the forearm of ulnar, median, and radial nerves, respectively. Mean fascicular diameters were inversely proportional to fascicle count. Detailed quantitative anatomy of upper extremity nerves is a resource for design of neural electrodes, guidance in extraneural procedures, and improved neurosurgical planning. Muscle Nerve 56: 463-471, 2017. © 2016 Wiley Periodicals, Inc.

  8. Absolute Quantification of Selected Proteins in the Human Osteoarthritic Secretome

    Directory of Open Access Journals (Sweden)

    Mandy J. Peffers

    2013-10-01

    Full Text Available Osteoarthritis (OA is characterized by a loss of extracellular matrix which is driven by catabolic cytokines. Proteomic analysis of the OA cartilage secretome enables the global study of secreted proteins. These are an important class of molecules with roles in numerous pathological mechanisms. Although cartilage studies have identified profiles of secreted proteins, quantitative proteomics techniques have been implemented that would enable further biological questions to be addressed. To overcome this limitation, we used the secretome from human OA cartilage explants stimulated with IL-1β and compared proteins released into the media using a label-free LC-MS/MS-based strategy. We employed QconCAT technology to quantify specific proteins using selected reaction monitoring. A total of 252 proteins were identified, nine were differentially expressed by IL-1 β stimulation. Selected protein candidates were quantified in absolute amounts using QconCAT. These findings confirmed a significant reduction in TIMP-1 in the secretome following IL-1β stimulation. Label-free and QconCAT analysis produced equivocal results indicating no effect of cytokine stimulation on aggrecan, cartilage oligomeric matrix protein, fibromodulin, matrix metalloproteinases 1 and 3 or plasminogen release. This study enabled comparative protein profiling and absolute quantification of proteins involved in molecular pathways pertinent to understanding the pathogenesis of OA.

  9. Real-time PCR assays for hepatitis B virus DNA quantification may require two different targets.

    Science.gov (United States)

    Liu, Chao; Chang, Le; Jia, Tingting; Guo, Fei; Zhang, Lu; Ji, Huimin; Zhao, Junpeng; Wang, Lunan

    2017-05-12

    Quantification Hepatitis B virus (HBV) DNA plays a critical role in the management of chronic HBV infections. However, HBV is a DNA virus with high levels of genetic variation, and drug-resistant mutations have emerged with the use of antiviral drugs. If a mutation caused a sequence mismatched in the primer or probe of a commercial DNA quantification kit, this would lead to an underestimation of the viral load of the sample. The aim of this study was to determine whether commercial kits, which use only one pair of primers and a single probe, accurately quantify the HBV DNA levels and to develop an improved duplex real-time PCR assay. We developed a new duplex real-time PCR assay that used two pairs of primers and two probes based on the conserved S and C regions of the HBV genome. We performed HBV DNA quantitative detection of HBV samples and compared the results of our duplex real-time PCR assays with the COBAS TaqMan HBV Test version 2 and Daan real-time PCR assays. The target region of the discordant sample was amplified, sequenced, and validated using plasmid. The results of the duplex real-time PCR were in good accordance with the commercial COBAS TaqMan HBV Test version 2 and Daan real-time PCR assays. We showed that two samples from Chinese HBV infections underestimated viral loads when quantified by the Roche kit because of a mismatch between the viral sequence and the reverse primer of the Roche kit. The HBV DNA levels of six samples were undervalued by duplex real-time PCR assays of the C region because of mutations in the primer of C region. We developed a new duplex real-time PCR assay, and the results of this assay were similar to the results of commercial kits. The HBV DNA level could be undervalued when using the COBAS TaqMan HBV Test version 2 for Chinese HBV infections owing to a mismatch with the primer/probe. A duplex real-time PCR assay based on the S and C regions could solve this problem to some extent.

  10. DNA Methylation Landscapes of Human Fetal Development

    NARCIS (Netherlands)

    Slieker, Roderick C.; Roost, Matthias S.; van Iperen, Liesbeth; Suchiman, H. Eka D; Tobi, Elmar W.; Carlotti, Françoise; de Koning, Eelco J P; Slagboom, P. Eline; Heijmans, Bastiaan T.; Chuva de Sousa Lopes, Susana M.

    2015-01-01

    Remodelling the methylome is a hallmark of mammalian development and cell differentiation. However, current knowledge of DNA methylation dynamics in human tissue specification and organ development largely stems from the extrapolation of studies in vitro and animal models. Here, we report on the DNA

  11. Mitochondrial DNA as a non-invasive biomarker: Accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias

    Energy Technology Data Exchange (ETDEWEB)

    Malik, Afshan N., E-mail: afshan.malik@kcl.ac.uk [King' s College London, Diabetes Research Group, Division of Diabetes and Nutritional Sciences, School of Medicine (United Kingdom); Shahni, Rojeen; Rodriguez-de-Ledesma, Ana; Laftah, Abas; Cunningham, Phil [King' s College London, Diabetes Research Group, Division of Diabetes and Nutritional Sciences, School of Medicine (United Kingdom)

    2011-08-19

    Highlights: {yields} Mitochondrial dysfunction is central to many diseases of oxidative stress. {yields} 95% of the mitochondrial genome is duplicated in the nuclear genome. {yields} Dilution of untreated genomic DNA leads to dilution bias. {yields} Unique primers and template pretreatment are needed to accurately measure mitochondrial DNA content. -- Abstract: Circulating mitochondrial DNA (MtDNA) is a potential non-invasive biomarker of cellular mitochondrial dysfunction, the latter known to be central to a wide range of human diseases. Changes in MtDNA are usually determined by quantification of MtDNA relative to nuclear DNA (Mt/N) using real time quantitative PCR. We propose that the methodology for measuring Mt/N needs to be improved and we have identified that current methods have at least one of the following three problems: (1) As much of the mitochondrial genome is duplicated in the nuclear genome, many commonly used MtDNA primers co-amplify homologous pseudogenes found in the nuclear genome; (2) use of regions from genes such as {beta}-actin and 18S rRNA which are repetitive and/or highly variable for qPCR of the nuclear genome leads to errors; and (3) the size difference of mitochondrial and nuclear genomes cause a 'dilution bias' when template DNA is diluted. We describe a PCR-based method using unique regions in the human mitochondrial genome not duplicated in the nuclear genome; unique single copy region in the nuclear genome and template treatment to remove dilution bias, to accurately quantify MtDNA from human samples.

  12. Assessment of DNA degradation induced by thermal and UV radiation processing: implications for quantification of genetically modified organisms.

    Science.gov (United States)

    Ballari, Rajashekhar V; Martin, Asha

    2013-12-01

    DNA quality is an important parameter for the detection and quantification of genetically modified organisms (GMO's) using the polymerase chain reaction (PCR). Food processing leads to degradation of DNA, which may impair GMO detection and quantification. This study evaluated the effect of various processing treatments such as heating, baking, microwaving, autoclaving and ultraviolet (UV) irradiation on the relative transgenic content of MON 810 maize using pRSETMON-02, a dual target plasmid as a model system. Amongst all the processing treatments examined, autoclaving and UV irradiation resulted in the least recovery of the transgenic (CaMV 35S promoter) and taxon-specific (zein) target DNA sequences. Although a profound impact on DNA degradation was seen during the processing, DNA could still be reliably quantified by Real-time PCR. The measured mean DNA copy number ratios of the processed samples were in agreement with the expected values. Our study confirms the premise that the final analytical value assigned to a particular sample is independent of the degree of DNA degradation since the transgenic and the taxon-specific target sequences possessing approximately similar lengths degrade in parallel. The results of our study demonstrate that food processing does not alter the relative quantification of the transgenic content provided the quantitative assays target shorter amplicons and the difference in the amplicon size between the transgenic and taxon-specific genes is minimal.

  13. Properties of targeted preamplification in DNA and cDNA quantification.

    Science.gov (United States)

    Andersson, Daniel; Akrap, Nina; Svec, David; Godfrey, Tony E; Kubista, Mikael; Landberg, Göran; Ståhlberg, Anders

    2015-01-01

    Quantification of small molecule numbers often requires preamplification to generate enough copies for accurate downstream enumerations. Here, we studied experimental parameters in targeted preamplification and their effects on downstream quantitative real-time PCR (qPCR). To evaluate different strategies, we monitored the preamplification reaction in real-time using SYBR Green detection chemistry followed by melting curve analysis. Furthermore, individual targets were evaluated by qPCR. The preamplification reaction performed best when a large number of primer pairs was included in the primer pool. In addition, preamplification efficiency, reproducibility and specificity were found to depend on the number of template molecules present, primer concentration, annealing time and annealing temperature. The amount of nonspecific PCR products could also be reduced about 1000-fold using bovine serum albumin, glycerol and formamide in the preamplification. On the basis of our findings, we provide recommendations how to perform robust and highly accurate targeted preamplification in combination with qPCR or next-generation sequencing.

  14. Real-time PCR quantification of human complement C4A and C4B genes

    Directory of Open Access Journals (Sweden)

    Fust George

    2006-01-01

    Full Text Available Abstract Background The fourth component of human complement (C4, an essential factor of the innate immunity, is represented as two isoforms (C4A and C4B in the genome. Although these genes differ only in 5 nucleotides, the encoded C4A and C4B proteins are functionally different. Based on phenotypic determination, unbalanced production of C4A and C4B is associated with several diseases, such as systemic lupus erythematosus, type 1 diabetes, several autoimmune diseases, moreover with higher morbidity and mortality of myocardial infarction and increased susceptibility for bacterial infections. Despite of this major clinical relevance, only low throughput, time and labor intensive methods have been used so far for the quantification of C4A and C4B genes. Results A novel quantitative real-time PCR (qPCR technique was developed for rapid and accurate quantification of the C4A and C4B genes applying a duplex, TaqMan based methodology. The reliable, single-step analysis provides the determination of the copy number of the C4A and C4B genes applying a wide range of DNA template concentration (0.3–300 ng genomic DNA. The developed qPCR was applied to determine C4A and C4B gene dosages in a healthy Hungarian population (N = 118. The obtained data were compared to the results of an earlier study of the same population. Moreover a set of 33 samples were analyzed by two independent methods. No significant difference was observed between the gene dosages determined by the employed techniques demonstrating the reliability of the novel qPCR methodology. A Microsoft Excel worksheet and a DOS executable are also provided for simple and automated evaluation of the measured data. Conclusion This report describes a novel real-time PCR method for single-step quantification of C4A and C4B genes. The developed technique could facilitate studies investigating disease association of different C4 isotypes.

  15. Human Insulin from Recombinant DNA Technology

    Science.gov (United States)

    Johnson, Irving S.

    1983-02-01

    Human insulin produced by recombinant DNA technology is the first commercial health care product derived from this technology. Work on this product was initiated before there were federal guidelines for large-scale recombinant DNA work or commercial development of recombinant DNA products. The steps taken to facilitate acceptance of large-scale work and proof of the identity and safety of such a product are described. While basic studies in recombinant DNA technology will continue to have a profound impact on research in the life sciences, commercial applications may well be controlled by economic conditions and the availability of investment capital.

  16. Apoptosis and DNA damage in human spermatozoa

    Institute of Scientific and Technical Information of China (English)

    R John Aitken; Adam J Koppers

    2011-01-01

    DNA damage is frequently encountered in spermatozoa of subfertile males and is correlated with a range of adverse clinical outcomes including impaired fertilization, disrupted preimplantation embryonic development, increased rates of miscarriage and an enhanced risk of disease in the progeny. The etiology of DNA fragmentation in human spermatozoa is closely correlated with the appearance of oxidative base adducts and evidence of impaired spermiogenesis. We hypothesize that oxidative stress impedes spermiogenesis,resulting in the generation of spermatozoa with poorly remodelled chromatin. These defective cells have a tendency to default to an apoptotic pathway associated with motility loss, caspase activation, phosphatidylserine exteriorization and the activation of free radical generation by the mitochondria. The latter induces lipid peroxidation and oxidative DNA damage, which then leads to DNA fragmentation and cell death. The physical architecture of spermatozoa prevents any nucleases activated as a result of this apoptotic process from gaining access to the nuclear DNA and inducing its fragmentation. It is for this reason that a majority of the DNA damage encountered in human spermatozoa seems to be oxidative. Given the important role that oxidative stress seems to have in the etiology of DNA damage, there should be an important role for antioxidants in the treatment of this condition. If oxidative DNA damage in spermatozoa is providing a sensitive readout of systemic oxidative stress, the implications of these findings could stretch beyond our immediate goal of trying to minimize DNA damage in spermatozoa as a prelude to assisted conception therapy.

  17. Quantification of Fewer than Ten Copies of a DNA Biomarker without Amplification or Labeling.

    Science.gov (United States)

    Lee, Yoonhee; Kim, Youngkyu; Lee, Donggyu; Roy, Dhruvajyoti; Park, Joon Won

    2016-06-08

    Polymerase chain reaction (PCR) is a highly sensitive diagnosis technique for detection of nucleic acids and for monitoring residual disease; however, PCR can be unreliable for samples containing very few target molecules. Here, we describe a quantification method, using force-distance (FD) curve based atomic force microscopy (AFM) to detect a target DNA bound to small (1.4-1.9 μm diameter) probe DNA spots, allowing mapping of entire spots to nanometer resolution. Using a synthetic BCR-ABL fusion gene sequence target, we examined samples containing between one and 10 target copies. A high degree of correlation (r(2) = 0.994) between numbers of target copies and detected probe clusters was observed, and the approach could detect the BCR-ABL biomarker when only a single copy was present, although multiple screens were required. Our results clearly demonstrate that FD curve-based imaging is suitable for quantitative analysis of fewer than 10 copies of DNA biomarkers without amplification, modification, or labeling.

  18. Absolute quantification of olive oil DNA by droplet digital-PCR (ddPCR): Comparison of isolation and amplification methodologies.

    Science.gov (United States)

    Scollo, Francesco; Egea, Leticia A; Gentile, Alessandra; La Malfa, Stefano; Dorado, Gabriel; Hernandez, Pilar

    2016-12-15

    Olive oil is considered a premium product for its nutritional value and health benefits, and the ability to define its origin and varietal composition is a key step towards ensuring the traceability of the product. However, isolating the DNA from such a matrix is a difficult task. In this study, the quality and quantity of olive oil DNA, isolated using four different DNA isolation protocols, was evaluated using the qRT-PCR and ddPCR techniques. The results indicate that CTAB-based extraction methods were the best for unfiltered oil, while Nucleo Spin-based extraction protocols showed greater overall reproducibility. The use of both qRT-PCR and ddPCR led to the absolute quantification of the DNA copy number. The results clearly demonstrate the importance of the choice of DNA-isolation protocol, which should take into consideration the qualitative aspects of DNA and the evaluation of the amplified DNA copy number.

  19. Real-Time PCR Quantification of Heteroplasmy in a Mouse Model with Mitochondrial DNA of C57BL/6 and NZB/BINJ Strains

    Science.gov (United States)

    Sangalli, Juliano Rodrigues; Rodrigues, Thiago Bittencourt; Smith, Lawrence Charles; Meirelles, Flávio Vieira; Chiaratti, Marcos Roberto

    2015-01-01

    Mouse models are widely employed to study mitochondrial inheritance, which have implications to several human diseases caused by mutations in the mitochondrial genome (mtDNA). These mouse models take advantage of polymorphisms between the mtDNA of the NZB/BINJ and the mtDNA of common inbred laboratory (i.e., C57BL/6) strains to generate mice with two mtDNA haplotypes (heteroplasmy). Based on PCR followed by restriction fragment length polymorphism (PCR-RFLP), these studies determine the level of heteroplasmy across generations and in different cell types aiming to understand the mechanisms underlying mitochondrial inheritance. However, PCR-RFLP is a time-consuming method of low sensitivity and accuracy that dependents on the use of restriction enzyme digestions. A more robust method to measure heteroplasmy has been provided by the use of real-time quantitative PCR (qPCR) based on allelic refractory mutation detection system (ARMS-qPCR). Herein, we report an ARMS-qPCR assay for quantification of heteroplasmy using heteroplasmic mice with mtDNA of NZB/BINJ and C57BL/6 origin. Heteroplasmy and mtDNA copy number were estimated in germline and somatic tissues, providing evidence of the reliability of the approach. Furthermore, it enabled single-step quantification of heteroplasmy, with sensitivity to detect as low as 0.1% of either NZB/BINJ or C57BL/6 mtDNA. These findings are relevant as the ARMS-qPCR assay reported here is fully compatible with similar heteroplasmic mouse models used to study mitochondrial inheritance in mammals. PMID:26274500

  20. Accurate and precise DNA quantification in the presence of different amplification efficiencies using an improved Cy0 method.

    Directory of Open Access Journals (Sweden)

    Michele Guescini

    Full Text Available Quantitative real-time PCR represents a highly sensitive and powerful technology for the quantification of DNA. Although real-time PCR is well accepted as the gold standard in nucleic acid quantification, there is a largely unexplored area of experimental conditions that limit the application of the Ct method. As an alternative, our research team has recently proposed the Cy0 method, which can compensate for small amplification variations among the samples being compared. However, when there is a marked decrease in amplification efficiency, the Cy0 is impaired, hence determining reaction efficiency is essential to achieve a reliable quantification. The proposed improvement in Cy0 is based on the use of the kinetic parameters calculated in the curve inflection point to compensate for efficiency variations. Three experimental models were used: inhibition of primer extension, non-optimal primer annealing and a very small biological sample. In all these models, the improved Cy0 method increased quantification accuracy up to about 500% without affecting precision. Furthermore, the stability of this procedure was enhanced integrating it with the SOD method. In short, the improved Cy0 method represents a simple yet powerful approach for reliable DNA quantification even in the presence of marked efficiency variations.

  1. DNA quantification via ICP-MS using lanthanide-labeled probes and ligation-mediated amplification.

    Science.gov (United States)

    Brückner, Kathrin; Schwarz, Kathleen; Beck, Sebastian; Linscheid, Michael W

    2014-01-07

    The combination of lanthanide-tagged oligonucleotide probes with inductively coupled plasma mass spectrometry (ICP-MS) as the detection technique is a novel labeling and analysis strategy for heterogeneous nucleic acid quantification assays. We describe a hybridization assay based on biotin-streptavidin affinity using lanthanide-labeled reporter probes and biotinylated capture probes. For the basic sandwich type assay, performed in streptavidin-coated microtitration wells, the limit of detection (LOD) was 7.2 fmol of DNA target, corresponding to a final concentration of 6 pM terbium-labeled probes detectable by ICP-MS after elution from the solid support. To improve the sensitivity and sequence specificity of the approach, it was combined with established molecular biological techniques, i.e., elution with a restriction endonuclease and signal and target amplification by the ligase detection reaction (LDR) and ligase chain reaction (LCR), respectively. Initial experiments showed that the enzymes facilitated the discrimination of single-base mismatches within the recognition or ligation site. Furthermore, LCR as a target amplification step resulted in a 6000-fold increase of sensitivity, and finally an LOD of 2.6 amol was achieved with an artificial double-stranded DNA target.

  2. Different Length (DL qPCR for Quantification of Cell Killing by UV-induced DNA Damage

    Directory of Open Access Journals (Sweden)

    Guro Skjefstad

    2010-08-01

    Full Text Available We describe the different length (DL qPCR method for quantification of UV induced DNA damage in cell killing. The principle of DL qPCR is that DNA damage inhibits PCR. Applications with different lengths can therefore be used to detect different levels of UV-induced DNA damage. The assay was evaluated on three strains of Escherichia coli exposed to varying levels of ultraviolet (UV radiation. We show that DL qPCR sensitivity and reproducibility are within the range of practical application to detect the effect of UV cell killing.

  3. DNA repair responses in human skin cells

    Energy Technology Data Exchange (ETDEWEB)

    Hanawalt, P.C.; Liu, S.C.; Parsons, C.S.

    1981-07-01

    Sunlight and some environmental chemical agents produce lesions in the DNA of human skin cells that if unrepaired may interfere with normal functioning of these cells. The most serious outcome of such interactions may be malignancy. It is therefore important to develop an understanding of mechanisms by which the lesions may be repaired or tolerated without deleterious consequences. Our models for the molecular processing of damaged DNA have been derived largely from the study of bacterial systems. Some similarities but significant differences are revealed when human cell responses are tested against these models. It is also of importance to learn DNA repair responses of epidermal keratinocytes for comparison with the more extensive studies that have been carried out with dermal fibroblasts. Our experimental results thus far indicate similarities for the excision-repair of ultraviolet-induced pyrimidine dimers in human keratinocytes and fibroblasts. Both the monoadducts and the interstrand crosslinks produced in DNA by photoactivated 8-methoxypsoralen (PUVA) can be repaired in normal human fibroblasts but not in those from xeroderma pigmentosum patients. The monoadducts, like pyrimidine dimers, are probably the more mutagenic/carcinogenic lesions while the crosslinks are less easily repaired and probably result in more effective blocking of DNA function. It is suggested that a split-dose protocol that maximizes the production of crosslinks while minimizing the yield of monoadducts may be more effective and potentially less carcinogenic than the single ultraviolet exposure regimen in PUVA therapy for psoriasis.

  4. Perinatal transmission of human papilomavirus DNA.

    Science.gov (United States)

    Rombaldi, Renato L; Serafini, Eduardo P; Mandelli, Jovana; Zimmermann, Edineia; Losquiavo, Kamille P

    2009-06-21

    The purpose was to study the perinatal transmission of human papillomavirus DNA (HPV-DNA) in 63 mother-newborn pairs, besides looking at the epidemiological factors involved in the viral DNA transmission. The following sampling methods were used: (1) in the pregnant woman, when was recruited, in cervix and clinical lesions of the vagina, vulva and perineal region; (2) in the newborn, (a) buccal, axillary and inguinal regions; (b) nasopharyngeal aspirate, and (c) cord blood; (3) in the children, buccal was repeated in the 4th week and 6th and 12th month of life. HPV-DNA was identified using two methodologies: multiplex PCR (PGMY09 and MY11 primers) and nested-PCR (genotypes 6/11, 16, 18, 31, 33, 42, 52 and 58). Perinatal transmission was considered when concordance was found in type-specific HPV between mother/newborn or mother/child. HPV-DNA genital was detected in 49 pregnant women submitted to delivery. Eleven newborns (22.4%, n = 11/49) were HPV-DNA positive. In 8 cases (16.3%, n = 8/49) there was type specific HPV concordance between mother/newborn samples. At the end of the first month of life three children (6.1%, n = 3/49) became HPV-DNA positive, while two remained positive from birth. In 3 cases (100%, n = 3/3) there was type specific HPV concordance between mother/newborn samples. In the 6th month, a child (2%, n = 1/49) had become HPV-DNA positive between the 1st and 6th month of life, and there was type specific HPV concordance of mother/newborn samples. All the HPV-DNA positive children (22.4%, n = 11/49) at birth and at the end first month of life (6.1%, n = 3/49) became HPV-DNA negative at the age of 6 months. The HPV-DNA positive child (2%, n = 1/49) from 1st to the 6th month of life became HPV-DNA negative between the 6th and 12th month of life and one child had anogenital warts. In the twelfth month all (100%, n = 49/49) the children studied were HPV-DNA negative. A positive and significant correlation was observed between perinatal transmission

  5. Perinatal transmission of human papilomavirus DNA

    Directory of Open Access Journals (Sweden)

    Serafini Eduardo P

    2009-06-01

    Full Text Available Abstract The purpose was to study the perinatal transmission of human papillomavirus DNA (HPV-DNA in 63 mother-newborn pairs, besides looking at the epidemiological factors involved in the viral DNA transmission. The following sampling methods were used: (1 in the pregnant woman, when was recruited, in cervix and clinical lesions of the vagina, vulva and perineal region; (2 in the newborn, (a buccal, axillary and inguinal regions; (b nasopharyngeal aspirate, and (c cord blood; (3 in the children, buccal was repeated in the 4th week and 6th and 12th month of life. HPV-DNA was identified using two methodologies: multiplex PCR (PGMY09 and MY11 primers and nested-PCR (genotypes 6/11, 16, 18, 31, 33, 42, 52 and 58. Perinatal transmission was considered when concordance was found in type-specific HPV between mother/newborn or mother/child. HPV-DNA genital was detected in 49 pregnant women submitted to delivery. Eleven newborns (22.4%, n = 11/49 were HPV-DNA positive. In 8 cases (16.3%, n = 8/49 there was type specific HPV concordance between mother/newborn samples. At the end of the first month of life three children (6.1%, n = 3/49 became HPV-DNA positive, while two remained positive from birth. In 3 cases (100%, n = 3/3 there was type specific HPV concordance between mother/newborn samples. In the 6th month, a child (2%, n = 1/49 had become HPV-DNA positive between the 1st and 6th month of life, and there was type specific HPV concordance of mother/newborn samples. All the HPV-DNA positive children (22.4%, n = 11/49 at birth and at the end first month of life (6.1%, n = 3/49 became HPV-DNA negative at the age of 6 months. The HPV-DNA positive child (2%, n = 1/49 from 1st to the 6th month of life became HPV-DNA negative between the 6th and 12th month of life and one child had anogenital warts. In the twelfth month all (100%, n = 49/49 the children studied were HPV-DNA negative. A positive and significant correlation was observed between perinatal

  6. Human DNA Ligase III Recognizes DNA Ends by Dynamic Switching between Two DNA-Bound States

    Energy Technology Data Exchange (ETDEWEB)

    Cotner-Gohara, Elizabeth; Kim, In-Kwon; Hammel, Michal; Tainer, John A.; Tomkinson, Alan E.; Ellenberger, Tom (Scripps); (Maryland-MED); (WU-MED); (LBNL)

    2010-09-13

    Human DNA ligase III has essential functions in nuclear and mitochondrial DNA replication and repair and contains a PARP-like zinc finger (ZnF) that increases the extent of DNA nick joining and intermolecular DNA ligation, yet the bases for ligase III specificity and structural variation among human ligases are not understood. Here combined crystal structure and small-angle X-ray scattering results reveal dynamic switching between two nick-binding components of ligase III: the ZnF-DNA binding domain (DBD) forms a crescent-shaped surface used for DNA end recognition which switches to a ring formed by the nucleotidyl transferase (NTase) and OB-fold (OBD) domains for catalysis. Structural and mutational analyses indicate that high flexibility and distinct DNA binding domain features in ligase III assist both nick sensing and the transition from nick sensing by the ZnF to nick joining by the catalytic core. The collective results support a 'jackknife model' in which the ZnF loads ligase III onto nicked DNA and conformational changes deliver DNA into the active site. This work has implications for the biological specificity of DNA ligases and functions of PARP-like zinc fingers.

  7. Quantification of pharmaceutical peptides in human plasma by LC-ICP-MS sulfur detection

    DEFF Research Database (Denmark)

    Møller, Laura Hyrup; Macherius, André; Hansen, Thomas Hesselhøj

    2016-01-01

    A method for quantification of a pharmaceutical peptide in human plasma was developed using gradient elution LC-ICP-MS. A membrane desolvation (MD) system was applied to remove organic solvents from the eluent prior to the detection as SO+ in the dynamic reaction cell (DRC) of the ICP-DRC-MS inst......A method for quantification of a pharmaceutical peptide in human plasma was developed using gradient elution LC-ICP-MS. A membrane desolvation (MD) system was applied to remove organic solvents from the eluent prior to the detection as SO+ in the dynamic reaction cell (DRC) of the ICP...

  8. Comparison of extraction and quantification methods of perfluorinated compounds in human plasma, serum, and whole blood

    Energy Technology Data Exchange (ETDEWEB)

    Reagen, William K. [3M Environmental Laboratory, 3M Center, Building 0260-05-N-17, St. Paul, MN 55144-1000 (United States)], E-mail: wkreagen@mmm.com; Ellefson, Mark E. [3M Environmental Laboratory, 3M Center, Building 0260-05-N-17, St. Paul, MN 55144-1000 (United States); Kannan, Kurunthachalam [Wadsworth Center, New York State Department of Health and Department of Environmental Health Sciences (United States); State University of New York at Albany, NY 12201-0509 (United States); Giesy, John P. [Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan, 44 Campus Drive, Saskatoon, SK (Canada); Department of Biology and Chemistry, Center for Coastal Pollution and Conservation, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong (China); Zoology Department, National Food Safety and Toxicology Center, Center for Integrative Toxicology, Michigan State University, E. Lansing, MI (United States); School of Environment, Nanjing University, Nanjing (China)

    2008-11-03

    Perfluorinated compounds are ubiquitous in the environment and have been reported to occur in human blood. Accurate risk assessments require accurate measurements of exposures, but identification and quantification of PFCs in biological matrices can be affected by both ion suppression and enhancement in liquid chromatography-tandem mass spectrometry techniques (LC/MS-MS). A study was conducted to quantify potential biases in LC/MS-MS quantification methods. Using isotopically labeled perfluorooctanoic acid ([{sup 13}C{sub 2}]-PFOA), perfluorononanoic acid ([{sup 13}C{sub 2}]-PFNA), and ammonium perfluorooctanesulfonate ([{sup 18}O{sub 2}]-PFOS) spiked tissues, ion-pairing extraction, solid-phase extraction, and protein precipitation sample preparation techniques were compared. Analytical accuracy was assessed using both solvent calibration and matrix-matched calibration for quantification. Data accuracy and precision of 100 {+-} 15% was demonstrated in both human sera and plasma for all three sample preparation techniques when matrix-matched calibration was used in quantification. In contrast, quantification of ion-pairing extraction data using solvent calibration in combination with a surrogate internal standard resulted in significant analytical biases for all target analytes. The accuracy of results, based on solvent calibration was highly variable and dependent on the serum and plasma matrices, the specific target analyte [{sup 13}C{sub 2}]-PFOA, [{sup 13}C{sub 2}]-PFNA, or [{sup 18}O{sub 2}]-PFOS, the target analyte concentration, the LC/MS-MS instrumentation used in data generation, and the specific surrogate internal standard used in quantification. These results suggest that concentrations of PFCs reported for human blood using surrogate internal standards in combination with external solvent calibration can be inaccurate unless biases are accounted for in data quantification.

  9. Quantifications and Modeling of Human Failure Events in a Fire PSA

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Dae Il; Kim, Kilyoo; Jang, Seung-Cheol [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2014-10-15

    USNRC and EPRI developed guidance, 'Fire Human Reliability Analysis Guidelines, NUREG-1921', for estimating human error probabilities (HEPs) for HFEs under fire conditions. NUREG-1921 classifies HFEs into four types associated with the following human actions: - Type 1: New and existing Main Control Room (MCR) actions - Type 2: New and existing ex-MCR actions - Type 3: Actions associated with using alternate shutdown means (ASD) - Type 4: Actions relating to the error of commissions (EOCs) or error of omissions (EOOs) as a result of incorrect indications (SPI) In this paper, approaches for the quantifications and modeling of HFEs related to Type 1, 2 and 3 human actions are introduced. This paper introduced the human reliability analysis process for a fire PSA of Hanul Unit 3. A multiplier of 10 was used to re-estimate the HEPs for the preexisting internal human actions. The HEPs for all ex- MCR actions were assumed to be one. New MCR human actions were quantified using the scoping analysis method of NUREG-1921. If the quantified human action were identified to be risk-significant, detailed approaches (modeling and quantification) were used for incorporating fire situations into them. Multiple HFEs for single human action were defined and they were separately and were separately quantified to incorporate the specific fire situations into them. From this study, we can confirm that the modeling as well as quantifications of human actions is very important to appropriately treat them in PSA logic structures.

  10. DNA-labelled cytidine assay for the quantification of CAG repeats.

    Science.gov (United States)

    Pérez-Bello, Dannelys; Xu, Z H; Higginson-Clarke, David; Rojas, Ana María Riverón; Le, Weidong; Rodríguez-Tanty, Chryslaine

    2008-03-30

    The sequencing procedure has been used to determine the size of the CAG repeat expansion for the diagnosis of genetic disorders. Likewise, standard polymerase chain reaction (PCR) and gel electrophoresis techniques are applied for screening large number of patients. The trinucleotide repeats (TNR) region amplification by means of the PCR procedure was initially performed using 32-P end-labelled primers and currently carried out with fluorescently end-labelled primers. The goal to obtain reliable TNR quantification assays, at low cost and short assay times, represents a challenge for the molecular diagnosis aimed at massive screening of affected populations. In the current work, we obtained preliminary results of a new methodology for the detection and size estimation of CAG expanded alleles. The assay was based on an indirect enzyme linked immunosorbent assay (ELISA) for quantifying the amount of labelled cytidines in DNA molecules. The label, 6-(p-bromobenzamido)caproyl radical, was introduced by the transamination and acylation reactions. A group of model sequences containing different numbers of CAG repeats, as well as the ATXN3 (ataxin 3) gene (from subjects suffering type 3 spinocerebellar ataxia SCA3) were used for assay standardization. The assay is simple, inexpensive, and easy to perform and differentiates distinct degrees of CAG expansions.

  11. A Method for Functional Quantification of the Reflex Effect of Human Peripheral Nerve Stimulation

    OpenAIRE

    Zehr, E. P.; Komiyama, Tomoyushi; Stein, R B

    2000-01-01

    E.P. Zehr, KOMIYAMA, T. and R.B. Stein. A Method for Functional Quantification of the Reflex Effect of Human Peripheral Nerve Stimulation. Adv. Exerc. Sports Physiol., Vol.6, No.1 pp25-32, 2000. We have developed simple method that accounts for the overrall function of reflex effects occurring in the surface electrimyogran (EMG) after human nerve stimulation. This method involves the subtraction of pre-stimulus EMG levels from EMG modulation curves obtained after human peripheral nerve stimul...

  12. Development of a novel DNA extraction method for identification and quantification of Mycobacterium avium subsp. paratuberculosis from tissue samples by real-time PCR.

    Science.gov (United States)

    Park, Kun Taek; Allen, Andrew J; Davis, William C

    2014-04-01

    Mycobacterium avium subsp. paratuberculosis (Map) is the causative agent of Johne's disease in ruminants and possibly associated with human Crohn's disease. One impediment in furthering our understanding of this potential association has been the lack of an accurate method for detection of Map in affected tissues. Real time polymerase chain reaction (RT-PCR) methods have been reported to have different sensitivities in detection of Map. This is in part attributable to the difficulties of extracting Map DNA and removing PCR inhibitors from the clinical specimens. The maximum efficiency of RT-PCR can only be achieved by using high quality DNA samples. In this study, we present a novel pre-treatment method which significantly increases Map DNA recovery and decreases PCR inhibitors (pDNA extracts prepared with pre-treatment method than without (5 vs 3). This improved Map DNA extraction method from tissue samples will make RT-PCR a more powerful tool for a wide range of applications for Map identification and quantification. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Quantification of HIV-1 proviral DNA from peripheral blood mononuclear cells using a high throughput four-competitor competitive PCR.

    Science.gov (United States)

    Comandini, U V; Sönnerborg, A; Vahlne, A; Yun, Z

    1997-12-01

    A multiple competitor PCR (mcPCR) was developed to quantify HIV-1 proviral DNA from peripheral blood mononuclear cells (PBMC). DNA extracted from a mixture of HIV infected PBMC and four size-mutated DNA competitors were co-amplified. The Cy5-fluorescence labelled PCR products were denatured by heating, separated using an automated DNA sequencer and quantified by a fragment analysis computer software. An internal standard was generated by plotting the peak areas of the four competitors against their inputs. Based on the internal standard, HIV sample DNA was quantified by extrapolating the corresponding signal. The linear range of the mcPCR was three log wide and the quantitation limit was about 20 copies of HIV DNA/10(6) PBMC. Using the mcPCR, HIV DNA was quantified from 14 long-term non progressors (LTNP) and 14 patients with advanced disease. A significantly lower copy number of HIV DNA was obtained in the LTNP (p = 0.018). These data suggest that the mcPCR is sensitive, reliable and especially useful for HIV DNA quantification of a large number of clinical samples.

  14. Human factors quantification via boundary identification of flight performance margin

    Directory of Open Access Journals (Sweden)

    Yang Changpeng

    2014-08-01

    Full Text Available A systematic methodology including a computational pilot model and a pattern recognition method is presented to identify the boundary of the flight performance margin for quantifying the human factors. The pilot model is proposed to correlate a set of quantitative human factors which represent the attributes and characteristics of a group of pilots. Three information processing components which are influenced by human factors are modeled: information perception, decision making, and action execution. By treating the human factors as stochastic variables that follow appropriate probability density functions, the effects of human factors on flight performance can be investigated through Monte Carlo (MC simulation. Kernel density estimation algorithm is selected to find and rank the influential human factors. Subsequently, human factors are quantified through identifying the boundary of the flight performance margin by the k-nearest neighbor (k-NN classifier. Simulation-based analysis shows that flight performance can be dramatically improved with the quantitative human factors.

  15. Quantification and Genotyping of Human Sapoviruses in the Llobregat River Catchment, Spain▿ †

    Science.gov (United States)

    Sano, Daisuke; Pérez-Sautu, Unai; Guix, Susana; Pintó, Rosa Maria; Miura, Takayuki; Okabe, Satoshi; Bosch, Albert

    2011-01-01

    Human sapoviruses (SaVs) were quantified and characterized in an 18-month survey conducted along the Llobregat river catchment area in Spain. Sample types included freshwater, untreated and treated wastewater, and drinking water. All genogroups were recovered, and a seasonal distribution was observed. This is the first report of SaV quantification and genotyping in the environment outside Japan. PMID:21148702

  16. Quantification and Genotyping of Human Sapoviruses in the Llobregat River Catchment, Spain▿ †

    OpenAIRE

    Sano, Daisuke; Pérez-Sautu, Unai; Guix, Susana; Pintó, Rosa Maria; Miura, Takayuki; Okabe, Satoshi; Bosch, Albert

    2010-01-01

    Human sapoviruses (SaVs) were quantified and characterized in an 18-month survey conducted along the Llobregat river catchment area in Spain. Sample types included freshwater, untreated and treated wastewater, and drinking water. All genogroups were recovered, and a seasonal distribution was observed. This is the first report of SaV quantification and genotyping in the environment outside Japan.

  17. Scientists Spot 15 Regions of Human DNA Linked to Depression

    Science.gov (United States)

    ... Spot 15 Regions of Human DNA Linked to Depression Many are located near genes involved in brain ... identified 15 regions of human DNA associated with depression. These regions may contain genes that increase the ...

  18. DNA purification from crude samples for human identification using gradient elution isotachophoresis.

    Science.gov (United States)

    Strychalski, Elizabeth A; Konek, Christopher; Butts, Erica L R; Vallone, Peter M; Henry, Alyssa C; Ross, David

    2013-09-01

    Gradient elution isotachophoresis (GEITP) was demonstrated for DNA purification, concentration, and quantification from crude samples, represented here by soiled buccal swabs, with minimal sample preparation prior to human identification using STR analysis. During GEITP, an electric field applied across leading and trailing electrolyte solutions resulted in isotachophoretic focusing of DNA at the interface between these solutions, while a pressure-driven counterflow controlled the movement of the interface from the sample reservoir into a microfluidic capillary. This counterflow also prevented particulates from fouling or clogging the capillary and reduced or eliminated contamination of the delivered DNA by PCR inhibitors. On-line DNA quantification using laser-induced fluorescence compared favorably with quantitative PCR measurements and potentially eliminates the need for quantitative PCR prior to STR analysis. GEITP promises to address the need for a rapid and robust method to deliver DNA from crude samples to aid the forensic community in human identification. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Admission Cell Free DNA as a Prognostic Factor in Burns: Quantification by Use of a Direct Rapid Fluorometric Technique

    Directory of Open Access Journals (Sweden)

    Yaron Shoham

    2014-01-01

    Full Text Available Background. Despite great advances in the treatment of burn patients, useful prognostic markers are sparse. During the past years there has been increasing interest in circulating plasma cell free DNA as a potential marker for tissue injury. We have developed a rapid direct fluorescent assay for cell free DNA quantification that allows obtaining accurate, fast, and inexpensive measurements. Objective. To use this technique for measuring plasma cell free DNA levels in burn patients and to further explore the use of cell free DNA as a potential marker of patient outcome in burns. Methods. Cell free DNA levels obtained from 14 burn victims within 6 hours of injury and 14 healthy controls were quantified by a direct rapid fluorometric assay. Results. Patient admission cell free DNA levels were significantly elevated compared with that of controls (1797 ± 1523 ng/mL versus 374 ± 245 ng/mL, P=0.004. There are statistically significant correlations between cell free DNA admission levels and burn degree (Spearman’s correlation = 0.78, P=0.001, total body surface area (Spearman’s correlation = 0.61, P=0.02, and total burn volume (Spearman’s correlation = 0.64, P=0.014. Conclusions. Admission cell free DNA levels can serve as a prognostic factor in burns and future routine use can be made possible by use of our direct rapid fluorometric assay.

  20. Hepatitis B virus DNA splicing in Lebanese blood donors and genotype A to E strains: implications for hepatitis B virus DNA quantification and infectivity.

    Science.gov (United States)

    El Chaar, Mira; El Jisr, Tamima; Allain, Jean-Pierre

    2012-10-01

    Hepatitis B virus (HBV) is one of the major viruses transmissible by blood that causes chronic infection in immunocompromised individuals. The study of 61 HBV carrier blood donors from Lebanon revealed multiple patterns of spliced HBV DNA. HBV DNA splicing was examined and quantified in samples of five genotypes and in seroconversion panels. The Lebanese sample median viral load was 1.5 ×10(2) IU/ml. All strains were genotype D, serotype ayw; 35 clustered as subgenotype D1 and 7 clustered as subgenotype D2. Three splice variants (SP1, SP1A, and Pol/S) were observed in 12 high-viral-load samples. Twenty samples of each genotype, A to E, were tested for the presence of HBV spliced DNA and SP1-specific splice variant. An unspliced HBV genome was dominant, but 100% of strains with a viral load of ≥10(5) copies/ml contained various proportions of spliced DNA. SP1 was detected in 56/100 (56%) samples in levels that correlated with the overall viral load. HBV DNA quantification with S (unspliced) and X (total DNA) regions provided different levels of viral load, with the difference corresponding to spliced DNA. During the highly infectious window period, the SP1 variant became detectable shortly after the hepatitis B surface antigen (HBsAg), suggesting a correlation between the initiation of splicing and the production of detectable levels of HBsAg. The quantification of HBV DNA with primers located outside and inside the spliced region might provide different estimations of viral load and differentiate between infectious and defective viral genomes. The role of splicing neoproteins in HBV replication and interaction with the host remains to be determined.

  1. Nanomagnetic competition assay for low-abundance protein biomarker quantification in unprocessed human sera.

    Science.gov (United States)

    Li, Yuanpeng; Srinivasan, Balasubramanian; Jing, Ying; Yao, Xiaofeng; Hugger, Marie A; Wang, Jian-Ping; Xing, Chengguo

    2010-03-31

    A novel giant magnetoresistive sensor and uniform high-magnetic-moment FeCo nanoparticles (12.8 nm)-based detecting platform with minimized detecting distance was developed for rapid biomolecule quantification from body fluids. Such a system demonstrates specific, accurate, and quick detection and quantification of interleukin-6, a low-abundance protein and a potential cancer biomarker, directly in 4 muL of unprocessed human sera. This platform is expected to facilitate the identification and validation of disease biomarkers. It may eventually lead to a low-cost personal medical device for chronic disease early detection, diagnosis, and prognosis.

  2. Development of a fast and reliable method for the assessment of microbial colonization and growth on textiles by DNA quantification.

    Science.gov (United States)

    Teufel, Linda; Schuster, K Christian; Merschak, Petra; Bechtold, Thomas; Redl, Bernhard

    2008-01-01

    There is a lack of relevant methods to assess the colonization of textiles by skin bacteria because present methods are mainly culture-based procedures. Therefore, the goal of this study was to develop a fast and sensitive culture-independent procedure for the quantification of microbial colonization and growth on textiles. We have established a suitable protocol to use DNA quantification as a reliable method for in vitroand in vivoinvestigations of textiles. For DNA extraction, a two-step procedure comprising treatment of the textile with a solution containing Triton X-100 and lysozyme for 1 h and a successive treatment by SDS and proteinase K for 2 h turned out to be most efficient. DNA extracted from textiles and fabrics was than quantified with the highly sensitive PicoGreen fluorescent dye. In vitrochallenge tests demonstrated a strong correlation between numbers of bacteria on textiles and amount of DNA extracted from textiles. Therefore, this method was used to compare different materials after in vivotrials for assessment of their susceptibility for microbial colonization and growth.

  3. Multiplex quantification of four DNA targets in one reaction with Bio-Rad droplet digital PCR system for GMO detection

    Science.gov (United States)

    Dobnik, David; Štebih, Dejan; Blejec, Andrej; Morisset, Dany; Žel, Jana

    2016-10-01

    The advantages of the digital PCR technology are already well documented until now. One way to achieve better cost efficiency of the technique is to use it in a multiplexing strategy. Droplet digital PCR platforms, which include two fluorescence filters, support at least duplex reactions and with some developments and optimization higher multiplexing is possible. The present study not only shows a development of multiplex assays in droplet digital PCR, but also presents a first thorough evaluation of several parameters in such multiplex digital PCR. Two 4-plex assays were developed for quantification of 8 different DNA targets (7 genetically modified maize events and maize endogene). Per assay, two of the targets were labelled with one fluorophore and two with another. As current analysis software does not support analysis of more than duplex, a new R- and Shiny-based web application analysis tool (http://bit.ly/ddPCRmulti) was developed that automates the analysis of 4-plex results. In conclusion, the two developed multiplex assays are suitable for quantification of GMO maize events and the same approach can be used in any other field with a need for accurate and reliable quantification of multiple DNA targets.

  4. Measurement and Quantification of Gross Human Shoulder Motion

    Directory of Open Access Journals (Sweden)

    Jeremy T. Newkirk

    2013-01-01

    Full Text Available The shoulder girdle plays an important role in the large pointing workspace that humans enjoy. The goal of this work was to characterize the human shoulder girdle motion in relation to the arm. The overall motion of the human shoulder girdle was characterized based on motion studies completed on test subjects during voluntary (natural/unforced motion. The collected data from the experiments were used to develop surface fit equations that represent the position and orientation of the glenohumeral joint for a given humeral pointing direction. These equations completely quantify gross human shoulder girdle motion relative to the humerus. The equations are presented along with goodness-of-fit results that indicate the equations well approximate the motion of the human glenohumeral joint. This is the first time the motion has been quantified for the entire workspace, and the equations provide a reference against which to compare future work.

  5. DNA methylation and healthy human aging.

    Science.gov (United States)

    Jones, Meaghan J; Goodman, Sarah J; Kobor, Michael S

    2015-12-01

    The process of aging results in a host of changes at the cellular and molecular levels, which include senescence, telomere shortening, and changes in gene expression. Epigenetic patterns also change over the lifespan, suggesting that epigenetic changes may constitute an important component of the aging process. The epigenetic mark that has been most highly studied is DNA methylation, the presence of methyl groups at CpG dinucleotides. These dinucleotides are often located near gene promoters and associate with gene expression levels. Early studies indicated that global levels of DNA methylation increase over the first few years of life and then decrease beginning in late adulthood. Recently, with the advent of microarray and next-generation sequencing technologies, increases in variability of DNA methylation with age have been observed, and a number of site-specific patterns have been identified. It has also been shown that certain CpG sites are highly associated with age, to the extent that prediction models using a small number of these sites can accurately predict the chronological age of the donor. Together, these observations point to the existence of two phenomena that both contribute to age-related DNA methylation changes: epigenetic drift and the epigenetic clock. In this review, we focus on healthy human aging throughout the lifetime and discuss the dynamics of DNA methylation as well as how interactions between the genome, environment, and the epigenome influence aging rates. We also discuss the impact of determining 'epigenetic age' for human health and outline some important caveats to existing and future studies.

  6. Accurate measurement of circulating mitochondrial DNA content from human blood samples using real-time quantitative PCR.

    Science.gov (United States)

    Ajaz, Saima; Czajka, Anna; Malik, Afshan

    2015-01-01

    We describe a protocol to accurately measure the amount of human mitochondrial DNA (MtDNA) in peripheral blood samples which can be modified to quantify MtDNA from other body fluids, human cells, and tissues. This protocol is based on the use of real-time quantitative PCR (qPCR) to quantify the amount of MtDNA relative to nuclear DNA (designated the Mt/N ratio). In the last decade, there have been increasing numbers of studies describing altered MtDNA or Mt/N in circulation in common nongenetic diseases where mitochondrial dysfunction may play a role (for review see Malik and Czajka, Mitochondrion 13:481-492, 2013). These studies are distinct from those looking at genetic mitochondrial disease and are attempting to identify acquired changes in circulating MtDNA content as an indicator of mitochondrial function. However, the methodology being used is not always specific and reproducible. As more than 95 % of the human mitochondrial genome is duplicated in the human nuclear genome, it is important to avoid co-amplification of nuclear pseudogenes. Furthermore, template preparation protocols can also affect the results because of the size and structural differences between the mitochondrial and nuclear genomes. Here we describe how to (1) prepare DNA from blood samples; (2) pretreat the DNA to prevent dilution bias; (3) prepare dilution standards for absolute quantification using the unique primers human mitochondrial genome forward primer (hMitoF3) and human mitochondrial genome reverse primer(hMitoR3) for the mitochondrial genome, and human nuclear genome forward primer (hB2MF1) and human nuclear genome reverse primer (hB2MR1) primers for the human nuclear genome; (4) carry out qPCR for either relative or absolute quantification from test samples; (5) analyze qPCR data; and (6) calculate the sample size to adequately power studies. The protocol presented here is suitable for high-throughput use.

  7. Absolute Quantification of the Host-To-Parasite DNA Ratio in Theileria parva-Infected Lymphocyte Cell Lines.

    Directory of Open Access Journals (Sweden)

    Hanzel T Gotia

    Full Text Available Theileria parva is a tick-transmitted intracellular apicomplexan pathogen of cattle in sub-Saharan Africa that causes East Coast fever (ECF. ECF is an acute fatal disease that kills over one million cattle annually, imposing a tremendous burden on African small-holder cattle farmers. The pathology and level of T. parva infections in its wildlife host, African buffalo (Syncerus caffer, and in cattle are distinct. We have developed an absolute quantification method based on quantitative PCR (qPCR in which recombinant plasmids containing single copy genes specific to the parasite (apical membrane antigen 1 gene, ama1 or the host (hypoxanthine phosphoribosyltransferase 1, hprt1 are used as the quantification reference standards. Our study shows that T. parva and bovine cells are present in similar numbers in T. parva-infected lymphocyte cell lines and that consequently, due to its much smaller genome size, T. parva DNA comprises between 0.9% and 3% of the total DNA samples extracted from these lines. This absolute quantification assay of parasite and host genome copy number in a sample provides a simple and reliable method of assessing T. parva load in infected bovine lymphocytes, and is accurate over a wide range of host-to-parasite DNA ratios. Knowledge of the proportion of target DNA in a sample, as enabled by this method, is essential for efficient high-throughput genome sequencing applications for a variety of intracellular pathogens. This assay will also be very useful in future studies of interactions of distinct host-T. parva stocks and to fully characterize the dynamics of ECF infection in the field.

  8. Cytochrome c oxidase subunit 1-based human RNA quantification to enhance mRNA profiling in forensic biology

    Directory of Open Access Journals (Sweden)

    Dong Zhao

    2017-01-01

    Full Text Available RNA analysis offers many potential applications in forensic science, and molecular identification of body fluids by analysis of cell-specific RNA markers represents a new technique for use in forensic cases. However, due to the nature of forensic materials that often admixed with nonhuman cellular components, human-specific RNA quantification is required for the forensic RNA assays. Quantification assay for human RNA has been developed in the present study with respect to body fluid samples in forensic biology. The quantitative assay is based on real-time reverse transcription-polymerase chain reaction of mitochondrial RNA cytochrome c oxidase subunit I and capable of RNA quantification with high reproducibility and a wide dynamic range. The human RNA quantification improves the quality of mRNA profiling in the identification of body fluids of saliva and semen because the quantification assay can exclude the influence of nonhuman components and reduce the adverse affection from degraded RNA fragments.

  9. Accurate episomal HIV 2-LTR circles quantification using optimized DNA isolation and droplet digital PCR

    OpenAIRE

    Eva Malatinkova; Maja Kiselinova; Pawel Bonczkowski; Wim Trypsteen; Peter Messiaen; Jolien Vermeire; Bruno Verhasselt; Karen Vervisch; Linos Vandekerckhove; Ward De Spiegelaere

    2014-01-01

    Introduction: In HIV-infected patients on combination antiretroviral therapy (cART), the detection of episomal HIV 2-LTR circles is a potential marker for ongoing viral replication. Quantification of 2-LTR circles is based on quantitative PCR or more recently on digital PCR assessment, but is hampered due to its low abundance. Sample pre-PCR processing is a critical step for 2-LTR circles quantification, which has not yet been sufficiently evaluated in patient derived samples. Materials and M...

  10. Non-invasive quantification of hemodynamics in human choriocapillaries

    Science.gov (United States)

    Yu, Huidan (Whitney); Chen, Rou; An, Senyou; McDonough, James; Gelfand, Bradley; Yao, Jun

    2016-11-01

    The development of retinal disease is inextricably linked to defects in the choroidal blood supply. However, to date a description of the hemodynamics in the human choroidal circulation is lacking. Through high resolution choroidal vascular network mapped from immunofluorescent labeling and confocal microscopy of human cadaver donor eyes. We noninvasively quantify hemodynamics including velocity, pressure, and wall-shear stress (WSS) in choriocapillaries through mesoscale modeling and GPU-accelerated fast computation. This is the first-ever map of hemodynamic parameters (WSS, pressure, and velocity) in anatomically accurate human choroidal vasculature in health and disease. The pore scale simulation results are used to evaluate porous media models with the same porosity and boundary conditions. School of Medicine, Indiana University.

  11. Sensitive targeted quantification of ERK phosphorylation dynamics and stoichiometry in human cells without affinity enrichment.

    Science.gov (United States)

    Shi, Tujin; Gao, Yuqian; Gaffrey, Matthew J; Nicora, Carrie D; Fillmore, Thomas L; Chrisler, William B; Gritsenko, Marina A; Wu, Chaochao; He, Jintang; Bloodsworth, Kent J; Zhao, Rui; Camp, David G; Liu, Tao; Rodland, Karin D; Smith, Richard D; Wiley, H Steven; Qian, Wei-Jun

    2015-01-20

    Targeted mass spectrometry is a promising technology for site-specific quantification of posttranslational modifications. However, a major constraint is the limited sensitivity for quantifying low-abundance PTMs, requiring the use of affinity reagents for enrichment. Herein, we demonstrate the direct site-specific quantification of ERK phosphorylation isoforms (pT, pY, pTpY) and their relative stoichiometry using a sensitive targeted MS approach termed high-pressure, high-resolution separations with intelligent selection, and multiplexing (PRISM). PRISM provides effective enrichment of target peptides into a given fraction from complex mixture, followed by selected reaction monitoring quantification. Direct quantification of ERK phosphorylation in human mammary epithelial cells (HMEC) was demonstrated from as little as 25 μg tryptic peptides from whole cell lysates. Compared to immobilized metal-ion affinity chromatography, PRISM provided ∼10-fold higher signal intensities, presumably due to the better peptide recovery of PRISM. This approach was applied to quantify ERK phosphorylation dynamics in HMEC treated by different doses of epidermal growth factor at both the peak activation (10 min) and steady state (2 h). The maximal ERK activation was observed with 0.3 and 3 ng/mL doses for 10 min and 2 h time points, respectively. The dose-response profiles of individual phosphorylated isoforms showed that singly phosphorylated pT-ERK never increases significantly, while the increase of pY-ERK paralleled that of pTpY-ERK. This data supports for a processive, rather than distributed model of ERK phosphorylation. The PRISM-SRM quantification of protein phosphorylation illustrates the potential for simultaneous quantification of multiple PTMs.

  12. Human error in strabismus surgery: Quantification with a sensitivity analysis

    NARCIS (Netherlands)

    S. Schutte (Sander); J.R. Polling; F.C.T. van der Helm (Frans); H.J. Simonsz (Huib)

    2009-01-01

    textabstractBackground: Reoperations are frequently necessary in strabismus surgery. The goal of this study was to analyze human-error related factors that introduce variability in the results of strabismus surgery in a systematic fashion. Methods: We identified the primary factors that influence th

  13. Human error in strabismus surgery: quantification with a sensitivity analysis

    NARCIS (Netherlands)

    Schutte, S.; Polling, J.R.; Van der Helm, F.C.T.; Simonsz, H.J.

    2008-01-01

    Background- Reoperations are frequently necessary in strabismus surgery. The goal of this study was to analyze human-error related factors that introduce variability in the results of strabismus surgery in a systematic fashion. Methods- We identified the primary factors that influence the outcome of

  14. Identification and quantification of diadenosine polyphosphate concentrations in human plasma

    DEFF Research Database (Denmark)

    Jankowski, Joachim; Jankowski, Vera; Laufer, Udo;

    2003-01-01

    Diadenosine polyphosphates have been demonstrated to be involved in the control of vascular tone as well as the growth of vascular smooth muscle cells and hence, possibly, in atherogenesis. In this study we investigated the question of whether diadenosine polyphosphates are present in human plasm...

  15. Quantification of sensory information in human balance control

    NARCIS (Netherlands)

    Kooij, van der Herman; Koopman, Bart; Jacobs, Ron; Mergner, Thomas; Grootenboer, Henk

    1998-01-01

    A human balance control model is developed, which includes the different sensory systems as well as neural time delays. The model is based on optimal control theory. Platform perturbation experiments were done to quantify the precision of the different sensory systems by matching model predictions w

  16. Rational design of human DNA ligase inhibitors that target cellular DNA replication and repair.

    Science.gov (United States)

    Chen, Xi; Zhong, Shijun; Zhu, Xiao; Dziegielewska, Barbara; Ellenberger, Tom; Wilson, Gerald M; MacKerell, Alexander D; Tomkinson, Alan E

    2008-05-01

    Based on the crystal structure of human DNA ligase I complexed with nicked DNA, computer-aided drug design was used to identify compounds in a database of 1.5 million commercially available low molecular weight chemicals that were predicted to bind to a DNA-binding pocket within the DNA-binding domain of DNA ligase I, thereby inhibiting DNA joining. Ten of 192 candidates specifically inhibited purified human DNA ligase I. Notably, a subset of these compounds was also active against the other human DNA ligases. Three compounds that differed in their specificity for the three human DNA ligases were analyzed further. L82 inhibited DNA ligase I, L67 inhibited DNA ligases I and III, and L189 inhibited DNA ligases I, III, and IV in DNA joining assays with purified proteins and in cell extract assays of DNA replication, base excision repair, and nonhomologous end-joining. L67 and L189 are simple competitive inhibitors with respect to nicked DNA, whereas L82 is an uncompetitive inhibitor that stabilized complex formation between DNA ligase I and nicked DNA. In cell culture assays, L82 was cytostatic whereas L67 and L189 were cytotoxic. Concordant with their ability to inhibit DNA repair in vitro, subtoxic concentrations of L67 and L189 significantly increased the cytotoxicity of DNA-damaging agents. Interestingly, the ligase inhibitors specifically sensitized cancer cells to DNA damage. Thus, these novel human DNA ligase inhibitors will not only provide insights into the cellular function of these enzymes but also serve as lead compounds for the development of anticancer agents.

  17. Isolation and Quantification of Glycosaminoglycans from Human Hair Shaft

    Science.gov (United States)

    Bonovas, Stefanos; Sitaras, Nikolaos

    2016-01-01

    Background There is evidence that glycosaminoglycans (GAGs) are present in the hair shaft within the follicle but there are no studies regarding GAGs isolation and measurement in the human hair shaft over the scalp surface, it means, in the free hair shaft. Objective The purpose of our research was to isolate and measure the total GAGs from human free hair shaft. Methods Seventy-five healthy individuals participated in the study, 58 adults, men and women over the age of 50 and 17 children (aged 4~9). GAGs in hair samples, received from the parietal and the occipital areas, were isolated with 4 M guanidine HCl and measured by the uronic acid-carbazole reaction assay. Results GAGs concentration was significantly higher in the occipital area than in the parietal area, in all study groups. GAG levels from both areas were significantly higher in children than in adults. GAG levels were not associated with gender, hair color or type. Conclusion We report the presence of GAGs in the human free hair shaft and the correlation of hair GAG levels with the scalp area and participants' age. PMID:27746630

  18. Liquid chromatography-tandem mass spectrometry quantification of 6-thioguanine in DNA using endogenous guanine as internal standard

    DEFF Research Database (Denmark)

    Jacobsen, Jack Hummeland; Schmiegelow, Kjeld; Nersting, Jakob

    2012-01-01

    Thiopurines are S-substituted antimetabolites that are widely used in the treatment of hematological malignancies and as immunosuppressants. Because of extensive inter-individual variation in drug disposition and the significant toxicity associated with thiopurine therapy, there is a need...... was estimated at 63% (RSD 26%), which is corrected for by the internal standard resulting in stable quantification. The TG levels found were above the LOQ in 18 out of 18 childhood leukemia patients on 6-mercaptopurine/methotrexate maintenance therapy (median 377, range 45-1190 fmol/μg DNA) with intra...

  19. DNA quantification as prognostic factor in a case of acinar cell carcinoma of the parotid gland, diagnosed by FNA.

    Science.gov (United States)

    Azúa-Romeo, Javier; Sánchez-Garnica, Juan Carlos; Azúa-Blanco, Javier; Tovar-Lázaro, Mayte

    2005-01-01

    Hereby we present a case of a 43-years-old male who complained of a three years history preauricular painful mass. Fine needle aspiration cytology was performed, diagnosing of compatible with acinar cell carcinoma, thus DNA quantification by image cytometry was carried out. Biological parameters studied (ploidy, S-phase, 5-c exceeding rate) showed that it is a low grade of malignancy lesion. Total parotidectomy conservative of facial nerve was recommended, without regional lymphadenectomy. Patient remains, one year later, asymptomatic and free of disease.

  20. Micropatterned comet assay enables high throughput and sensitive DNA damage quantification.

    Science.gov (United States)

    Ge, Jing; Chow, Danielle N; Fessler, Jessica L; Weingeist, David M; Wood, David K; Engelward, Bevin P

    2015-01-01

    The single cell gel electrophoresis assay, also known as the comet assay, is a versatile method for measuring many classes of DNA damage, including base damage, abasic sites, single strand breaks and double strand breaks. However, limited throughput and difficulties with reproducibility have limited its utility, particularly for clinical and epidemiological studies. To address these limitations, we created a microarray comet assay. The use of a micrometer scale array of cells increases the number of analysable comets per square centimetre and enables automated imaging and analysis. In addition, the platform is compatible with standard 24- and 96-well plate formats. Here, we have assessed the consistency and sensitivity of the microarray comet assay. We showed that the linear detection range for H2O2-induced DNA damage in human lymphoblastoid cells is between 30 and 100 μM, and that within this range, inter-sample coefficient of variance was between 5 and 10%. Importantly, only 20 comets were required to detect a statistically significant induction of DNA damage for doses within the linear range. We also evaluated sample-to-sample and experiment-to-experiment variation and found that for both conditions, the coefficient of variation was lower than what has been reported for the traditional comet assay. Finally, we also show that the assay can be performed using a 4× objective (rather than the standard 10× objective for the traditional assay). This adjustment combined with the microarray format makes it possible to capture more than 50 analysable comets in a single image, which can then be automatically analysed using in-house software. Overall, throughput is increased more than 100-fold compared to the traditional assay. Together, the results presented here demonstrate key advances in comet assay technology that improve the throughput, sensitivity, and robustness, thus enabling larger scale clinical and epidemiological studies.

  1. Two DNA-binding and Nick Recognition Modules in Human DNA Ligase III*

    OpenAIRE

    Cotner-Gohara, Elizabeth; Kim, In-Kwon; Tomkinson, Alan E.; Ellenberger, Tom

    2008-01-01

    Human DNA ligase III contains an N-terminal zinc finger domain that binds to nicks and gaps in DNA. This small domain has been described as a DNA nick sensor, but it is not required for DNA nick joining activity in vitro. In light of new structural information for mammalian ligases, we measured the DNA binding affinity and specificity of each domain of DNA ligase III. These studies identified two separate, independent DNA-binding modules in DNA ligase III that each bin...

  2. Investigation of DNA repair in human oocytes and preimplantation embryos

    OpenAIRE

    Jaroudi, S.

    2010-01-01

    DNA repair genes are expressed in mammalian embryos and in human germinal vesicles, however, little is known about DNA repair in human preimplantation embryos. This project had three aims: 1) to produce a DNA repair profile of human MII oocytes and blastocysts using expression arrays and identify repair pathways that may be active before and after embryonic genome activation; 2) to design an in vitro functional assay that targeted mismatch repair and which could be applied to human oocytes...

  3. Correction of the lack of commutability between plasmid DNA and genomic DNA for quantification of genetically modified organisms using pBSTopas as a model.

    Science.gov (United States)

    Zhang, Li; Wu, Yuhua; Wu, Gang; Cao, Yinglong; Lu, Changming

    2014-10-01

    Plasmid calibrators are increasingly applied for polymerase chain reaction (PCR) analysis of genetically modified organisms (GMOs). To evaluate the commutability between plasmid DNA (pDNA) and genomic DNA (gDNA) as calibrators, a plasmid molecule, pBSTopas, was constructed, harboring a Topas 19/2 event-specific sequence and a partial sequence of the rapeseed reference gene CruA. Assays of the pDNA showed similar limits of detection (five copies for Topas 19/2 and CruA) and quantification (40 copies for Topas 19/2 and 20 for CruA) as those for the gDNA. Comparisons of plasmid and genomic standard curves indicated that the slopes, intercepts, and PCR efficiency for pBSTopas were significantly different from CRM Topas 19/2 gDNA for quantitative analysis of GMOs. Three correction methods were used to calibrate the quantitative analysis of control samples using pDNA as calibrators: model a, or coefficient value a (Cva); model b, or coefficient value b (Cvb); and the novel model c or coefficient formula (Cf). Cva and Cvb gave similar estimated values for the control samples, and the quantitative bias of the low concentration sample exceeded the acceptable range within ±25% in two of the four repeats. Using Cfs to normalize the Ct values of test samples, the estimated values were very close to the reference values (bias -13.27 to 13.05%). In the validation of control samples, model c was more appropriate than Cva or Cvb. The application of Cf allowed pBSTopas to substitute for Topas 19/2 gDNA as a calibrator to accurately quantify the GMO.

  4. Discrimination and quantification of autofluorescence spectra of human lung cells

    Science.gov (United States)

    Rahmani, Mahya; Khani, Mohammad Mehdi; Khazaei Koohpar, Zeinab; Molik, Paria

    2016-10-01

    To study laser-induced autofluorescence spectroscopy of the human lung cell line, we evaluated the native fluorescence properties of cancer QU-DB and normal MRC-5 human lung cells during continuous exposure to 405 nm laser light. Two emission bands centered at ~470 nm and ~560 nm were observed. These peaks are most likely attributable to mitochondrial fluorescent reduced nicotinamide adenine dinucleotide and riboflavin fluorophores, respectively. This article highlights lung cell autofluorescence characterization and signal discrimination by collective investigation of different spectral features. The absolute intensity, the spectral shape factor or redox ratio, the full width of half-maximum and the full width of quarter maximum was evaluated. Moreover, the intensity ratio, the area under the peak and the area ratio as a contrast factor for normal and cancerous cells were also calculated. Among all these features it seems that the contrast factor precisely and significantly discriminates the spectral differences of normal and cancerous lung cells. On the other hand, the relative quantum yield for both cell types were found by comparing the quantum yield of an unknown compound with known fluorescein sodium as a reference solution.

  5. Quantitation of the residual DNA from rice-derived recombinant human serum albumin.

    Science.gov (United States)

    Chen, Zhen; Dai, Huixia; Liu, Zhenwei; Zhang, Liping; Pang, Jianlei; Ou, Jiquan; Yang, Daichang

    2014-04-01

    Residual DNA in recombinant protein pharmaceuticals can potentially cause safety issues in clinical applications; thus, maximum residual limit has been established by drug safety authorities. Assays for residual DNA in Escherichia coli, yeast, and Chinese hamster ovary (CHO) cell expression systems have been established, but no rice residual DNA assay for rice expression systems has been designed. To develop an assay for the quantification of residual DNA that is produced from rice seed, we established a sensitive assay using quantitative real-time polymerase chain reaction (qPCR) based on the 5S ribosomal RNA (rRNA) genes. We found that a 40-cycle qPCR exhibited a linear response when the template concentration was in the range of 2×10(4) to 0.2pg of DNA per reaction in TaqMan and SYBR Green I assays. The amplification efficiency was 103 to 104%, and the amount of residual DNA from recombinant human serum albumin from Oryza sativa (OsrHSA) was less than 3.8ng per dosage, which was lower than that recommended by the World Health Organization (WHO). Our results indicate that the current purification protocol could efficiently remove residual DNA during manufacturing and processing. Furthermore, this protocol could be viable in other cereal crop endosperm expression systems for developing a residual DNA quantitation assay using the highly conserved 5S rRNA gene of the crops.

  6. A VISION OF QUANTIFICATION OF SUSTAINABLE HUMAN DEVELOPMENT

    Directory of Open Access Journals (Sweden)

    Neyda Ibáñez

    2015-07-01

    Full Text Available The present study aimed to propose new measurement components of sustainable human development based on the historical and theoretical development path. The research assumes ontoepistemological position based on positivism, addressing technical survey and instrument of written questionnaire administered to thirty-one (31 experts in the field of knowledge, and whose analysis allowed us to conclude that traditional models to measure development are insufficient to demonstrate the reality of nations. Therefore, the proposed measure is derived in seven components: ethical, spiritual and cultural addition to the formally established by Munasinghe (1993, 2011 and the UN (2012: economic, social, environmental, institutional constituted in total, by selecting one hundred fifty five (155 variables.

  7. Quantification of dsDNA using the Hitachi F-7000 Fluorescence Spectrophotometer and PicoGreen dye.

    Science.gov (United States)

    Moreno, Luis A; Cox, Kendra L

    2010-11-05

    Quantification of DNA, especially in small concentrations, is an important task with a wide range of biological applications including standard molecular biology assays such as synthesis and purification of DNA, diagnostic applications such as quantification of DNA amplification products, and detection of DNA molecules in drug preparations. During this video we will demonstrate the capability of the Hitachi F-7000 Fluorescence Spectrophotometer equipped with a Micro Plate Reader accessory to perform dsDNA quantification using Molecular Probes Quant-it PicoGreen dye reagent kit. The F-7000 Fluorescence Spectrophotometer offers high sensitivity and high speed measurements. It is a highly flexible system capable of measuring fluorescence, luminescence, and phosphorescence. Several measuring modes are available, including wavelength scan, time scan, photometry and 3-D scan measurement. The spectrophotometer has sensitivity in the range of 50 picomoles of fluorescein when using a 300 μL sample volume in the microplate, and is capable of measuring scan speeds of 60,000 nm/minute. It also has a wide dynamic range of up to 5 orders of magnitude which allows for the use of calibration curves over a wide range of concentrations. The optical system uses all reflective optics for maximum energy and sensitivity. The standard wavelength range is 200 to 750 nm, and can be extended to 900 nm when using one of the optional near infrared photomultipliers. The system allows optional temperature control for the plate reader from 5 to 60 degrees Celsius using an optional external temperature controlled liquid circulator. The microplate reader allows for the use of 96 well microplates, and the measuring speed for 96 wells is less than 60 seconds when using the kinetics mode. Software controls for the F-7000 and Microplate Reader are also highly flexible. Samples may be set in either column or row formats, and any combination of wells may be chosen for sample measurements. This allows

  8. Homologous DNA strand exchange activity of the human mitochondrial DNA helicase TWINKLE

    OpenAIRE

    Sen, Doyel; Patel, Gayatri; Smita S Patel

    2016-01-01

    A crucial component of the human mitochondrial DNA replisome is the ring-shaped helicase TWINKLE—a phage T7-gene 4-like protein expressed in the nucleus and localized in the human mitochondria. Our previous studies showed that despite being a helicase, TWINKLE has unique DNA annealing activity. At the time, the implications of DNA annealing by TWINKLE were unclear. Herein, we report that TWINKLE uses DNA annealing function to actively catalyze strand-exchange reaction between the unwinding su...

  9. A sensitive high performance liquid chromatography assay for the quantification of doxorubicin associated with DNA in tumor and tissues.

    Science.gov (United States)

    Lucas, Andrew T; O'Neal, Sara K; Santos, Charlene M; White, Taylor F; Zamboni, William C

    2016-02-01

    Doxorubicin, a widely used anticancer agent, exhibits antitumor activity against a wide variety of malignancies. The drug exerts its cytotoxic effects by binding to and intercalating within the DNA of tumor and tissue cells. However, current assays are unable to accurately determine the concentration of the intracellular active form of doxorubicin. Thus, the development of a sample processing method and a high-performance liquid chromatography (HPLC) methodology was performed in order to quantify doxorubicin that is associated with DNA in tumors and tissues, which provided an intracellular cytotoxic measure of doxorubicin exposure after administration of small molecule and nanoparticle formulations of doxorubicin. The assay uses daunorubicin as an internal standard; liquid-liquid phase extraction to isolate drug associated with DNA; a Shimadzu HPLC with fluorescence detection equipped with a Phenomenex Luna C18 (2μm, 2.0×100mm) analytical column and a gradient mobile phase of 0.1% formic acid in water or acetonitrile for separation and quantification. The assay has a lower limit of detection (LLOQ) of 10ng/mL and is shown to be linear up to 3000ng/mL. The intra- and inter-day precision of the assay expressed as a coefficient of variation (CV%) ranged from 4.01 to 8.81%. Furthermore, the suitability of this assay for measuring doxorubicin associated with DNA in vivo was demonstrated by using it to quantify the doxorubicin concentration within tumor samples from SKOV3 and HEC1A mice obtained 72h after administration of PEGylated liposomal doxorubicin (Doxil(®); PLD) at 6mg/kg IV x 1. This HPLC assay allows for sensitive intracellular quantification of doxorubicin and will be an important tool for future studies evaluating intracellular pharmacokinetics of doxorubicin and various nanoparticle formulations of doxorubicin.

  10. Comparison of droplet digital PCR to real-time PCR for quantification of hepatitis B virus DNA.

    Science.gov (United States)

    Tang, Hui; Cai, Qingchun; Li, Hu; Hu, Peng

    2016-06-16

    Quantitative real-time PCR (qPCR) has been widely implemented for clinical hepatitis B viral load testing, but a lack of standardization and relatively poor precision hinder its usefulness. Droplet digital PCR (ddPCR) is a promising tool that offers high precision and direct quantification. In this study, we compared the ddPCR QX100 platform by Bio-Rad with the CFX384 Touch Real-Time PCR Detection System (Bio-Rad, USA) to detect serial plasmid DNA dilutions of known concentrations as well as HBV DNA extracted from patient serum samples. Both methods showed a high degree of linearity and quantitative correlation. However, ddPCR assays generated more reproducible results and detected lower copy numbers than qPCR assays. Patient sample quantifications by ddPCR and qPCR were highly agreeable based on the Bland-Altman analysis. Collectively, our findings demonstrate that ddPCR offers improved analytical sensitivity and specificity for HBV measurements and is suitable for clinical HBV detection.

  11. Automated quantification of aligned collagen for human breast carcinoma prognosis

    Directory of Open Access Journals (Sweden)

    Jeremy S Bredfeldt

    2014-01-01

    Full Text Available Background: Mortality in cancer patients is directly attributable to the ability of cancer cells to metastasize to distant sites from the primary tumor. This migration of tumor cells begins with a remodeling of the local tumor microenvironment, including changes to the extracellular matrix and the recruitment of stromal cells, both of which facilitate invasion of tumor cells into the bloodstream. In breast cancer, it has been proposed that the alignment of collagen fibers surrounding tumor epithelial cells can serve as a quantitative image-based biomarker for survival of invasive ductal carcinoma patients. Specific types of collagen alignment have been identified for their prognostic value and now these tumor associated collagen signatures (TACS are central to several clinical specimen imaging trials. Here, we implement the semi-automated acquisition and analysis of this TACS candidate biomarker and demonstrate a protocol that will allow consistent scoring to be performed throughout large patient cohorts. Methods: Using large field of view high resolution microscopy techniques, image processing and supervised learning methods, we are able to quantify and score features of collagen fiber alignment with respect to adjacent tumor-stromal boundaries. Results: Our semi-automated technique produced scores that have statistically significant correlation with scores generated by a panel of three human observers. In addition, our system generated classification scores that accurately predicted survival in a cohort of 196 breast cancer patients. Feature rank analysis reveals that TACS positive fibers are more well-aligned with each other, are of generally lower density, and terminate within or near groups of epithelial cells at larger angles of interaction. Conclusion: These results demonstrate the utility of a supervised learning protocol for streamlining the analysis of collagen alignment with respect to tumor stromal boundaries.

  12. QUANTIFICATION OF GLOMERULAR EPITHELIAL-CELL ADHESION BY USING ANTI-DNA ANTIBODIES IN ELISA

    NARCIS (Netherlands)

    COERS, W; SMEENK, RJT; SALANT, DJ; WEENING, JJ

    1992-01-01

    A sensitive and reproducible microassay is described for quantification of adhesion of cells to matrix-coated 96-wells plates under different experimental conditions. For this purpose glomerular visceral epithelial cells (GVEC) were used. Attached GVEC were fixed with methanol and incubated with a m

  13. Quantification of cellular uptake of DNA nanostructures by qPCR

    DEFF Research Database (Denmark)

    Okholm, Anders Hauge; Nielsen, Jesper Sejrup; Vinther, Mathias

    2014-01-01

    DNA nanostructures facilitating drug delivery are likely soon to be realized. In the past few decades programmed self-assembly of DNA building blocks have successfully been employed to construct sophisticated nanoscale objects. By conjugating functionalities to DNA, other molecules such as peptid...

  14. Diagnosis of human herpes virus 1 and 2 (HHV-1 and HHV-2): use of a synthetic standard curve for absolute quantification by real time polymerase chain reaction.

    Science.gov (United States)

    Lima, Lyana Rodrigues Pinto; Silva, Amanda Perse da; Schmidt-Chanasit, Jonas; Paula, Vanessa Salete de

    2017-03-01

    The use of quantitative real time polymerase chain reaction (qPCR) for herpesvirus detection has improved the sensitivity and specificity of diagnosis, as it is able to detect shedding episodes in the absence of clinical lesions and diagnose clinical specimens that have low viral loads. With an aim to improve the detection and quantification of herpesvirus by qPCR, synthetic standard curves for human herpesvirus 1 and 2 (HHV-1 and HHV-2) targeting regions gD and gG, respectively, were designed and evaluated. The results show that synthetic curves can replace DNA standard curves in diagnostic herpes qPCR.

  15. Diagnosis of human herpes virus 1 and 2 (HHV-1 and HHV-2): use of a synthetic standard curve for absolute quantification by real time polymerase chain reaction

    Science.gov (United States)

    Lima, Lyana Rodrigues Pinto; da Silva, Amanda Perse; Schmidt-Chanasit, Jonas; de Paula, Vanessa Salete

    2017-01-01

    The use of quantitative real time polymerase chain reaction (qPCR) for herpesvirus detection has improved the sensitivity and specificity of diagnosis, as it is able to detect shedding episodes in the absence of clinical lesions and diagnose clinical specimens that have low viral loads. With an aim to improve the detection and quantification of herpesvirus by qPCR, synthetic standard curves for human herpesvirus 1 and 2 (HHV-1 and HHV-2) targeting regions gD and gG, respectively, were designed and evaluated. The results show that synthetic curves can replace DNA standard curves in diagnostic herpes qPCR. PMID:28225902

  16. Translesion synthesis mechanisms depend on the nature of DNA damage in UV-irradiated human cells

    Science.gov (United States)

    Quinet, Annabel; Martins, Davi Jardim; Vessoni, Alexandre Teixeira; Biard, Denis; Sarasin, Alain; Stary, Anne; Menck, Carlos Frederico Martins

    2016-01-01

    Ultraviolet-induced 6-4 photoproducts (6-4PP) and cyclobutane pyrimidine dimers (CPD) can be tolerated by translesion DNA polymerases (TLS Pols) at stalled replication forks or by gap-filling. Here, we investigated the involvement of Polη, Rev1 and Rev3L (Polζ catalytic subunit) in the specific bypass of 6-4PP and CPD in repair-deficient XP-C human cells. We combined DNA fiber assay and novel methodologies for detection and quantification of single-stranded DNA (ssDNA) gaps on ongoing replication forks and postreplication repair (PRR) tracts in the human genome. We demonstrated that Rev3L, but not Rev1, is required for postreplicative gap-filling, while Polη and Rev1 are responsible for TLS at stalled replication forks. Moreover, specific photolyases were employed to show that in XP-C cells, CPD arrest replication forks, while 6-4PP are responsible for the generation of ssDNA gaps and PRR tracts. On the other hand, in the absence of Polη or Rev1, both types of lesion block replication forks progression. Altogether, the data directly show that, in the human genome, Polη and Rev1 bypass CPD and 6-4PP at replication forks, while only 6-4PP are also tolerated by a Polζ-dependent gap-filling mechanism, independent of S phase. PMID:27095204

  17. Comparison of the ABI 7700 System (TaqMan) and Competitive PCR for Quantification of IS6110 DNA in Sputum during Treatment of Tuberculosis

    Science.gov (United States)

    Desjardin, L. e.; Chen, Y.; Perkins, M. D.; Teixeira, L.; Cave, M. D.; Eisenach, K. D.

    1998-01-01

    Mycobacterium tuberculosis can persist in sputum for long periods of time after the initiation of antituberculosis chemotherapy. The purpose of this study was to determine whether quantitative estimates of M. tuberculosis DNA in sputum correlate with the numbers of viable bacilli and thus measure the therapeutic response of patients during treatment. Two methods of M. tuberculosis DNA quantification were examined by using DNA isolated from sputum specimens serially collected during the course of chemotherapy. A competitive PCR assay was compared to an automated system of real-time quantification with the ABI Prism 7700 Sequence Detection System (TaqMan). The ABI 7700 system uses standard PCR in conjunction with a fluorogenic probe in which the intensity of fluorescence is proportional to the amount of target DNA present. The results showed that both PCR systems are reproducible and accurate. The amounts of M. tuberculosis DNA quantified in sputum corresponded well with the numbers of acid-fast bacilli (AFB) counted by microscopy. Before initiation of antituberculosis therapy, measures of AFB, M. tuberculosis DNA, and cultivable bacilli were similar, suggesting that quantification of DNA is a good method for measuring the initial bacillary load. However, the rate of disappearance of both AFB and M. tuberculosis DNA did not correlate with the decline in cultivable bacilli in the specimen; therefore, these tests are not appropriate for monitoring treatment efficacy. PMID:9650945

  18. Distortion of genetically modified organism quantification in processed foods: influence of particle size compositions and heat-induced DNA degradation.

    Science.gov (United States)

    Moreano, Francisco; Busch, Ulrich; Engel, Karl-Heinz

    2005-12-28

    Milling fractions from conventional and transgenic corn were prepared at laboratory scale and used to study the influence of sample composition and heat-induced DNA degradation on the relative quantification of genetically modified organisms (GMO) in food products. Particle size distributions of the obtained fractions (coarse grits, regular grits, meal, and flour) were characterized using a laser diffraction system. The application of two DNA isolation protocols revealed a strong correlation between the degree of comminution of the milling fractions and the DNA yield in the extracts. Mixtures of milling fractions from conventional and transgenic material (1%) were prepared and analyzed via real-time polymerase chain reaction. Accurate quantification of the adjusted GMO content was only possible in mixtures containing conventional and transgenic material in the form of analogous milling fractions, whereas mixtures of fractions exhibiting different particle size distributions delivered significantly over- and underestimated GMO contents depending on their compositions. The process of heat-induced nucleic acid degradation was followed by applying two established quantitative assays showing differences between the lengths of the recombinant and reference target sequences (A, deltal(A) = -25 bp; B, deltal(B) = +16 bp; values related to the amplicon length of the reference gene). Data obtained by the application of method A resulted in underestimated recoveries of GMO contents in the samples of heat-treated products, reflecting the favored degradation of the longer target sequence used for the detection of the transgene. In contrast, data yielded by the application of method B resulted in increasingly overestimated recoveries of GMO contents. The results show how commonly used food technological processes may lead to distortions in the results of quantitative GMO analyses.

  19. Cell-free DNA in human follicular fluid as a biomarker of embryo quality.

    Science.gov (United States)

    Scalici, E; Traver, S; Molinari, N; Mullet, T; Monforte, M; Vintejoux, E; Hamamah, S

    2014-12-01

    Could cell-free DNA (cfDNA) quantification in individual human follicular fluid (FF) samples become a new non-invasive predictive biomarker for in vitro fertilization (IVF) outcomes? CfDNA level in human follicular fluid samples was significantly correlated with embryo quality and could be used as an innovative non-invasive biomarker to improve IVF outcomes. CfDNA fragments, resulting from apoptotic or necrotic events, are present in the bloodstream and their quantification is already used as a biomarker for gynaecological and pregnancy disorders. Follicular fluid is important for oocyte development and contains plasma components and factors secreted by granulosa cells during folliculogenesis. CfDNA presence in follicular fluid and its potential use as an IVF outcome biomarker have never been investigated. One hundred individual follicular fluid samples were collected from 43 female patients undergoing conventional IVF (n = 26) or ICSI (n = 17). CfDNA level was quantified in each individual follicular fluid sample. At oocyte collection day, follicles were aspirated individually. Only blood-free follicular fluid samples were included in the study. Follicle size was calculated based on the follicular fluid volume. Each corresponding cumulus-oocyte complex was isolated for IVF or ICSI procedures. Follicular fluid cfDNA was measured by quantitative PCR with ALU-specific primers. Human follicular fluid samples from individual follicles contain measurable amounts of cfDNA (mean ± SD, 1.62 ± 2.08 ng/µl). CfDNA level was significantly higher in small follicles (8-12 mm in diameter) than in large ones (>18 mm) (mean ± SD, 2.54 ± 0.78 ng/µl versus 0.71 ± 0.44 ng/µl, respectively, P = 0.007). Moreover, cfDNA concentration was significantly and negatively correlated with follicle size (r = -0.34; P = 0.003). A weak significant negative correlation between DNA integrity and 17β-estradiol level in follicular fluid samples at oocyte collection day was observed (r = -0

  20. Human identification & forensic analyses of degraded or low level DNA

    NARCIS (Netherlands)

    Westen, Antoinette-Andrea

    2013-01-01

    DNA-based human identification is employed in varying situations, such as disaster victim identification, relationship testing and forensic analyses. When DNA is of low quality and/or quantity, standard methods for DNA profiling may not suffice. The research described in this thesis is aimed at the

  1. Human identification & forensic analyses of degraded or low level DNA

    NARCIS (Netherlands)

    Westen, Antoinette-Andrea

    2013-01-01

    DNA-based human identification is employed in varying situations, such as disaster victim identification, relationship testing and forensic analyses. When DNA is of low quality and/or quantity, standard methods for DNA profiling may not suffice. The research described in this thesis is aimed at the

  2. Rapid quantification of hepatitis B virus DNA by real-time PCR using efficient TaqMan probe and extraction of virus DNA

    Institute of Scientific and Technical Information of China (English)

    Yan-Qin Lu; Jin-Xiang Han; Peng Qi; Wei Xu; Yan-Hui Zu; Bo Zhu

    2006-01-01

    AIM: To rapidly quantify hepatitis B virus (HBV) DNA by real-time PCR using efficient TaqMan probe and extraction methods of virus DNA.METHODS: Three standards were prepared by cloning PCR products which targeted S, C and X region of HBV genome into pGEM-T vector respectively. A pair of primers and matched TaqMan probe were selected by comparing the copy number and the Ct values of HBV serum samples derived from the three different standard curves using certain serum DNA. Then the efficiency of six HBV DNA extraction methods including guanidinium isothiocyanate, proteinase K, NaI, NaOH lysis, alkaline lysis and simple boiling was analyzed in sample A, B and C by real-time PCR. Meanwhile, 8 clinical HBV serum samples were quantified.RESULTS: The copy number of the same HBV serum sample originated from the standard curve of S, C and Xregions was 5.7 x 104/mL, 6.3 x 102/mL and 1.6 x 103/mL respectively. The relative Ct value was 26.6, 31.8 and 29.5 respectively. Therefore, primers and matched probe from S region were chosen for further optimization of six extraction methods. The copy number of HBV serum samples A, B and C was 3.49 x 109/mL, 2.08 x 106/mL and 4.40 x 107/mL respectively, the relative Ct value was 19.9, 30 and 26.2 in the method of NaOH lysis,which was the efficientest among six methods. Simple boiling showed a slightly lower efficiency than NaOH lysis. Guanidinium isothiocyanate, proteinase K and NaI displayed that the copy number of HBV serum sample A,B and C was around 105/mL, meanwhile the Ct value was about 30. Alkaline failed to quantify the copy number of three HBV serum samples. Standard deviation (SD) and coefficient variation (CV) were very low in all 8 clinical HBV serum samples, showing that quantification of HBV DNA in triplicate was reliable and accurate.CONCLUSION: Real-time PCR based on optimized primers and TaqMan probe from S region in combination with NaOH lysis is a simple, rapid and accurate method for quantification of HBV serum DNA.

  3. HUMAN ERROR QUANTIFICATION USING PERFORMANCE SHAPING FACTORS IN THE SPAR-H METHOD

    Energy Technology Data Exchange (ETDEWEB)

    Harold S. Blackman; David I. Gertman; Ronald L. Boring

    2008-09-01

    This paper describes a cognitively based human reliability analysis (HRA) quantification technique for estimating the human error probabilities (HEPs) associated with operator and crew actions at nuclear power plants. The method described here, Standardized Plant Analysis Risk-Human Reliability Analysis (SPAR-H) method, was developed to aid in characterizing and quantifying human performance at nuclear power plants. The intent was to develop a defensible method that would consider all factors that may influence performance. In the SPAR-H approach, calculation of HEP rates is especially straightforward, starting with pre-defined nominal error rates for cognitive vs. action-oriented tasks, and incorporating performance shaping factor multipliers upon those nominal error rates.

  4. Quantification of DNA repair protein kinetics after γ-irradiation using number and brightness analysis

    Science.gov (United States)

    Abdisalaam, Salim; Poudel, Milan; Chen, David J.; Alexandrakis, George

    2011-03-01

    The kinetics of most proteins involved in DNA damage sensing, signaling and repair following ionizing radiation exposure cannot be quantified by current live cell fluorescence microscopy methods. This is because most of these proteins, with only few notable exceptions, do not attach in large numbers at DNA damage sites to form easily detectable foci in microscopy images. As a result a high fluorescence background from freely moving and immobile fluorescent proteins in the nucleus masks the aggregation of proteins at sparse DNA damage sites. Currently, the kinetics of these repair proteins are studied by laser-induced damage and Fluorescence Recovery After Photobleaching that rely on the detectability of high fluorescence intensity spots of clustered DNA damage. We report on the use of Number and Brightness (N&B) analysis methods as a means to monitor kinetics of DNA repair proteins during sparse DNA damage created by γ-irradiation, which is more relevant to cancer treatment than laser-induced clustered damage. We use two key double strand break repair proteins, namely Ku 70/80 and the DNA-dependent protein kinase catalytic subunit (DNA-PKCS), as specific examples to showcase the feasibility of the proposed methods to quantify dose-dependent kinetics for DNA repair proteins after exposure to γ-rays.

  5. UV-Visible Spectroscopy-Based Quantification of Unlabeled DNA Bound to Gold Nanoparticles.

    Science.gov (United States)

    Baldock, Brandi L; Hutchison, James E

    2016-12-20

    DNA-functionalized gold nanoparticles have been increasingly applied as sensitive and selective analytical probes and biosensors. The DNA ligands bound to a nanoparticle dictate its reactivity, making it essential to know the type and number of DNA strands bound to the nanoparticle surface. Existing methods used to determine the number of DNA strands per gold nanoparticle (AuNP) require that the sequences be fluorophore-labeled, which may affect the DNA surface coverage and reactivity of the nanoparticle and/or require specialized equipment and other fluorophore-containing reagents. We report a UV-visible-based method to conveniently and inexpensively determine the number of DNA strands attached to AuNPs of different core sizes. When this method is used in tandem with a fluorescence dye assay, it is possible to determine the ratio of two unlabeled sequences of different lengths bound to AuNPs. Two sizes of citrate-stabilized AuNPs (5 and 12 nm) were functionalized with mixtures of short (5 base) and long (32 base) disulfide-terminated DNA sequences, and the ratios of sequences bound to the AuNPs were determined using the new method. The long DNA sequence was present as a lower proportion of the ligand shell than in the ligand exchange mixture, suggesting it had a lower propensity to bind the AuNPs than the short DNA sequence. The ratio of DNA sequences bound to the AuNPs was not the same for the large and small AuNPs, which suggests that the radius of curvature had a significant influence on the assembly of DNA strands onto the AuNPs.

  6. Quantification of Human Kallikrein-Related Peptidases in Biological Fluids by Multiplatform Targeted Mass Spectrometry Assays.

    Science.gov (United States)

    Karakosta, Theano D; Soosaipillai, Antoninus; Diamandis, Eleftherios P; Batruch, Ihor; Drabovich, Andrei P

    2016-09-01

    Human kallikrein-related peptidases (KLKs) are a group of 15 secreted serine proteases encoded by the largest contiguous cluster of protease genes in the human genome. KLKs are involved in coordination of numerous physiological functions including regulation of blood pressure, neuronal plasticity, skin desquamation, and semen liquefaction, and thus represent promising diagnostic and therapeutic targets. Until now, quantification of KLKs in biological and clinical samples was accomplished by enzyme-linked immunosorbent assays (ELISA). Here, we developed multiplex targeted mass spectrometry assays for the simultaneous quantification of all 15 KLKs. Proteotypic peptides for each KLK were carefully selected based on experimental data and multiplexed in single assays. Performance of assays was evaluated using three different mass spectrometry platforms including triple quadrupole, quadrupole-ion trap, and quadrupole-orbitrap instruments. Heavy isotope-labeled synthetic peptides with a quantifying tag were used for absolute quantification of KLKs in sweat, cervico-vaginal fluid, seminal plasma, and blood serum, with limits of detection ranging from 5 to 500 ng/ml. Analytical performance of assays was evaluated by measuring endogenous KLKs in relevant biological fluids, and results were compared with selected ELISAs. The multiplex targeted proteomic assays were demonstrated to be accurate, reproducible, sensitive, and specific alternatives to antibody-based assays. Finally, KLK4, a highly prostate-specific protein and a speculated biomarker of prostate cancer, was unambiguously detected and quantified by immunoenrichment-SRM assay in seminal plasma and blood serum samples from individuals with confirmed prostate cancer and negative biopsy. Mass spectrometry revealed exclusively the presence of a secreted isoform and thus unequivocally resolved earlier disputes about KLK4 identity in seminal plasma. Measurements of KLK4 in either 41 seminal plasma or 58 blood serum samples

  7. Persistent organic pollutants alter DNA methylation during human adipocyte differentiation

    NARCIS (Netherlands)

    Dungen, van den Myrthe; Murk, Tinka; Steegenga, Wilma; Gils-Kok, van Dieuwertje

    2016-01-01

    Genome-wide DNA methylation profiling was performed in human mesenchymal stem cells (hMSCs) differentiated into adipocytes (day 10) while being continuously exposed to either one of three different persistent organic pollutants (POPs), namely TCDD, PFOS, and TBT. The Illumina Infinium 450K Human DNA

  8. The DNA methylome of human peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Li, Yingrui; Zhu, Jingde; Tian, Geng;

    2010-01-01

    DNA methylation plays an important role in biological processes in human health and disease. Recent technological advances allow unbiased whole-genome DNA methylation (methylome) analysis to be carried out on human cells. Using whole-genome bisulfite sequencing at 24.7-fold coverage (12.3-fold pe...

  9. Direct Quantification of Campylobacter jejuni in Chicken Fecal Samples Using Real-Time PCR: Evaluation of Six Rapid DNA Extraction Methods

    DEFF Research Database (Denmark)

    Garcia Clavero, Ana Belén; Kamara, Judy N.; Vigre, Håkan

    2013-01-01

    Direct and accurate quantification of Campylobacter in poultry is crucial for the assessment of public health risks and the evaluation of the effectiveness of control measures against Campylobacter in poultry. The aim of this study was to assess several rapid DNA extraction methods...... DNA extraction methods were compared based on their limit of detection, efficiency, reproducibility, and precision. Standard curves were designed for all the methods tested in order to assess their performance on the direct quantification of C. jejuni in chicken fecal samples. As a result...... efficiency was not optimal (AE = 139.5 %). DNA extraction methods Easy-DNA Invitrogen, MiniMAG® and NucleoSpin® Tissue produced good real-time PCR reproducibility generating standard deviations from 0.3 to 0.8 between replicates....

  10. Rapid Extraction of Human DNA Containing Humic Acid

    OpenAIRE

    Sutlović, Davorka; Definis Gojanović, Marija; Anđelinović, Šimun

    2007-01-01

    The identification process of dead bodies or human remains is nowadays conducted in numerous fields of forensic science, archeology and other judicial cases. A particular problem is the isolation and DNA typing of human remains found in mass graves, due to the degradation process, as well as post mortal DNA contamination with bacteria, fungi, humic acids, metals, etc. In this study, the influence of humic acid (HA) on the DNA extraction and typing is investigated. If present in...

  11. Structural basis of human PCNA sliding on DNA

    Science.gov (United States)

    de March, Matteo; Merino, Nekane; Barrera-Vilarmau, Susana; Crehuet, Ramon; Onesti, Silvia; Blanco, Francisco J.; de Biasio, Alfredo

    2017-01-01

    Sliding clamps encircle DNA and tether polymerases and other factors to the genomic template. However, the molecular mechanism of clamp sliding on DNA is unknown. Using crystallography, NMR and molecular dynamics simulations, here we show that the human clamp PCNA recognizes DNA through a double patch of basic residues within the ring channel, arranged in a right-hand spiral that matches the pitch of B-DNA. We propose that PCNA slides by tracking the DNA backbone via a `cogwheel' mechanism based on short-lived polar interactions, which keep the orientation of the clamp invariant relative to DNA. Mutation of residues at the PCNA-DNA interface has been shown to impair the initiation of DNA synthesis by polymerase δ (pol δ). Therefore, our findings suggest that a clamp correctly oriented on DNA is necessary for the assembly of a replication-competent PCNA-pol δ holoenzyme.

  12. Detection and quantification of DNA strand breaks using the ROPS (random oligonucleotide primed synthesis) assay.

    Science.gov (United States)

    Boyko, Alex; Kovalchuk, Igor

    2010-01-01

    DNA double strand breaks (DSBs) arise from spontaneous DNA damage due to metabolic activities or from direct and indirect damaging effects of stress. DSBs are also formed transiently during such processes as replication, transcription, and DNA repair. The level of DSBs positively correlates with the activities of homologous and nonhomologous DNA repair pathways, which in turn inversely correlate with methylation levels and chromatin structure. Thus, measurement of strand breaks can provide an informative picture of genome stability of a given cell. The use of random oligonucleotide-primed synthesis for the analysis of DSB levels is described. Applications of the assay for quantitative detection of 3'OH, 3'P, or DNA strand breaks at a cleavage site of the deoxyribose residue are discussed.

  13. Quantification of DNA fragmentation in processed foods using real-time PCR.

    Science.gov (United States)

    Mano, Junichi; Nishitsuji, Yasuyuki; Kikuchi, Yosuke; Fukudome, Shin-Ichi; Hayashida, Takuya; Kawakami, Hiroyuki; Kurimoto, Youichi; Noguchi, Akio; Kondo, Kazunari; Teshima, Reiko; Takabatake, Reona; Kitta, Kazumi

    2017-07-01

    DNA analysis of processed foods is performed widely to detect various targets, such as genetically modified organisms (GMOs). Food processing often causes DNA fragmentation, which consequently affects the results of PCR analysis. In order to assess the effects of DNA fragmentation on the reliability of PCR analysis, we investigated a novel methodology to quantify the degree of DNA fragmentation. We designed four real-time PCR assays that amplified 18S ribosomal RNA gene sequences common to various plants at lengths of approximately 100, 200, 400, and 800 base pairs (bp). Then, we created an indicator value, "DNA fragmentation index (DFI)", which is calculated from the Cq values derived from the real-time PCR assays. Finally, we demonstrated the efficacy of this method for the quality control of GMO detection in processed foods by evaluating the relationship between the DFI and the limit of detection. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Ion-pair HPLC method for the quantification of metformin in human urine

    Directory of Open Access Journals (Sweden)

    Eva Troja

    2016-01-01

    Full Text Available The aim of this study was to develop and validate a selective and sensitive ion-pairing HPLC–UV method for the determination of metformin in human urine using a conventional reversed-phase column. The bioanalytical method was carried out using a RP-C18 column (250×4.0 mm; 5 μm. A mobile phase consisting of acetonitrile and 10 mM sodium phosphate buffer (pH=6.0, 30:70 v/v and sodium dodecyl sulfate (0.3% was pumped at an isocratic flow rate of 1.00 mL/minute and quantification was achieved at 236 nm using using a UV/VIS DAD. The calibration curves were linear (r2 >0.999 in the concentration ranges of 62.5–2000 μg/mL for metformin in urine. LOD and LOQ were found to be 12 μg/mL and 35 μg/mL, respectively. The method was found to be rapid, precise and accurate for quantification of metformin in human urine. This method was successfully applied to a pharmacokinetic study of humans through oral administration.

  15. Simultaneous liquid chromatography-mass spectrometry quantification of cefixime and clavulanic acid in human plasma.

    Science.gov (United States)

    Dubala, Anil; Nagarajan, Janaki Sankarachari Krishnan; Vimal, Chandran Sathish; George, Renjith

    2015-01-01

    A simple and specific liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (LC-APCI-MS) assay method has been developed and fully validated for the simultaneous quantification of cefixime (CX) and clavulanic acid (CA) in human plasma. Analytes and internal standard were extracted from human plasma by a solid phase extraction technique using a Sam prep (3 mL, 100 mg) extraction cartridge. The extracted samples were chromatographed on a reverse phase C18 column using a mixture of methanol : acetonitrile : 2 mM ammonium acetate (pH 3.5) (25 : 25 : 50, v/v/v) as the mobile phase at a flow rate of 0.8 mL/min. Quantification of the analytes were carried out using single quadrupole LC-APCI-MS through selected ion monitoring at m/z 452 and 198, respectively, for CX and CA. The assay was linear over the concentration range of 0.05-10.0 and 0.1-10.0 μg/mL, respectively, for CX and CA. The mean plasma extraction recoveries of the CX and CA were found to be 95.20-96.27% and 94.67-95.58%, respectively. The method was successfully applied for the determination of pharmacokinetics of CX and CA after oral administration of single dosage CX/CA (200/125 mg) pill to the humans (n = 12).

  16. Human DNA ligase and DNA polymerase as molecular targets for heavy metals and anticancer drugs

    Energy Technology Data Exchange (ETDEWEB)

    Yang, S.

    1992-01-01

    DNA ligase and DNA polymerase play important roles in DNA replication, repair, and recombination. Frequencies of spontaneous and chemical- and physical-induced mutations are correlated to the fidelity of DNA replication. This dissertation elucidates the mechanisms of the DNA ligation reaction by DNA ligases and demonstrates that human DNA ligase I and DNA polymerase [alpha] are the molecular targets for two metal ions, Zn[sup 2+] and Cd[sup 2+], and an anticancer drug, F-ara-ATP. The formation of the AMP-DNA intermediate and the successive ligation reaction by human DNA ligases were analyzed. Both reactions showed their substrate specificity for ligases I and II, required Mg2+, and were inhibited by ATP. A protein inhibitor from HeLa cells and specific for human DNA ligase I but not ligase II and T4 ligase was discovered. It reversibly inhibited DNA ligation activity but not the AMP-binding activity due to the formation of a reversible ligase I-inhibitor complex. F-ara-ATP inhibited human DNA ligase I activity by competing with ATP for the AMP-binding site of DNA ligase I, forming a ligase I-F-ara-AMP complex, as well as when it was incorporated at 3[prime]-terminus of DNA nick by DNA polymerase [alpha]. All steps of the DNA ligation reaction were inhibited by Zn[sup 2+] and Cd[sup 2+] in a concentration-dependent manner. Both ions did not show the ability to change the fidelity of DNA ligation reaction catalyzed by human DNA ligase I. However, Zn[sup 2+] and Cd[sup 2+] showed their contradictory effects on the fidelity of the reaction by human DNA polymerase [alpha]. Zn[sup 2+] decreased the frequency of misinsertion but less affected that of mispair extension. On the contrary, Cd[sup 2+] increased the frequencies of both misinsertion and mispair extension at very low concentration. The data provided strong evidence in the molecular mechanisms for the mutagenicity of zinc and cadmium, and were comparable with the results previously reported.

  17. Hydrophobic ionic liquids for quantitative bacterial cell lysis with subsequent DNA quantification.

    Science.gov (United States)

    Fuchs-Telka, Sabine; Fister, Susanne; Mester, Patrick-Julian; Wagner, Martin; Rossmanith, Peter

    2017-02-01

    DNA is one of the most frequently analyzed molecules in the life sciences. In this article we describe a simple and fast protocol for quantitative DNA isolation from bacteria based on hydrophobic ionic liquid supported cell lysis at elevated temperatures (120-150 °C) for subsequent PCR-based analysis. From a set of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide was identified as the most suitable for quantitative cell lysis and DNA extraction because of limited quantitative PCR inhibition by the aqueous eluate as well as no detectable DNA uptake. The newly developed method was able to efficiently lyse Gram-negative bacterial cells, whereas Gram-positive cells were protected by their thick cell wall. The performance of the final protocol resulted in quantitative DNA extraction efficiencies for Gram-negative bacteria similar to those obtained with a commercial kit, whereas the number of handling steps, and especially the time required, was dramatically reduced. Graphical Abstract After careful evaluation of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide ([BMPyr (+) ][Ntf 2(-) ]) was identified as the most suitable ionic liquid for quantitative cell lysis and DNA extraction. When used for Gram-negative bacteria, the protocol presented is simple and very fast and achieves DNA extraction efficiencies similar to those obtained with a commercial kit. ddH 2 O double-distilled water, qPCR quantitative PCR.

  18. Label-free electrical quantification of the dielectrophoretic response of DNA

    CERN Document Server

    Henning, Anja; Bier, Frank F; Hoelzel, Ralph; 10.1186/1757-5036-1-4

    2009-01-01

    A purely electrical sensing scheme is presented that determines the concentration of macromolecules in solution by measuring the capacitance between planar microelectrodes. Concentrations of DNA in the ng/mL range have been used in samples of 1 microL volume. The method has been applied to the characterisation of the dielectrophoretic response of DNA without the need for any chemical modifications. The influence of electrical parameters like duty cycle, voltage and frequency has been investigated. The results are in good agreement with data from dielectrophoretic studies on fluorescently labelled DNA. Extension of the method down to the single molecule level appears feasible.

  19. Comparative Study of Seven Commercial Kits for Human DNA Extraction from Urine Samples Suitable for DNA Biomarker-Based Public Health Studies

    Science.gov (United States)

    El Bali, Latifa; Diman, Aurélie; Bernard, Alfred; Roosens, Nancy H. C.; De Keersmaecker, Sigrid C. J.

    2014-01-01

    Human genomic DNA extracted from urine could be an interesting tool for large-scale public health studies involving characterization of genetic variations or DNA biomarkers as a result of the simple and noninvasive collection method. These studies, involving many samples, require a rapid, easy, and standardized extraction protocol. Moreover, for practicability, there is a necessity to collect urine at a moment different from the first void and to store it appropriately until analysis. The present study compared seven commercial kits to select the most appropriate urinary human DNA extraction procedure for epidemiological studies. DNA yield has been determined using different quantification methods: two classical, i.e., NanoDrop and PicoGreen, and two species-specific real-time quantitative (q)PCR assays, as DNA extracted from urine contains, besides human, microbial DNA also, which largely contributes to the total DNA yield. In addition, the kits giving a good yield were also tested for the presence of PCR inhibitors. Further comparisons were performed regarding the sampling time and the storage conditions. Finally, as a proof-of-concept, an important gene related to smoking has been genotyped using the developed tools. We could select one well-performing kit for the human DNA extraction from urine suitable for molecular diagnostic real-time qPCR-based assays targeting genetic variations, applicable to large-scale studies. In addition, successful genotyping was possible using DNA extracted from urine stored at −20°C for several months, and an acceptable yield could also be obtained from urine collected at different moments during the day, which is particularly important for public health studies. PMID:25365790

  20. Comparative study of seven commercial kits for human DNA extraction from urine samples suitable for DNA biomarker-based public health studies.

    Science.gov (United States)

    El Bali, Latifa; Diman, Aurélie; Bernard, Alfred; Roosens, Nancy H C; De Keersmaecker, Sigrid C J

    2014-12-01

    Human genomic DNA extracted from urine could be an interesting tool for large-scale public health studies involving characterization of genetic variations or DNA biomarkers as a result of the simple and noninvasive collection method. These studies, involving many samples, require a rapid, easy, and standardized extraction protocol. Moreover, for practicability, there is a necessity to collect urine at a moment different from the first void and to store it appropriately until analysis. The present study compared seven commercial kits to select the most appropriate urinary human DNA extraction procedure for epidemiological studies. DNA yield has been determined using different quantification methods: two classical, i.e., NanoDrop and PicoGreen, and two species-specific real-time quantitative (q)PCR assays, as DNA extracted from urine contains, besides human, microbial DNA also, which largely contributes to the total DNA yield. In addition, the kits giving a good yield were also tested for the presence of PCR inhibitors. Further comparisons were performed regarding the sampling time and the storage conditions. Finally, as a proof-of-concept, an important gene related to smoking has been genotyped using the developed tools. We could select one well-performing kit for the human DNA extraction from urine suitable for molecular diagnostic real-time qPCR-based assays targeting genetic variations, applicable to large-scale studies. In addition, successful genotyping was possible using DNA extracted from urine stored at -20°C for several months, and an acceptable yield could also be obtained from urine collected at different moments during the day, which is particularly important for public health studies.

  1. Oxidized Extracellular DNA as a Stress Signal in Human Cells

    Directory of Open Access Journals (Sweden)

    Aleksei V. Ermakov

    2013-01-01

    Full Text Available The term “cell-free DNA” (cfDNA was recently coined for DNA fragments from plasma/serum, while DNA present in in vitro cell culture media is known as extracellular DNA (ecDNA. Under oxidative stress conditions, the levels of oxidative modification of cellular DNA and the rate of cell death increase. Dying cells release their damaged DNA, thus, contributing oxidized DNA fragments to the pool of cfDNA/ecDNA. Oxidized cell-free DNA could serve as a stress signal that promotes irradiation-induced bystander effect. Evidence points to TLR9 as a possible candidate for oxidized DNA sensor. An exposure to oxidized ecDNA stimulates a synthesis of reactive oxygen species (ROS that evokes an adaptive response that includes transposition of the homologous loci within the nucleus, polymerization and the formation of the stress fibers of the actin, as well as activation of the ribosomal gene expression, and nuclear translocation of NF-E2 related factor-2 (NRF2 that, in turn, mediates induction of phase II detoxifying and antioxidant enzymes. In conclusion, the oxidized DNA is a stress signal released in response to oxidative stress in the cultured cells and, possibly, in the human body; in particular, it might contribute to systemic abscopal effects of localized irradiation treatments.

  2. Electrochemical branched-DNA assay for polymerase chain reaction-free detection and quantification of oncogenes in messenger RNA.

    Science.gov (United States)

    Lee, Ai-Cheng; Dai, Ziyu; Chen, Baowei; Wu, Hong; Wang, Jun; Zhang, Aiguo; Zhang, Lurong; Lim, Tit-Meng; Lin, Yuehe

    2008-12-15

    We describe a novel electrochemical branched-DNA (bDNA) assay for polymerase chain reaction (PCR)-free detection and quantification of p185 BCR-ABL leukemia fusion transcripts in the population of messenger ribonucleic acid (mRNA) extracted from cell lines. The bDNA amplifier carrying high loading of alkaline phosphatase (ALP) tracers was used to amplify the target signal. The targets were captured on microplate well surfaces through cooperative sandwich hybridization prior to the labeling of bDNA. The activity of captured ALP was monitored by square-wave voltammetric (SWV) analysis of the electroactive enzymatic product in the presence of 1-naphthyl phosphate. The voltammetric characteristics of substrate and enzymatic product as well as the parameters of SWV analysis were systematically optimized. A detection limit of 1 fM (1 x 10(-19) mol of target transcripts in 100 microL) and a 3-order-wide dynamic range of target concentration were achieved by the electrochemical bDNA assay. Such limit corresponded to approximately 17 fg of the p185 BCR-ABL fusion transcripts. The specificity and sensitivity of assay enabled direct detection of target transcripts in as little as 4.6 ng of mRNA population without PCR amplification. In combination with the use of a well-quantified standard, the electrochemical bDNA assay was capable of direct use for a PCR-free quantitative analysis of target transcripts in mRNA population. A mean transcript copy number of 62,900/ng of mRNA was determined, which was at least 50-fold higher than that of real-time quantitative PCR (qPCR). The finding was consistent with the underestimation of targets by qPCR reported earlier. In addition, the unique design based on bDNA technology increases the assay specificity as only the p185 BCR-ABL fusion transcripts will respond to the detection. The approach thus provides a simple, sensitive, accurate, and quantitative tool alternative to the qPCR for early disease diagnosis.

  3. Oxidized DNA induces an adaptive response in human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Kostyuk, Svetlana V., E-mail: svet.kostyuk@gmail.com [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation); Tabakov, Viacheslav J.; Chestkov, Valerij V.; Konkova, Marina S.; Glebova, Kristina V.; Baydakova, Galina V.; Ershova, Elizaveta S.; Izhevskaya, Vera L. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation); Baranova, Ancha, E-mail: abaranov@gmu.edu [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation); Center for the Study of Chronic Metabolic Diseases, School of System Biology, George Mason University, Fairfax, VA 22030 (United States); Veiko, Natalia N. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation)

    2013-07-15

    Highlights: • We describe the effects of gDNAOX on human fibroblasts cultivated in serum withdrawal conditions. • gDNAOX evokes an adaptive response in human fibroblasts. • gDNAOX increases the survival rates in serum starving cell populations. • gDNAOX enhances the survival rates in cell populations irradiated at 1.2 Gy dose. • gDNAOX up-regulates NRF2 and inhibits NF-kappaB-signaling. - Abstract: Cell-free DNA (cfDNA) released from dying cells contains a substantial proportion of oxidized nucleotides, thus, forming cfDNA{sup OX}. The levels of cfDNA{sup OX} are increased in the serum of patients with chronic diseases. Oxidation of DNA turns it into a stress signal. The samples of genomic DNA (gDNA) oxidized by H{sub 2}O{sub 2}in vitro (gDNA{sup OX}) induce effects similar to that of DNA released from damaged cells. Here we describe the effects of gDNA{sup OX} on human fibroblasts cultivated in the stressful conditions of serum withdrawal. In these cells, gDNA{sup OX} evokes an adaptive response that leads to an increase in the rates of survival in serum starving cell populations as well as in populations irradiated at the dose of 1.2 Gy. These effects are not seen in control populations of fibroblasts treated with non-modified gDNA. In particular, the exposure to gDNA{sup OX} leads to a decrease in the expression of the proliferation marker Ki-67 and an increase in levels of PSNA, a decrease in the proportion of subG1- and G2/M cells, a decrease in proportion of cells with double strand breaks (DSBs). Both gDNA{sup OX} and gDNA suppress the expression of DNA sensors TLR9 and AIM2 and up-regulate nuclear factor-erythroid 2 p45-related factor 2 (NRF2), while only gDNA{sup OX} inhibits NF-κB signaling. gDNA{sup OX} is a model for oxidized cfDNA{sup OX} that is released from the dying tumor cells and being carried to the distant organs. The systemic effects of oxidized DNA have to be taken into account when treating tumors. In particular, the damaged DNA

  4. High precision quantification of human plasma proteins using the automated SISCAPA Immuno-MS workflow.

    Science.gov (United States)

    Razavi, Morteza; Leigh Anderson, N; Pope, Matthew E; Yip, Richard; Pearson, Terry W

    2016-09-25

    Efficient robotic workflows for trypsin digestion of human plasma and subsequent antibody-mediated peptide enrichment (the SISCAPA method) were developed with the goal of improving assay precision and throughput for multiplexed protein biomarker quantification. First, an 'addition only' tryptic digestion protocol was simplified from classical methods, eliminating the need for sample cleanup, while improving reproducibility, scalability and cost. Second, methods were developed to allow multiplexed enrichment and quantification of peptide surrogates of protein biomarkers representing a very broad range of concentrations and widely different molecular masses in human plasma. The total workflow coefficients of variation (including the 3 sequential steps of digestion, SISCAPA peptide enrichment and mass spectrometric analysis) for 5 proteotypic peptides measured in 6 replicates of each of 6 different samples repeated over 6 days averaged 3.4% within-run and 4.3% across all runs. An experiment to identify sources of variation in the workflow demonstrated that MRM measurement and tryptic digestion steps each had average CVs of ∼2.7%. Because of the high purity of the peptide analytes enriched by antibody capture, the liquid chromatography step is minimized and in some cases eliminated altogether, enabling throughput levels consistent with requirements of large biomarker and clinical studies.

  5. Performance of a Taqman Assay for Improved Detection and Quantification of Human Rhinovirus Viral Load

    Science.gov (United States)

    Ng, Kim Tien; Chook, Jack Bee; Oong, Xiang Yong; Chan, Yoke Fun; Chan, Kok Gan; Hanafi, Nik Sherina; Pang, Yong Kek; Kamarulzaman, Adeeba; Tee, Kok Keng

    2016-01-01

    Human rhinovirus (HRV) is the major aetiology of respiratory tract infections. HRV viral load assays are available but limitations that affect accurate quantification exist. We developed a one-step Taqman assay using oligonucleotides designed based on a comprehensive list of global HRV sequences. The new oligonucleotides targeting the 5′-UTR region showed high PCR efficiency (E = 99.6%, R2 = 0.996), with quantifiable viral load as low as 2 viral copies/μl. Assay evaluation using an External Quality Assessment (EQA) panel yielded a detection rate of 90%. When tested on 315 human enterovirus-positive specimens comprising at least 84 genetically distinct HRV types/serotypes (determined by the VP4/VP2 gene phylogenetic analysis), the assay detected all HRV species and types, as well as other non-polio enteroviruses. A commercial quantification kit, which failed to detect any of the EQA specimens, produced a detection rate of 13.3% (42/315) among the clinical specimens. Using the improved assay, we showed that HRV sheds in the upper respiratory tract for more than a week following acute infection. We also showed that HRV-C had a significantly higher viral load at 2–7 days after the onset of symptoms (p = 0.001). The availability of such assay is important to facilitate disease management, antiviral development, and infection control. PMID:27721388

  6. DNA Aptamers in the Diagnosis and Treatment of Human Diseases

    Directory of Open Access Journals (Sweden)

    Qinchang Zhu

    2015-11-01

    Full Text Available Aptamers have a promising role in the field of life science and have been extensively researched for application as analytical tools, therapeutic agents and as vehicles for targeted drug delivery. Compared with RNA aptamers, DNA aptamers have inherent advantages in stability and facility of generation and synthesis. To better understand the specific potential of DNA aptamers, an overview of the progress in the generation and application of DNA aptamers in human disease diagnosis and therapy are presented in this review. Special attention is given to researches that are relatively close to practical application. DNA aptamers are expected to have great potential in the diagnosis and treatment of human diseases.

  7. Quantification of Circulating Free DNA as a Diagnostic Marker in Gall Bladder Cancer.

    Science.gov (United States)

    Kumari, Swati; Tewari, Shikha; Husain, Nuzhat; Agarwal, Akash; Pandey, Anshuman; Singhal, Ashish; Lohani, Mohtashim

    2017-01-01

    Gall bladder Carcinoma (GBC) is the fifth most common cancer of the digestive tract and frequently diagnosed in late stage of disease. Estimation of circulating free DNA (cfDNA) in serum has been applied as a "liquid biopsy" in several deep seated malignancies. Its value in diagnosis of gall bladder carcinoma has not been studied. The present study was designed to assess the role of cfDNA in the diagnosis of GBC and correlate levels with the TNM stage. Serum was collected from 34 patients with GBC and 39 age and sex matched controls including 22 cholecystitis and 17 healthy individuals. Serum cfDNA levels were measured through quantitative polymerase chain reaction (qPCR) by amplification of β-globin gene. Performance of the assay was calculated through the receiver operating characteristic (ROC) curve. The cfDNA level was significantly lower in healthy controls and cholecystitis (89.32 ± 59.76 ng/ml, 174.21 ± 99.93 ng/ml) compared to GBC (1245.91 ± 892.46 ng/ml, p = <0.001). The cfDNA level was significantly associated with TNM stage, lymph node involvement and jaundice (0.002, 0.027, and 0.041, respectively). Area under curve of ROC analysis for cancer group versus healthy and cholecystitis group was 1.00 and 0.983 with sensitivity of 100 %, 88.24 % and specificity of 100 % respectively. Quantitative analysis of cfDNA may distinguish cholecystitis and gall bladder carcinoma and may serve as new diagnostic, noninvasive marker adjunct to imaging for the diagnosis of GBC.

  8. Sperm DNA fragmentation affects epigenetic feature in human male pronucleus.

    Science.gov (United States)

    Rajabi, H; Mohseni-Kouchesfehani, H; Eslami-Arshaghi, T; Salehi, M

    2017-03-06

    To evaluate whether the sperm DNA fragmentation affects male pronucleus epigenetic factors, semen analysis was performed and DNA fragmentation was assessed by the method of sperm chromatin structure assay (SCSA). Human-mouse interspecies fertilisation was used to create human male pronucleus. Male pronucleus DNA methylation and H4K12 acetylation were evaluated by immunostaining. Results showed a significant positive correlation between the level of sperm DNA fragmentation and DNA methylation in male pronuclei. In other words, an increase in DNA damage caused an upsurge in DNA methylation. In the case of H4K12 acetylation, no correlation was detected between DNA damage and the level of histone acetylation in the normal group, but results for the group in which male pronuclei were derived from sperm cells with DNA fragmentation, increased DNA damage led to a decreased acetylation level. Sperm DNA fragmentation interferes with the active demethylation process and disrupts the insertion of histones into the male chromatin in the male pronucleus, following fertilisation. © 2017 Blackwell Verlag GmbH.

  9. Human RAD52 Captures and Holds DNA Strands, Increases DNA Flexibility, and Prevents Melting of Duplex DNA: Implications for DNA Recombination

    Directory of Open Access Journals (Sweden)

    Ineke Brouwer

    2017-03-01

    Full Text Available Human RAD52 promotes annealing of complementary single-stranded DNA (ssDNA. In-depth knowledge of RAD52-DNA interaction is required to understand how its activity is integrated in DNA repair processes. Here, we visualize individual fluorescent RAD52 complexes interacting with single DNA molecules. The interaction with ssDNA is rapid, static, and tight, where ssDNA appears to wrap around RAD52 complexes that promote intra-molecular bridging. With double-stranded DNA (dsDNA, interaction is slower, weaker, and often diffusive. Interestingly, force spectroscopy experiments show that RAD52 alters the mechanics dsDNA by enhancing DNA flexibility and increasing DNA contour length, suggesting intercalation. RAD52 binding changes the nature of the overstretching transition of dsDNA and prevents DNA melting, which is advantageous for strand clamping during or after annealing. DNA-bound RAD52 is efficient at capturing ssDNA in trans. Together, these effects may help key steps in DNA repair, such as second-end capture during homologous recombination or strand annealing during RAD51-independent recombination reactions.

  10. Quantification of Plasmodiophora brassicae Using a DNA-Based Soil Test Facilitates Sustainable Oilseed Rape Production

    Directory of Open Access Journals (Sweden)

    Ann-Charlotte Wallenhammar

    2016-04-01

    Full Text Available Outbreaks of clubroot disease caused by the soil-borne obligate parasite Plasmodiophora brassicae are common in oilseed rape (OSR in Sweden. A DNA-based soil testing service that identifies fields where P. brassicae poses a significant risk of clubroot infection is now commercially available. It was applied here in field surveys to monitor the prevalence of P. brassicae DNA in field soils intended for winter OSR production and winter OSR field experiments. In 2013 in Scania, prior to planting, P. brassicae DNA was detected in 60% of 45 fields on 10 of 18 farms. In 2014, P. brassicae DNA was detected in 44% of 59 fields in 14 of 36 farms, in the main winter OSR producing region in southern Sweden. P. brassicae was present indicative of a risk for >10% yield loss with susceptible cultivars (>1300 DNA copies g soil−1 in 47% and 44% of fields in 2013 and 2014 respectively. Furthermore, P. brassicae DNA was indicative of sites at risk of complete crop failure if susceptible cultivars were grown (>50 000 copies g−1 soil in 14% and 8% of fields in 2013 and 2014, respectively. A survey of all fields at Lanna research station in western Sweden showed that P. brassicae was spread throughout the farm, as only three of the fields (20% showed infection levels below the detection limit for P.brassicae DNA, while the level was >50,000 DNA copies g−1 soil in 20% of the fields. Soil-borne spread is of critical importance and soil scraped off footwear showed levels of up to 682 million spores g−1 soil. Soil testing is an important tool for determining the presence of P. brassicae and providing an indication of potential yield loss, e.g., in advisory work on planning for a sustainable OSR crop rotation. This soil test is gaining acceptance as a tool that increases the likelihood of success in precision agriculture and in applied research conducted in commercial oilseed fields and at research stations. The present application highlights the importance of

  11. Changes in neuronal DNA content variation in the human brain during aging.

    Science.gov (United States)

    Fischer, Hans-Georg; Morawski, Markus; Brückner, Martina K; Mittag, Anja; Tarnok, Attila; Arendt, Thomas

    2012-08-01

    The human brain has been proposed to represent a genetic mosaic, containing a small but constant number of neurons with an amount of DNA exceeding the diploid level that appear to be generated through various chromosome segregation defects initially. While a portion of these cells apparently die during development, neurons with abnormal chromosomal copy number have been identified in the mature brain. This genomic alteration might to lead to chromosomal instability affecting neuronal viability and could thus contribute to age-related mental disorders. Changes in the frequency of neurons with such structural genomic variation in the adult and aging brain, however, are unknown. Here, we quantified the frequency of neurons with a more than diploid DNA content in the cerebral cortex of normal human brain and analyzed its changes between the fourth and ninth decades of life. We applied a protocol of slide-based cytometry optimized for DNA quantification of single identified neurons, which allowed to analyze the DNA content of about 500 000 neurons for each brain. On average, 11.5% of cortical neurons showed DNA content above the diploid level. The frequency of neurons with this genomic alteration was highest at younger age and declined with age. Our results indicate that the genomic variation associated with DNA content exceeding the diploid level might compromise viability of these neurons in the aging brain and might thus contribute to susceptibilities for age-related CNS disorders. Alternatively, a potential selection bias of "healthy aging brains" needs to be considered, assuming that DNA content variation above a certain threshold associates with Alzheimer's disease.

  12. [DNA image-fluorimetry of individual human chromosomes].

    Science.gov (United States)

    Agafonova, N A; Sakuta, G A; Rozanov, Iu M; Shteĭn, G I; Kudriavtsev, B N

    2013-01-01

    Mucrofluorimetric method for the determination of DNA content in individual human chromosomes has been developed. The method is based on a preliminary identification of chromosomes with Hoechst 33258, followed by staining of the chromosomes with Feulgen reaction using Schiffs reagent type ethidium bromide-SO2, then measuring the fluorescence intensity of the chromosomes using an image analyzer. The method allows to determine the DNA content of individual chromosomes with accuracy up to 4.5 fg. DNA content of individual human chromosomes, their p-and q-arms as well as homologous chromosomes were measured using the developed method. It has been shown that the DNA content in the chromosomes of normal human karyotype is unstable. Fluctuations in the DNA content in some chromosomes can vary 35-40 fg.

  13. DNA methylation profiling of human chromosomes 6, 20 and 22

    OpenAIRE

    Eckhardt, Florian; Lewin, Joern; Cortese, Rene; Rakyan, Vardhman K.; Attwood, John; Burger, Matthias; Burton, John; Cox, Tony V.; Davies, Rob; Down, Thomas A; Haefliger, Carolina; Horton, Roger; Howe, Kevin; Jackson, David K.; Kunde, Jan

    2006-01-01

    DNA methylation constitutes the most stable type of epigenetic modifications modulating the transcriptional plasticity of mammalian genomes. Using bisulfite DNA sequencing, we report high-resolution methylation reference profiles of human chromosomes 6, 20 and 22, providing a resource of about 1.9 million CpG methylation values derived from 12 different tissues. Analysis of 6 annotation categories, revealed evolutionary conserved regions to be the predominant sites for differential DNA methyl...

  14. Quantification of Leishmania infantumDNA in the bone marrow, lymph node and spleen of dogs

    Directory of Open Access Journals (Sweden)

    Rafael Antonio Nascimento Ramos

    Full Text Available The aim of the present study was to quantify the parasite load ofLeishmania infantum in dogs using real-time PCR (qPCR. Bone marrow, lymph node and spleen samples were taken from 24 dogs serologically positive for L. infantum that had been put down by the official epidemiological surveillance service. According to the clinical signs the dogs were classified as asymptomatic or symptomatic. After DNA extraction, the samples were subjected to qPCR to detect and quantify L. infantum DNA. Out of the 24 dogs, 12.5% (3/24 were classified as asymptomatic and 87.5% (21/24 as symptomatic. Real-time PCR detected L. infantum DNA in all the animals, in at least one biological sample. In particular, 100% of bone marrow and lymph node scored positive, whereas in spleen, the presence of DNA was detected in 95.9% (23/24. In addition, out of 24 animals, 15 were microscopically positive to amastigote forms of L. infantum in bone marrow. No statistical significant difference was found in the overall mean quantity of DNA among the different biological samples (P = 0.518. Considering each organ separately, there was 100% positivity in bone marrow and lymph nodes, while among the spleen samples, 95.9% (23/24 were positive. Regarding the different clinical groups, the overall mean parasite load varied significantly (P = 0.022. According to the results obtained, it was not possible determine which biological sample was most suitable tissue for the diagnosis, based only on the parasite load. Therefore, other characteristics such as convenience and easily of obtaining samples should be taken into consideration.

  15. Adjusting MtDNA Quantification in Whole Blood for Peripheral Blood Platelet and Leukocyte Counts.

    Science.gov (United States)

    Hurtado-Roca, Yamilee; Ledesma, Marta; Gonzalez-Lazaro, Monica; Moreno-Loshuertos, Raquel; Fernandez-Silva, Patricio; Enriquez, Jose Antonio; Laclaustra, Martin

    2016-01-01

    Alterations of mitochondrial DNA copy number (mtDNAcn) in the blood (mitochondrial to nuclear DNA ratio) appear associated with several systemic diseases, including primary mitochondrial disorders, carcinogenesis, and hematologic diseases. Measuring mtDNAcn in DNA extracted from whole blood (WB) instead of from peripheral blood mononuclear cells or buffy coat may yield different results due to mitochondrial DNA present in platelets. The aim of this work is to quantify the contribution of platelets to mtDNAcn in whole blood [mtDNAcn(WB)] and to propose a correction formula to estimate leukocytes' mtDNAcn [mtDNAcn(L)] from mtDNAcn(WB). Blood samples from 10 healthy adults were combined with platelet-enriched plasma and saline solution to produce artificial blood preparations. Aliquots of each sample were combined with five different platelet concentrations. In 46 of these blood preparations, mtDNAcn was measured by qPCR. MtDNAcn(WB) increased 1.07 (95%CI 0.86, 1.29; p<0.001) per 1000 platelets present in the preparation. We proved that leukocyte count should also be taken into account as mtDNAcn(WB) was inversely associated with leukocyte count; it increased 1.10 (95%CI 0.95, 1.25, p<0.001) per unit increase of the ratio between platelet and leukocyte counts. If hematological measurements are available, subtracting 1.10 the platelets/leukocyte ratio from mtDNAcn(WB) may serve as an estimation for mtDNAcn(L). Both platelet and leukocyte counts in the sample are important sources of variation if comparing mtDNAcn among groups of patients when mtDNAcn is measured in DNA extracted from whole blood. Not taking the platelet/leukocyte ratio into account in whole blood measurements, may lead to overestimation and misclassification if interpreted as leukocytes' mtDNAcn.

  16. Adjusting MtDNA Quantification in Whole Blood for Peripheral Blood Platelet and Leukocyte Counts

    Science.gov (United States)

    Gonzalez-Lazaro, Monica; Moreno-Loshuertos, Raquel; Fernandez-Silva, Patricio; Enriquez, Jose Antonio; Laclaustra, Martin

    2016-01-01

    Alterations of mitochondrial DNA copy number (mtDNAcn) in the blood (mitochondrial to nuclear DNA ratio) appear associated with several systemic diseases, including primary mitochondrial disorders, carcinogenesis, and hematologic diseases. Measuring mtDNAcn in DNA extracted from whole blood (WB) instead of from peripheral blood mononuclear cells or buffy coat may yield different results due to mitochondrial DNA present in platelets. The aim of this work is to quantify the contribution of platelets to mtDNAcn in whole blood [mtDNAcn(WB)] and to propose a correction formula to estimate leukocytes' mtDNAcn [mtDNAcn(L)] from mtDNAcn(WB). Blood samples from 10 healthy adults were combined with platelet-enriched plasma and saline solution to produce artificial blood preparations. Aliquots of each sample were combined with five different platelet concentrations. In 46 of these blood preparations, mtDNAcn was measured by qPCR. MtDNAcn(WB) increased 1.07 (95%CI 0.86, 1.29; p<0.001) per 1000 platelets present in the preparation. We proved that leukocyte count should also be taken into account as mtDNAcn(WB) was inversely associated with leukocyte count; it increased 1.10 (95%CI 0.95, 1.25, p<0.001) per unit increase of the ratio between platelet and leukocyte counts. If hematological measurements are available, subtracting 1.10 the platelets/leukocyte ratio from mtDNAcn(WB) may serve as an estimation for mtDNAcn(L). Both platelet and leukocyte counts in the sample are important sources of variation if comparing mtDNAcn among groups of patients when mtDNAcn is measured in DNA extracted from whole blood. Not taking the platelet/leukocyte ratio into account in whole blood measurements, may lead to overestimation and misclassification if interpreted as leukocytes' mtDNAcn. PMID:27736919

  17. DNA and bone structure preservation in medieval human skeletons.

    Science.gov (United States)

    Coulson-Thomas, Yvette M; Norton, Andrew L; Coulson-Thomas, Vivien J; Florencio-Silva, Rinaldo; Ali, Nadir; Elmrghni, Samir; Gil, Cristiane D; Sasso, Gisela R S; Dixon, Ronald A; Nader, Helena B

    2015-06-01

    Morphological and ultrastructural data from archaeological human bones are scarce, particularly data that have been correlated with information on the preservation of molecules such as DNA. Here we examine the bone structure of macroscopically well-preserved medieval human skeletons by transmission electron microscopy and immunohistochemistry, and the quantity and quality of DNA extracted from these skeletons. DNA technology has been increasingly used for analyzing physical evidence in archaeological forensics; however, the isolation of ancient DNA is difficult since it is highly degraded, extraction yields are low and the co-extraction of PCR inhibitors is a problem. We adapted and optimised a method that is frequently used for isolating DNA from modern samples, Chelex(®) 100 (Bio-Rad) extraction, for isolating DNA from archaeological human bones and teeth. The isolated DNA was analysed by real-time PCR using primers targeting the sex determining region on the Y chromosome (SRY) and STR typing using the AmpFlSTR(®) Identifiler PCR Amplification kit. Our results clearly show the preservation of bone matrix in medieval bones and the presence of intact osteocytes with well preserved encapsulated nuclei. In addition, we show how effective Chelex(®) 100 is for isolating ancient DNA from archaeological bones and teeth. This optimised method is suitable for STR typing using kits aimed specifically at degraded and difficult DNA templates since amplicons of up to 250bp were successfully amplified.

  18. Markov chain for estimating human mitochondrial DNA mutation pattern

    Science.gov (United States)

    Vantika, Sandy; Pasaribu, Udjianna S.

    2015-12-01

    The Markov chain was proposed to estimate the human mitochondrial DNA mutation pattern. One DNA sequence was taken randomly from 100 sequences in Genbank. The nucleotide transition matrix and mutation transition matrix were estimated from this sequence. We determined whether the states (mutation/normal) are recurrent or transient. The results showed that both of them are recurrent.

  19. Effects of Captan on DNA and DNA metabolic processes in human diploid fibroblasts.

    Science.gov (United States)

    Snyder, R D

    1992-01-01

    The fungicide Captan has been examined for its effects on DNA and DNA processing in order to better understand the genotoxicity associated with this agent. Captan treatment resulted in production of DNA single strand breaks and DNA-protein cross-links and elicited an excision repair response in human diploid fibroblasts. Captan was also shown to inhibit cellular DNA synthesis and to form stable adducts in herring sperm and human cellular DNA. Misincorporation of nucleotides into Captan-treated synthetic DNA templates was significantly elevated in an in vitro assay using E. coli DNA polymerase I, suggesting that DNA adduct formation by Captan could have mutagenic consequences. In sum, these studies demonstrate that Captan is capable of interacting with DNA at a number of levels and that these interactions could provide the basis for Captan's genotoxicity. The extreme cytotoxicity of this fungicide, however, could be due to other cellular effects since at the IC50 for cell killing, approximately 0.8 microM, none of the above genotoxic events could be detected by the methods employed.

  20. Quantification of false positive reduction in nucleic acid purification on hemorrhagic fever DNA.

    Energy Technology Data Exchange (ETDEWEB)

    James, Conrad D.; Pohl, Kenneth Roy; Derzon, Mark Steven; McClain, Jaime; Achyuthan, Komandoor

    2006-11-01

    Columbia University has developed a sensitive highly multiplexed system for genetic identification of nucleic acid targets. The primary obstacle to implementing this technology is the high rate of false positives due to high levels of unbound reporters that remain within the system after hybridization. The ability to distinguish between free reporters and reporters bound to targets limits the use of this technology. We previously demonstrated a new electrokinetic method for binary separation of kb pair long DNA molecules and oligonucleotides. The purpose of this project 99864 is to take these previous demonstrations and further develop the technique and hardware for field use. Specifically, our objective was to implement separation in a heterogeneous sample (containing target DNA and background oligo), to perform the separation in a flow-based device, and to develop all of the components necessary for field testing a breadboard prototype system.

  1. Nanobiosensor for Detection and Quantification of DNA Sequences in Degraded Mixed Meats

    Directory of Open Access Journals (Sweden)

    M. E. Ali

    2011-01-01

    Full Text Available A novel class of nanobiosensor was developed by integrating a 27-nucleotide AluI fragment of swine cytochrome b (cytb gene to a 3-nm diameter citrate-tannate coated gold nanoparticle (GNP. The biosensor detected 0.5% and 1% pork in raw and 2.5-h autoclaved pork-beef binary admixtures in a single step without any separation or washing. The hybridization kinetics of the hybrid sensor was studied with synthetic and AluI digested real pork targets from moderate to extreme target concentrations and a sigmoidal relationship was found. Using the kinetic curve, a convenient method for quantifying and counting target DNA copy number was developed. The accuracy of the method was over 90% and 80% for raw and autoclaved pork-beef binary admixtures in the range of 5–100% pork adulteration. The biosensor probe identified a target DNA sequence that was several-folds shorter than a typical PCR-template. This offered the detection and quantitation of potential targets in highly processed or degraded samples where PCR amplification was not possible due to template crisis. The assay was a viable alternative approach of qPCR for detecting, quantifying and counting copy number of shorter size DNA sequences to address a wide ranging biological problem in food industry, diagnostic laboratories and forensic medicine.

  2. Ionizing radiation-induced DNA damage and its repair in human cells. Final performance report, July 1992--June 1995

    Energy Technology Data Exchange (ETDEWEB)

    Dizdaroglu, M.

    1995-12-31

    The studies of DNA damage in living cells in vitro and in vivo were continued. A variety of systems including cultured mammalian cells, animals, and human tissues were used to conduct these studies. In addition, enzymatic repair of DNA base damage was studied using several DNA glycosylases. To this end, substrate specificities of these enzymes were examined in terms of a large number of base lesions in DNA. In the first phase of the studies, the author sought to introduce improvements to his methodologies for measurement of DNA damage using the technique of gas chromatography/mass spectrometry (GC/MS). In particular, the quantitative measurement of DNA base damage and DNA-protein crosslinks was improved by incorporation of isotope-dilution mass spectrometry into the methodologies. This is one of the most accurate techniques for quantification of organic compounds. Having improved the measurement technique, studies of DNA damage in living cells and DNA repair by repair enzymes were pursued. This report provides a summary of these studies with references to the original work.

  3. Expression of DNA-dependent protein kinase in human granulocytes

    Institute of Scientific and Technical Information of China (English)

    Annahita SALLMYR; Anna MILLER; Aida GABDOULKHAKOVA; Valentina SAFRONOVA; Gunnel HENRIKSSON; Anders BREDBERG

    2004-01-01

    Human polymorphonuclear leukocytes (PMN) have been reported to completely lack of DNA-dependent protein kinase (DNA-PK) which is composed of Ku protein and the catalytic subunit DNA-PKcs, needed for nonhomologous end-joining (NHEJ) of DNA double-strand breaks. Promyelocytic HL-60 cells express a variant form of Ku resulting in enhanced radiation sensitivity. This raises the question if low efficiency of NHEJ, instrumental for the cellular repair of oxidative damage, is a normal characteristic of myeloid differentiation. Here we confirmed the complete lack of DNAPK in P MN protein extracts, and the expression of the truncated Ku86 variant form in HL-60. However, this degradation of DNA-PK was shown to be due to a DNA-PK-degrading protease in PMN and HL-60. In addition, by using a protease-resistant whole cell assay, both Ku86 and DNA-PKcs could be demonstrated in PMN, suggesting the previously reported absence in PMN of DNA-PK to be an artefact. The levels of Ku86 and DNA-PKcs were much reduced in PMN, as compared with that of the lymphocytes, whereas HL-60 displayed a markedly elevated DNA-PK concentration.In conclusion, our findings provide evidence of reduced, not depleted expression of DNA-PK during the mature stages of myeloid differentiation.

  4. Human papillomavirus DNA in plasma of patients with cervical cancer

    Directory of Open Access Journals (Sweden)

    Voravud Narin

    2001-03-01

    Full Text Available Abstract Background Human papillomavirus (HPV is a crucial etiological factor for cervical cancer (CC development. From a diagnostic view-point, the consistent presence of HPV in CC allows the viral DNA to be used as a genetic marker. The aims of this study were to evaluate the presence, physical status and clinical significant of HPV DNA in circulation of CC patients. Results Whereas 6 out of 50 (12% HPV positive CC patients revealed plasma HPV DNA, it was detected in none of 20 normal controls or 13 HPV negative CC cases. The plasma DNA exhibited an HPV type identical to the HPV in the primary tumors and the DNA from both sources was integrated into host genome. Interestingly, several findings suggested an association between plasma HPV DNA and metastasis. First, three of the HPV DNA positive cases were CC patients with clinical stage IVB or recurrence with distance metastases (P = 0.001, RR = 15.67. Second, the amount of plasma HPV DNA from metastatic patients to be three times more than three other patients without metastases. Finally, the later cases had tendency to develop recurrence distant metastases within one year after complete treatment when compared with other HPV associated CC patients with the same stage but without the present of plasma HPV DNA. Conclusions The plasma HPV DNA originated from the CC, was associated with metastasis and could be used as a marker representing the circulating free CC DNA.

  5. Human papillomavirus DNA in plasma of patients with HPV16 DNA-positive uterine cervical cancer.

    OpenAIRE

    Shimada, Takako; Yamaguchi, Naohiro; Nishida, Noriyuki; Yamasaki, Kentaro; Miura, Kiyonori; Katamine, Shigeru; Masuzaki, Hideaki

    2010-01-01

    OBJECTIVES: The squamous cell carcinoma antigen is considered the most accurate serologic tumor marker for uterine cervical carcinoma. However, serum squamous cell carcinoma antigen levels were found to correlate significantly with clinical severity of atopic dermatitis and chronic renal failure. The present study was conducted in patients with human papillomavirus 16 DNA-positive uterine cervical cancer to determine the plasma level of human papillomavirus 16 DNA and the diagnostic values of...

  6. A DNA methylation fingerprint of 1628 human samples

    Science.gov (United States)

    Fernandez, Agustin F.; Assenov, Yassen; Martin-Subero, Jose Ignacio; Balint, Balazs; Siebert, Reiner; Taniguchi, Hiroaki; Yamamoto, Hiroyuki; Hidalgo, Manuel; Tan, Aik-Choon; Galm, Oliver; Ferrer, Isidre; Sanchez-Cespedes, Montse; Villanueva, Alberto; Carmona, Javier; Sanchez-Mut, Jose V.; Berdasco, Maria; Moreno, Victor; Capella, Gabriel; Monk, David; Ballestar, Esteban; Ropero, Santiago; Martinez, Ramon; Sanchez-Carbayo, Marta; Prosper, Felipe; Agirre, Xabier; Fraga, Mario F.; Graña, Osvaldo; Perez-Jurado, Luis; Mora, Jaume; Puig, Susana; Prat, Jaime; Badimon, Lina; Puca, Annibale A.; Meltzer, Stephen J.; Lengauer, Thomas; Bridgewater, John; Bock, Christoph; Esteller, Manel

    2012-01-01

    Most of the studies characterizing DNA methylation patterns have been restricted to particular genomic loci in a limited number of human samples and pathological conditions. Herein, we present a compromise between an extremely comprehensive study of a human sample population with an intermediate level of resolution of CpGs at the genomic level. We obtained a DNA methylation fingerprint of 1628 human samples in which we interrogated 1505 CpG sites. The DNA methylation patterns revealed show this epigenetic mark to be critical in tissue-type definition and stemness, particularly around transcription start sites that are not within a CpG island. For disease, the generated DNA methylation fingerprints show that, during tumorigenesis, human cancer cells underwent a progressive gain of promoter CpG-island hypermethylation and a loss of CpG methylation in non-CpG-island promoters. Although transformed cells are those in which DNA methylation disruption is more obvious, we observed that other common human diseases, such as neurological and autoimmune disorders, had their own distinct DNA methylation profiles. Most importantly, we provide proof of principle that the DNA methylation fingerprints obtained might be useful for translational purposes by showing that we are able to identify the tumor type origin of cancers of unknown primary origin (CUPs). Thus, the DNA methylation patterns identified across the largest spectrum of samples, tissues, and diseases reported to date constitute a baseline for developing higher-resolution DNA methylation maps and provide important clues concerning the contribution of CpG methylation to tissue identity and its changes in the most prevalent human diseases. PMID:21613409

  7. Mapping and Quantification of Vascular Branching in Plants, Animals and Humans by VESGEN Software

    Science.gov (United States)

    Parsons-Wingerter, P. A.; Vickerman, M. B.; Keith, P. A.

    2010-01-01

    Humans face daunting challenges in the successful exploration and colonization of space, including adverse alterations in gravity and radiation. The Earth-determined biology of plants, animals and humans is significantly modified in such extraterrestrial environments. One physiological requirement shared by larger plants and animals with humans is a complex, highly branching vascular system that is dynamically responsive to cellular metabolism, immunological protection and specialized cellular/tissue function. VESsel GENeration (VESGEN) Analysis has been developed as a mature beta version, pre-release research software for mapping and quantification of the fractal-based complexity of vascular branching. Alterations in vascular branching pattern can provide informative read-outs of altered vascular regulation. Originally developed for biomedical applications in angiogenesis, VESGEN 2D has provided novel insights into the cytokine, transgenic and therapeutic regulation of angiogenesis, lymphangiogenesis and other microvascular remodeling phenomena. Vascular trees, networks and tree-network composites are mapped and quantified. Applications include disease progression from clinical ophthalmic images of the human retina; experimental regulation of vascular remodeling in the mouse retina; avian and mouse coronary vasculature, and other experimental models in vivo. We envision that altered branching in the leaves of plants studied on ISS such as Arabidopsis thaliana cans also be analyzed.

  8. Direct quantification of brown algae-derived fucoidans in human plasma by a fluorescent probe assay

    CERN Document Server

    Warttinger, Ulrich; Harenberg, Job; Krämer, Roland

    2016-01-01

    Fucoidan is a generic term for a class of fucose rich, structurally diverse sulfated polysaccharides that are found in brown algae and other marine organisms. Depending on the species from which the fucoidan is extracted, a wide variety of biological activities including antitumor, antiinflammatory, immune-modulating, antiviral, antibacterial and pro- and anticoagulant activities has been described. Fucoidans have the advantage of low toxicity and oral bioavailibiity and are viable drug candidates, preclinical and pilot clinical trials show promising results. The availability of robust assays, in particular for analysing the blood levels of fucoidan, is a fundamental requirement for pharmacokinetic analysis in drug development projects. This contribution describes the application of a commercially availbale, protein-free fluorescent probe assay (Heparin Red) for the direct quantification of several fucoidans (from Fucus vesiculosus, Macrocystis pyrifera, and Undaria pinnatifida) in human plasma. By only minor...

  9. Magnetic particle spectroscopy allows precise quantification of nanoparticles after passage through human brain microvascular endothelial cells

    Science.gov (United States)

    Gräfe, C.; Slabu, I.; Wiekhorst, F.; Bergemann, C.; von Eggeling, F.; Hochhaus, A.; Trahms, L.; Clement, J. H.

    2016-06-01

    Crossing the blood-brain barrier is an urgent requirement for the treatment of brain disorders. Superparamagnetic iron oxide nanoparticles (SPIONs) are a promising tool as carriers for therapeutics because of their physical properties, biocompatibility, and their biodegradability. In order to investigate the interaction of nanoparticles with endothelial cell layers in detail, in vitro systems are of great importance. Human brain microvascular endothelial cells are a well-suited blood-brain barrier model. Apart from generating optimal conditions for the barrier-forming cell units, the accurate detection and quantification of SPIONs is a major challenge. For that purpose we use magnetic particle spectroscopy to sensitively and directly quantify the SPION-specific iron content. We could show that SPION concentration depends on incubation time, nanoparticle concentration and location. This model system allows for further investigations on particle uptake and transport at cellular barriers with regard to parameters including particles’ shape, material, size, and coating.

  10. Simultaneous Quantification of Antidiabetic Agents in Human Plasma by a UPLC–QToF-MS Method

    Science.gov (United States)

    Fachi, Mariana Millan; Cerqueira, Letícia Bonancio; Leonart, Letícia Paula; de Francisco, Thais Martins Guimarães

    2016-01-01

    An ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry method for the simultaneous quantification of chlorpropamide, glibenclamide, gliclazide, glimepiride, metformin, nateglinide, pioglitazone, rosiglitazone, and vildagliptin in human plasma was developed and validated, using isoniazid and sulfaquinoxaline as internal standards. Following plasma protein precipitation using acetonitrile with 1% formic acid, chromatographic separation was performed on a cyano column using gradient elution with water and acetonitrile, both containing 0.1% formic acid. Detection was performed in a quadrupole time-of-flight analyzer, using electrospray ionization operated in the positive mode. Data from validation studies demonstrated that the new method is highly sensitive, selective, precise (RSD 0.99), free of matrix and has no residual effects. The developed method was successfully applied to volunteers’ plasma samples. Hence, this method was demonstrated to be appropriate for clinical monitoring of antidiabetic agents. PMID:27930700

  11. Buspirone, fexofenadine, and omeprazole: quantification of probe drugs and their metabolites in human plasma.

    Science.gov (United States)

    Gor, Parul; Alnouti, Yazen; Reed, Gregory A

    2011-07-15

    Probe drugs are critical tools for the measurement of drug metabolism and transport activities in human subjects. Often several probe drugs are administered simultaneously in a "cocktail". This cocktail approach requires efficient analytical methods for the simultaneous quantitation of multiple analytes. We have developed and validated a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of three probe drugs and their metabolites in human plasma. The analytes include omeprazole and its metabolites omeprazole sulfone and 5'-hydroxyomeprazole; buspirone and its metabolite 1-[2-pyrimidyl]-piperazine (1PP); and fexofenadine. These analytes and the internal standard lansoprazole were extracted from plasma using protein precipitation with acetonitrile. Gradient reverse-phase chromatography was performed with 7.5mM ammonium bicarbonate and acetonitrile, and the analytes were quantified in positive ion electrospray mode with multiple reaction monitoring. The method was validated to quantify the concentration ranges of 1.0-1000ng/ml for omeprazole, omeprazole sulfone, 5'-hydroxyomeprazole, and fexofenadine; 0.1-100ng/ml for buspirone, and 1.0-100ng/ml for 1PP. These linear ranges span the plasma concentrations for all of the analytes from probe drug studies. The intra-day precision was between 2.1 and 16.1%, and the accuracy ranged from 86 to 115% for all analytes. Inter-day precision and accuracy ranged from 0.3 to 14% and from 90 to 110%, respectively. The lower limits of quantification were 0.1ng/ml for buspirone and 1ng/ml for all other analytes. This method provides a fast, sensitive, and selective analytical tool for quantification of the six analytes in plasma necessary to support the use of this probe drug cocktail in clinical studies.

  12. Infection Efficiency of Four Phytophthora infestans Clonal Lineages and DNA-Based Quantification of Sporangia.

    Science.gov (United States)

    Fall, Mamadou Lamine; Tremblay, David Mathieu; Gobeil-Richard, Mélanie; Couillard, Julie; Rocheleau, Hélène; Van der Heyden, Hervé; Lévesque, Camile André; Beaulieu, Carole; Carisse, Odile

    2015-01-01

    The presence and abundance of pathogen inoculum is with host resistance and environmental conditions a key factor in epidemic development. Therefore, several spore-sampling devices have been proposed to monitor pathogen inoculum above fields. However, to make spore sampling more reliable as a management tool and to facilitate its adoption, information on infection efficiency and molecular tools for estimating airborne sporangia concentration are needed. Experiments were thus undertaken in a growth chamber to study the infection efficiency of four clonal lineages of P. infestans (US-8, US-11, US-23, and US-24) by measuring the airborne sporangia concentration and resulting disease intensity. The relationship between the airborne sporangia concentration and the number of lesions per leaf was exponential. For the same concentration, the sporangia of US-23 caused significantly more lesions than the sporangia of the other clonal lineages did. Under optimal conditions, an airborne sporangia concentration of 10 sporangia m-3 for US-23 was sufficient to cause one lesion per leaf, whereas for the other clonal lineages, it took 15 to 25 sporangia m-3 to reach the same disease intensity. However, in terms of diseased leaf area, there was no difference between clonal lineages US-8, US-23 and US-24. Also, a sensitive quantitative real-time polymerase chain reaction (qPCR) tool was developed to quantify P. infestans airborne sporangia with detection sensitivity of one sporangium. The specificity of the qPCR assay was rigorously tested for airborne inoculum and was either similar to, or an improvement on, other published PCR assays. This assay allows rapid and reliable detection and quantification of P. infestans airborne sporangia and thereby, facilitates the implementation of spores-sampling network.

  13. Translesion synthesis mechanisms depend on the nature of DNA damage in UV-irradiated human cells.

    Science.gov (United States)

    Quinet, Annabel; Martins, Davi Jardim; Vessoni, Alexandre Teixeira; Biard, Denis; Sarasin, Alain; Stary, Anne; Menck, Carlos Frederico Martins

    2016-07-08

    Ultraviolet-induced 6-4 photoproducts (6-4PP) and cyclobutane pyrimidine dimers (CPD) can be tolerated by translesion DNA polymerases (TLS Pols) at stalled replication forks or by gap-filling. Here, we investigated the involvement of Polη, Rev1 and Rev3L (Polζ catalytic subunit) in the specific bypass of 6-4PP and CPD in repair-deficient XP-C human cells. We combined DNA fiber assay and novel methodologies for detection and quantification of single-stranded DNA (ssDNA) gaps on ongoing replication forks and postreplication repair (PRR) tracts in the human genome. We demonstrated that Rev3L, but not Rev1, is required for postreplicative gap-filling, while Polη and Rev1 are responsible for TLS at stalled replication forks. Moreover, specific photolyases were employed to show that in XP-C cells, CPD arrest replication forks, while 6-4PP are responsible for the generation of ssDNA gaps and PRR tracts. On the other hand, in the absence of Polη or Rev1, both types of lesion block replication forks progression. Altogether, the data directly show that, in the human genome, Polη and Rev1 bypass CPD and 6-4PP at replication forks, while only 6-4PP are also tolerated by a Polζ-dependent gap-filling mechanism, independent of S phase. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Dental DNA fingerprinting in identification of human remains

    Directory of Open Access Journals (Sweden)

    K L Girish

    2010-01-01

    Full Text Available The recent advances in molecular biology have revolutionized all aspects of dentistry. DNA, the language of life yields information beyond our imagination, both in health or disease. DNA fingerprinting is a tool used to unravel all the mysteries associated with the oral cavity and its manifestations during diseased conditions. It is being increasingly used in analyzing various scenarios related to forensic science. The technical advances in molecular biology have propelled the analysis of the DNA into routine usage in crime laboratories for rapid and early diagnosis. DNA is an excellent means for identification of unidentified human remains. As dental pulp is surrounded by dentin and enamel, which forms dental armor, it offers the best source of DNA for reliable genetic type in forensic science. This paper summarizes the recent literature on use of this technique in identification of unidentified human remains.

  15. Rapid extraction and preservation of genomic DNA from human samples.

    Science.gov (United States)

    Kalyanasundaram, D; Kim, J-H; Yeo, W-H; Oh, K; Lee, K-H; Kim, M-H; Ryew, S-M; Ahn, S-G; Gao, D; Cangelosi, G A; Chung, J-H

    2013-02-01

    Simple and rapid extraction of human genomic DNA remains a bottleneck for genome analysis and disease diagnosis. Current methods using microfilters require cumbersome, multiple handling steps in part because salt conditions must be controlled for attraction and elution of DNA in porous silica. We report a novel extraction method of human genomic DNA from buccal swab and saliva samples. DNA is attracted onto a gold-coated microchip by an electric field and capillary action while the captured DNA is eluted by thermal heating at 70 °C. A prototype device was designed to handle four microchips, and a compatible protocol was developed. The extracted DNA using microchips was characterized by qPCR for different sample volumes, using different lengths of PCR amplicon, and nuclear and mitochondrial genes. In comparison with a commercial kit, an equivalent yield of DNA extraction was achieved with fewer steps. Room-temperature preservation for 1 month was demonstrated for captured DNA, facilitating straightforward collection, delivery, and handling of genomic DNA in an environment-friendly protocol.

  16. Princess takamatsu symposium on DNA repair and human cancers.

    Science.gov (United States)

    Loeb, Lawrence A; Nishimura, Susumu

    2010-06-01

    The 40th International Symposium of the Princess Takamatsu Cancer Research Fund, entitled "DNA Repair and Human Cancers," was held on November 10-12, 2009 at Hotel Grand Palace, Tokyo, Japan. The meeting focused on the role of DNA repair in preventing mutations by endogenous and exogenous DNA damage and increasing the efficacy of chemotherapeutic agents by interfering with DNA repair. The 14 presentations by the speakers from the United States, four from the United Kingdom, one each from Italy, The Netherlands, and France, and 13 from Japan, covered most aspects of DNA repair, spanning DNA damage, molecular structures of repair enzymes, and clinical studies on inhibition of DNA repair processes. Extensive time was reserved for discussions with the active participation of the 150 invited Japanese scientists. The choice of a symposium on DNA repair in human cancers resulted in part from the excellent basic and clinical studies that have been carried out for many years in Japan, and the general lack of recognition versus the importance of DNA repair in understanding carcinogenesis. Copyright 2010 AACR.

  17. Comparison of the quantification of KRAS mutations by digital PCR and E-ice-COLD-PCR in circulating-cell-free DNA from metastatic colorectal cancer patients.

    Science.gov (United States)

    Sefrioui, David; Mauger, Florence; Leclere, Laurence; Beaussire, Ludivine; Di Fiore, Frédéric; Deleuze, Jean-François; Sarafan-Vasseur, Nasrin; Tost, Jörg

    2017-02-01

    Circulating cell-free DNA (ccfDNA) bears great promise as biomarker for personalized medicine, but ccfDNA is present only at low levels in the plasma or serum of cancer patients. E-ice-COLD-PCR is a recently developed enrichment method to detect and identify mutations present at low-abundance in clinical samples. However, recent studies have shown the importance to accurately quantify low-abundance mutations as clinically important decisions will depend on certain mutation thresholds. The possibility for an enrichment method to accurately quantify the mutation levels remains a point of concern and might limit its clinical applicability. In the present study, we compared the quantification of KRAS mutations in ccfDNA from metastatic colorectal cancer patients by E-ice-COLD-PCR with two digital PCR approaches. For the quantification of mutations by E-ice-COLD-PCR, cell lines with known mutations diluted into WT genomic DNA were used for calibration. E-ice-COLD-PCR and the two digital PCR approaches showed the same range of the mutation level and were concordant for mutation levels below the clinical relevant threshold. E-ice-COLD-PCR can accurately detect and quantify low-abundant mutations in ccfDNA and has a shorter time to results making it compatible with the requirements of analyses in a clinical setting without the loss of quantitative accuracy. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  18. Development of a new ultra sensitive real-time PCR assay (ultra sensitive RTQ-PCR for the quantification of HBV-DNA

    Directory of Open Access Journals (Sweden)

    Varaklioti Agoritsa

    2010-03-01

    Full Text Available Abstract Background Improved sensitivity of HBV-DNA tests is of critical importance for the management of HBV infection. Our aim was to develop and assess a new ultra sensitive in-house real-time PCR assay for HBV-DNA quantification (ultra sensitive RTQ-PCR. Results Previously used HBV-DNA standards were calibrated against the WHO 1st International Standard for HBV-DNA (OptiQuant® HBV-DNA Quantification Panel, Accrometrix Europe B.V.. The 95% and 50% HBV-DNA detection end-point of the assay were 22.2 and 8.4 IU/mL. According to the calibration results, 1 IU/mL equals 2.8 copies/mL. Importantly the clinical performance of the ultra sensitive real-time PCR was tested similar (67% to the Procleix Ultrio discriminatory HBV test (dHBV (70% in low-titer samples from patients with occult Hepatitis B. Finally, in the comparison of ultra sensitive RTQ-PCR with the commercially available COBAS TaqMan HBV Test, the in-house assay identified 94.7% of the 94 specimens as positive versus 90.4% identified by TaqMan, while the quantitative results that were positive by both assay were strongly correlated (r = 0.979. Conclusions We report a new ultra sensitive real time PCR molecular beacon based assay with remarkable analytical and clinical sensitivity, calibrated against the WHO 1st International standard.

  19. Sensitive and simultaneous quantification of zinc pyrithione and climbazole deposition from anti-dandruff shampoos onto human scalp

    NARCIS (Netherlands)

    Chen, G.; Miao, M.; Hoptroff, M.; Fei, X.; Collins, L.Z.; Jones, A.; Janssen, H.G.

    2015-01-01

    A sensitive ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed and validated for simultaneous quantification of zinc pyrithione (ZPT) and climbazole (CBZ) deposited onto human scalp from anti-dandruff (AD) shampoos. Scrubbing with a buffer so

  20. Sensitive and simultaneous quantification of zinc pyrithione and climbazole deposition from anti-dandruff shampoos onto human scalp

    NARCIS (Netherlands)

    G. Chen; M. Miao; M. Hoptroff; X. Fei; L.Z. Collins; A. Jones; H.G. Janssen

    2015-01-01

    A sensitive ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed and validated for simultaneous quantification of zinc pyrithione (ZPT) and climbazole (CBZ) deposited onto human scalp from anti-dandruff (AD) shampoos. Scrubbing with a buffer so

  1. Detection and quantification of EBV, HHV-6 and CMV DNA in the gastrointestinal tract of HIV-positive patients.

    Science.gov (United States)

    Falasca, F; Maida, P; Gaeta, A; Verzaro, S; Mezzaroma, I; Fantauzzi, A; Donato, G; Bonci, E; Castilletti, C; Antonelli, G; Turriziani, O

    2014-12-01

    Human herpes viruses (HHVs) have been frequently detected in the gastrointestinal (GI) tract and may contribute to the development of gastric cancer. In the present study, the detection rate and viral load of Epstein Barr virus (EBV), HHV-6 and Cytomegalovirus (CMV) were assessed in the GI tract of human immunodeficiency virus (HIV) positive patients and of uninfected patients. The analysis revealed a significantly higher detection rate of EBV and HHV-6 in HIV-infected individuals than in uninfected subjects (88.5 vs 63%; p = 0.03). Moreover, EBV DNA load was significantly higher in the stomach of HIV patients than in controls. These data suggest that the HIV infection status may increase the persistence of these viruses in the GI compartment. Intriguingly, CMV DNA was undetectable in all biopsy specimens analyzed.

  2. Detection and DNA quantification of Enterococcus casseliflavus in a foal with septic meningitis.

    Science.gov (United States)

    Stefanetti, Valentina; Beccati, Francesca; Passamonti, Fabrizio; Sgariglia, Elisa; Coletti, Mauro; Vuerich, Matteo; Marenzoni, Maria Luisa

    2016-07-01

    CASE DESCRIPTION A 3-month-old 180-kg (396-lb) Hanoverian colt was examined because of fever, lethargy, inappetence, drooping of the left ear, and stiff neck posture. Initial treatment included empirical antimicrobial treatment and NSAIDs. CLINICAL FINDINGS Initial findings were consistent with CNS anomalies. Endoscopy revealed hyperemia, ecchymosis, and some mucopurulent exudate in the right guttural pouch. Hematologic findings were consistent with neutrophilic inflammation. On the third day of hospitalization, severe neurologic signs were observed. Computed tomography of the skull revealed a comminuted fracture of the axial aspect of the right mandibular condyle. Examination of CSF revealed turbidity, xanthochromia, and intracellular and extracellular cocci, consistent with septic meningitis. After DNA extraction from blood and CSF, sequenced products from a PCR assay for the bacterial 16S rRNA gene were 99% identical to Enterococcus casseliflavus. Microbial culture of CSF and blood samples yielded bacteria with Enterococcus spp morphology; antimicrobials were selected on the basis of susceptibility testing that identified the isolate as vancomycin resistant. A quantitative PCR assay was used to estimate Enterococcus DNA concentrations in CSF and blood. TREATMENT AND OUTCOME Treatment for E casseliflavus meningitis, including trimethoprim-sulfadiazine and ampicillin sodium administration, resulted in resolution of clinical signs. Culture of CSF and blood samples after 12 days of the targeted treatment yielded no growth. CLINICAL RELEVANCE To the authors' knowledge, this was the first report of E casseliflavus meningitis in a horse. Treatment was successful; vancomycin-resistant enterococci can be a clinical problem and may potentially be zoonotic.

  3. CCQM-K86/P113.1: Relative quantification of genomic DNA fragments extracted from a biological tissue

    Science.gov (United States)

    Corbisier, P.; Vincent, S.; Schimmel, H.; Kortekaas, A.-M.; Trapmann, S.; Burns, M.; Bushell, C.; Akgoz, M.; Akyürek, S.; Dong, L.; Fu, B.; Zhang, L.; Wang, J.; Pérez Urquiza, M.; Bautista, J. L.; Garibay, A.; Fuller, B.; Baoutina, A.; Partis, L.; Emslie, K.; Holden, M.; Chum, W. Y.; Kim, H.-H.; Phunbua, N.; Milavec, M.; Zel, J.; Vonsky, M.; Konopelko, L. A.; Lau, T. L. T.; Yang, B.; Hui, M. H. K.; Yu, A. C. H.; Viroonudomphol, D.; Prawettongsopon, C.; Wiangnon, K.; Takabatake, R.; Kitta, K.; Kawaharasaki, M.; Parkes, H.

    2012-01-01

    Key comparison CCQM-K86 was performed to demonstrate and document the capacity of interested national metrology institutes (NMIs) and designated institutes (DIs) in the determination of the relative quantity of two specific genomic DNA fragments present in a biological tissue. The study provides the support for the following measurement claim: "Quantification of the ratio of the number of copies of specified intact sequence fragments of a length in the range of 70 to 100 nucleotides in a single genomic DNA extract from ground maize seed materials". The study was carried out under the auspices of the Bioanalysis Working Group (BAWG) of the Comité Consultatif pour la Quantité de Matière (CCQM) and was piloted by the Institute for Reference Materials and Methods (IRMM) in Geel (Belgium). The following laboratories (in alphabetical order) participated in this key comparison: AIST (Japan), CENAM (Mexico), DMSc (Thailand), GLHK (Hong Kong), IRMM (European Union), KRISS (Republic of Korea), LGC (United Kingdom), MIRS/NIB (Slovenia), NIM (PR China), NIST (USA), NMIA (Australia), TÜBITAK UME (Turkey) and VNIIM (Russian Federation). The following laboratories (in alphabetical order) participated in a pilot study that was organized in parallel: LGC (United Kingdom), PKU (PR China), NFRI (Japan) and NIMT (Thailand). Good agreement was observed between the reported results of eleven participants. Main text. To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (MRA).

  4. The linguistics of DNA. [HUMAN GENOME PROJECT

    Energy Technology Data Exchange (ETDEWEB)

    Searls, D.B. (Univ. of Pennsylvania, Philadelphia (United States))

    Discusses the structure of DNA and RNA and the mechanisms of transcription and translation in relation to the grammatical rules of language. The ultimate purpose is to design a grammar which can be used to write flexible, adaptive computer programs for searching nucleotide sequences, with the goal of being able to search large sequences for gene-coding regions. 11 refs., 16 figs.

  5. The dynamic DNA methylomes of double-stranded DNA viruses associated with human cancer

    Science.gov (United States)

    Fernandez, Agustin F.; Rosales, Cecilia; Lopez-Nieva, Pilar; Graña, Osvaldo; Ballestar, Esteban; Ropero, Santiago; Espada, Jesus; Melo, Sonia A.; Lujambio, Amaia; Fraga, Mario F.; Pino, Irene; Javierre, Biola; Carmona, Francisco J.; Acquadro, Francesco; Steenbergen, Renske D.M.; Snijders, Peter J.F.; Meijer, Chris J.; Pineau, Pascal; Dejean, Anne; Lloveras, Belen; Capella, Gabriel; Quer, Josep; Buti, Maria; Esteban, Juan-Ignacio; Allende, Helena; Rodriguez-Frias, Francisco; Castellsague, Xavier; Minarovits, Janos; Ponce, Jordi; Capello, Daniela; Gaidano, Gianluca; Cigudosa, Juan Cruz; Gomez-Lopez, Gonzalo; Pisano, David G.; Valencia, Alfonso; Piris, Miguel Angel; Bosch, Francesc X.; Cahir-McFarland, Ellen; Kieff, Elliott; Esteller, Manel

    2009-01-01

    The natural history of cancers associated with virus exposure is intriguing, since only a minority of human tissues infected with these viruses inevitably progress to cancer. However, the molecular reasons why the infection is controlled or instead progresses to subsequent stages of tumorigenesis are largely unknown. In this article, we provide the first complete DNA methylomes of double-stranded DNA viruses associated with human cancer that might provide important clues to help us understand the described process. Using bisulfite genomic sequencing of multiple clones, we have obtained the DNA methylation status of every CpG dinucleotide in the genome of the Human Papilloma Viruses 16 and 18 and Human Hepatitis B Virus, and in all the transcription start sites of the Epstein-Barr Virus. These viruses are associated with infectious diseases (such as hepatitis B and infectious mononucleosis) and the development of human tumors (cervical, hepatic, and nasopharyngeal cancers, and lymphoma), and are responsible for 1 million deaths worldwide every year. The DNA methylomes presented provide evidence of the dynamic nature of the epigenome in contrast to the genome. We observed that the DNA methylome of these viruses evolves from an unmethylated to a highly methylated genome in association with the progression of the disease, from asymptomatic healthy carriers, through chronically infected tissues and pre-malignant lesions, to the full-blown invasive tumor. The observed DNA methylation changes have a major functional impact on the biological behavior of the viruses. PMID:19208682

  6. Quantification of organellar DNA and RNA using real-time PCR.

    Science.gov (United States)

    Weihe, Andreas

    2014-01-01

    Quantitative (real-time) polymerase chain reaction (PCR) allows the measurement of relative organellar gene copy numbers as well as transcript abundance of individual mitochondrial or plastidial genes. Requiring only minute amounts of total DNA or RNA, the described method can replace traditional analyses like Southern or Northern hybridization which require large amounts of organellar nucleic acids and usually provide only semiquantitative data. Here we describe prerequisites, reaction conditions, and data analysis principles, which should be applicable for a wide range of plant species and experimental situations where comparative and precise determination of gene copy numbers or transcript abundance is requested. Sequences of amplification primers for qPCR of organellar genes from Arabidopsis are provided.

  7. Exploiting Nanobodies in the Detection and Quantification of Human Growth Hormone via Phage-Sandwich Enzyme-Linked Immunosorbent Assay

    Directory of Open Access Journals (Sweden)

    Hossam Murad

    2017-05-01

    Full Text Available BackgroundMonitoring blood levels of human growth hormone (hGH in most children with short stature deficiencies is crucial for taking a decision of treatment with extended course of daily and expensive doses of recombinant hGH (rhGH or Somatropin®. Besides, misusing of rhGH by sportsmen is banned by the World Anti-Doping Agency and thus sensitive GH-detecting methods are highly welcome in this field. Nanobodies are the tiniest antigen-binding entity derived from camel heavy chain antibodies. They were successfully generated against numerous antigens including hormones.MethodsA fully nanobody-based sandwich ELISA method was developed in this work for direct measurement of GH in biological samples.ResultsTwo major characteristics of nanobody were exploited for this goal: the robust and stable structure of the nanobody (NbGH04 used to capture hGH from tested samples, and the great ability of tailoring, enabling the display of the anti-GH detector nanobody (NbGH07 on the tip of M13-phage. Such huge, stable, and easy-to-prepare phage-Nb was used in ELISA to provide an amplified signal. Previously, NbGH04 was retrieved on immobilized hGH by phage display from a wide “immune” cDNA library prepared from a hGH-immunized camel. Here, and in order to assure epitope heterogeneity, NbGH07 was isolated from the same library using NbGH04-captured hGH as bait. Interaction of both nanobodies with hGH was characterized and compared with different anti-GH nanobodies and antibodies. The sensitivity (~0.5 ng/ml and stability of the nanobody-base sandwich ELISA were assessed using rhGH before testing in the quantification of hGH in blood sera and cell culture supernatants.ConclusionIn regard to all advantages of nanobodies; stability, solubility, production affordability in Escherichia coli, and gene tailoring, nanobody-based phage sandwich ELISA developed here would provide a valuable method for hGH detection and quantification.

  8. Exploiting Nanobodies in the Detection and Quantification of Human Growth Hormone via Phage-Sandwich Enzyme-Linked Immunosorbent Assay.

    Science.gov (United States)

    Murad, Hossam; Assaad, Jana Mir; Al-Shemali, Rasha; Abbady, Abdul Qader

    2017-01-01

    Monitoring blood levels of human growth hormone (hGH) in most children with short stature deficiencies is crucial for taking a decision of treatment with extended course of daily and expensive doses of recombinant hGH (rhGH or Somatropin(®)). Besides, misusing of rhGH by sportsmen is banned by the World Anti-Doping Agency and thus sensitive GH-detecting methods are highly welcome in this field. Nanobodies are the tiniest antigen-binding entity derived from camel heavy chain antibodies. They were successfully generated against numerous antigens including hormones. A fully nanobody-based sandwich ELISA method was developed in this work for direct measurement of GH in biological samples. Two major characteristics of nanobody were exploited for this goal: the robust and stable structure of the nanobody (NbGH04) used to capture hGH from tested samples, and the great ability of tailoring, enabling the display of the anti-GH detector nanobody (NbGH07) on the tip of M13-phage. Such huge, stable, and easy-to-prepare phage-Nb was used in ELISA to provide an amplified signal. Previously, NbGH04 was retrieved on immobilized hGH by phage display from a wide "immune" cDNA library prepared from a hGH-immunized camel. Here, and in order to assure epitope heterogeneity, NbGH07 was isolated from the same library using NbGH04-captured hGH as bait. Interaction of both nanobodies with hGH was characterized and compared with different anti-GH nanobodies and antibodies. The sensitivity (~0.5 ng/ml) and stability of the nanobody-base sandwich ELISA were assessed using rhGH before testing in the quantification of hGH in blood sera and cell culture supernatants. In regard to all advantages of nanobodies; stability, solubility, production affordability in Escherichia coli, and gene tailoring, nanobody-based phage sandwich ELISA developed here would provide a valuable method for hGH detection and quantification.

  9. Nonneutral mitochondrial DNA variation in humans and chimpanzees

    Energy Technology Data Exchange (ETDEWEB)

    Nachman, M.W.; Aquadro, C.F. [Cornell Univ., Ithaca, NY (United States); Brown, W.M. [Univ. of Michigan, Ann Arbor, MI (United States)] [and others

    1996-03-01

    We sequenced the NADH dehydrogenase subunit 3 (ND3) gene from a sample of 61 humans, five common chimpanzees, and one gorilla to test whether patterns of mitochondrial DNA (mtDNA) variation are consistent with a neutral model of molecular evolution. Within humans and within chimpanzees, the ratio of replacement to silent nucleotide substitutions was higher than observed in comparisons between species, contrary to neutral expectations. To test the generality of this result, we reanalyzed published human RFLP data from the entire mitochondrial genome. Gains of restriction sites relative to a known human mtDNA sequence were used to infer unambiguous nucleotide substitutions. We also compared the complete mtDNA sequences of three humans. Both the RFLP data and the sequence data reveal a higher ratio of replacement to silent nucleotide substitutions within humans than is seen between species. This pattern is observed at most or all human mitochondrial genes and is inconsistent with a strictly neutral model. These data suggest that many mitochondrial protein polymorphisms are slightly deleterious, consistent with studies of human mitochondrial diseases. 59 refs., 2 figs., 8 tabs.

  10. Effects of incense smoke on human lymphocyte DNA.

    Science.gov (United States)

    Szeto, Yim Tong; Sok Wa Leong, Kosca; Keong Lam, Kason; Min Min Hong, Cynthia; Kai Mui Lee, Daphne; Teng Fun Chan, Yui; Benzie, Iris F F

    2009-01-01

    Incense burning is common in Southeast Asia, where it is a traditional and ceremonial practice in deity worship and paying respect to ancestors. However, incense emissions are an important source of indoor air pollution in Asia, and may induce health problems to those exposed. In this in vitro study the effects of incense emissions on human DNA were investigated using the comet assay. Particulates in smoke from six kinds of incense were trapped in saline or ethanol and human lymphocytes were exposed under controlled conditions. Results showed that DNA damage, including strand breaks, was induced by both aqueous and ethanolic extracts of two samples. The ethanolic extract of one sample induced DNA damage, while no significant DNA damage was found in the remaining three samples. The mechanisms underlying DNA damage induced by incense emissions were also investigated. Catalase (CAT), sodium azide, and superoxide dismutase (SOD) were co-incubated with extract, which exerted significant DNA damaging effects. Results showed that CAT with or without SOD diminished DNA damage, whereas sodium azide did not seem able to reduce DNA damage. Data indicate there are potential adverse health effects of such exposure, particularly for temple workers.

  11. Analysis of Genomic DNA Methylation Levels in Human Placenta using Liquid Chromatography-Electrospray Ionization Tandem Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Sulistyo Emantoko Dwi Putra

    2014-03-01

    Full Text Available Background: DNA-methylation is a common epigenetic tool which plays a crucial role in gene regulation and is essential for cell differentiation and embryonic development. The placenta is an important organ where gene activity can be regulated by epigenetic DNA modifications, including DNA methylation. This is of interest as, the placenta is the interface between the fetus and its environment, the mother. Exposure to environmental toxins and nutrition during pregnancy may alter DNA methylation of the placenta and subsequently placental function and as a result the phenotype of the offspring. The aim of this study was to develop a reliable method to quantify DNA methylation in large clinical studies. This will be a tool to analyze the degree of DNA methylation in the human placenta in relationship to clinical readouts. Methods: Liquid chromatography-electrospray ionization/multi-stage mass spectrometry (LC-ESI/MS/MS technique was used for the quantification of the 5dmC/dG ratio in placentas from 248 healthy pregnancies. We were able to demonstrate that this method is a reliable and stable way to determine global placental DNA methylation in large clinical trials. Results/Conclusion: The degree of placental DNA methylation seen in our pilot study varies substantially from 2% to 5%. The clinical implications of this variation need to be demonstrated in adequately powered large studies.

  12. Validation of a PicoGreen-based DNA quantification integrated in an RNA extraction method for two-dimensional and three-dimensional cell cultures.

    Science.gov (United States)

    Chen, Yantian; Sonnaert, Maarten; Roberts, Scott J; Luyten, Frank P; Schrooten, Jan

    2012-06-01

    DNA measurement and RNA extraction are two frequently used methods for cell characterization. In the conventional protocols, they require similar, but separate samples and in most cases, different pretreatments. The few combined protocols that exist still include time-consuming steps. Hence, to establish an efficient combined RNA extraction and DNA measurement protocol for two-dimensional (2D) and three-dimensional (3D) cell cultures, a PicoGreen-based DNA measurement was integrated in an existing RNA extraction protocol. It was validated by analysis of the influence of different lysis buffers, RLT, RA1, or Trizol, used for RNA extraction on the measured DNA concentration. The DNA cell yield was evaluated both in cell suspensions (2D) and on 3D cell-seeded scaffolds. Results showed that the different RNA lysis buffers caused a concentration-dependent perturbation of the PicoGreen signal. The measured DNA concentrations in 2D and 3D using RLT and RA1 buffer were comparable, also to the positive control. We, therefore, concluded that RNA extraction protocols using RA1 or RLT buffer allow the integration of a DNA quantification step without the buffer influencing the results. Hence, the combined DNA measurement and RNA extraction offer an alternative for DNA measurement techniques that is time and sample saving, for both 2D cell cultures and specific 3D constructs.

  13. Structural and functional interaction between the human DNA repair proteins DNA ligase IV and XRCC4.

    Science.gov (United States)

    Wu, Peï-Yu; Frit, Philippe; Meesala, SriLakshmi; Dauvillier, Stéphanie; Modesti, Mauro; Andres, Sara N; Huang, Ying; Sekiguchi, JoAnn; Calsou, Patrick; Salles, Bernard; Junop, Murray S

    2009-06-01

    Nonhomologous end-joining represents the major pathway used by human cells to repair DNA double-strand breaks. It relies on the XRCC4/DNA ligase IV complex to reseal DNA strands. Here we report the high-resolution crystal structure of human XRCC4 bound to the carboxy-terminal tandem BRCT repeat of DNA ligase IV. The structure differs from the homologous Saccharomyces cerevisiae complex and reveals an extensive DNA ligase IV binding interface formed by a helix-loop-helix structure within the inter-BRCT linker region, as well as significant interactions involving the second BRCT domain, which induces a kink in the tail region of XRCC4. We further demonstrate that interaction with the second BRCT domain of DNA ligase IV is necessary for stable binding to XRCC4 in cells, as well as to achieve efficient dominant-negative effects resulting in radiosensitization after ectopic overexpression of DNA ligase IV fragments in human fibroblasts. Together our findings provide unanticipated insight for understanding the physical and functional architecture of the nonhomologous end-joining ligation complex.

  14. Structural and Functional Interaction Between the Human DNA Repair Proteins DNA ligase IV and XRCC4

    Energy Technology Data Exchange (ETDEWEB)

    Wu, P.; Meesala, S; Dauvillier, S; Modesti, M; Andres, S; Huang, Y; Sekiguchi, J; Calsou, P; Salles, B; Junop, M

    2009-01-01

    Nonhomologous end-joining represents the major pathway used by human cells to repair DNA double-strand breaks. It relies on the XRCC4/DNA ligase IV complex to reseal DNA strands. Here we report the high-resolution crystal structure of human XRCC4 bound to the carboxy-terminal tandem BRCT repeat of DNA ligase IV. The structure differs from the homologous Saccharomyces cerevisiae complex and reveals an extensive DNA ligase IV binding interface formed by a helix-loop-helix structure within the inter-BRCT linker region, as well as significant interactions involving the second BRCT domain, which induces a kink in the tail region of XRCC4. We further demonstrate that interaction with the second BRCT domain of DNA ligase IV is necessary for stable binding to XRCC4 in cells, as well as to achieve efficient dominant-negative effects resulting in radiosensitization after ectopic overexpression of DNA ligase IV fragments in human fibroblasts. Together our findings provide unanticipated insight for understanding the physical and functional architecture of the nonhomologous end-joining ligation complex.

  15. Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA

    DEFF Research Database (Denmark)

    Boessenkool, Sanne; Epp, Laura S.; Haile, James Seymour

    2012-01-01

    , or bias, during the PCR. In this study, we test the utility of human-specific blocking primers in mammal diversity analyses of ancient permafrost samples from Siberia. Using quantitative PCR (qPCR) on human and mammoth DNA, we first optimized the design and concentration of blocking primer in the PCR...

  16. Human DNA ligase III bridges two DNA ends to promote specific intermolecular DNA end joining.

    Science.gov (United States)

    Kukshal, Vandna; Kim, In-Kwon; Hura, Gregory L; Tomkinson, Alan E; Tainer, John A; Ellenberger, Tom

    2015-08-18

    Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells dependent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with this activity, LigIII acts in an alternative pathway of DNA double strand break repair that buttresses canonical non-homologous end joining (NHEJ) and is manifest in NHEJ-defective cancer cells, but how LigIII acts in joining intermolecular DNA ends versus nick ligation is unclear. To investigate how LigIII efficiently joins two DNAs, we developed a real-time, fluorescence-based assay of DNA bridging suitable for high-throughput screening. On a nicked duplex DNA substrate, the results reveal binding competition between the ZnF and the oligonucleotide/oligosaccharide-binding domain, one of three domains constituting the LigIII catalytic core. In contrast, these domains collaborate and are essential for formation of a DNA-bridging intermediate by adenylated LigIII that positions a pair of blunt-ended duplex DNAs for efficient and specific intermolecular ligation.

  17. Human DNA ligase III bridges two DNA ends to promote specific intermolecular DNA end joining

    Science.gov (United States)

    Kukshal, Vandna; Kim, In-Kwon; Hura, Gregory L.; Tomkinson, Alan E.; Tainer, John A.; Ellenberger, Tom

    2015-01-01

    Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells dependent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with this activity, LigIII acts in an alternative pathway of DNA double strand break repair that buttresses canonical non-homologous end joining (NHEJ) and is manifest in NHEJ-defective cancer cells, but how LigIII acts in joining intermolecular DNA ends versus nick ligation is unclear. To investigate how LigIII efficiently joins two DNAs, we developed a real-time, fluorescence-based assay of DNA bridging suitable for high-throughput screening. On a nicked duplex DNA substrate, the results reveal binding competition between the ZnF and the oligonucleotide/oligosaccharide-binding domain, one of three domains constituting the LigIII catalytic core. In contrast, these domains collaborate and are essential for formation of a DNA-bridging intermediate by adenylated LigIII that positions a pair of blunt-ended duplex DNAs for efficient and specific intermolecular ligation. PMID:26130724

  18. Persistent organic pollutants alter DNA methylation during human adipocyte differentiation.

    Science.gov (United States)

    van den Dungen, Myrthe W; Murk, Albertinka J; Kok, Dieuwertje E; Steegenga, Wilma T

    2017-04-01

    Ubiquitous persistent organic pollutants (POPs) can accumulate in humans where they might influence differentiation of adipocytes. The aim of this study was to investigate whether DNA methylation is one of the underlying mechanisms by which POPs affect adipocyte differentiation, and to what extent DNA methylation can be related to gene transcription. Adipocyte differentiation was induced in two human cell models with continuous exposure to different POPs throughout differentiation. From the seven tested POPs, perfluorooctanesulfonic acid (PFOS) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) decreased lipid accumulation, while tributyltin (TBT) increased lipid accumulation. In human mesenchymal stem cells (hMSCs), TCDD and TBT induced opposite gene expression profiles, whereas after PFOS exposure gene expression remained relatively stable. Genome-wide DNA methylation analysis showed that all three POPs affected DNA methylation patterns in adipogenic and other genes, possibly related to the phenotypic outcome, but without concomitant gene expression changes. Differential methylation was predominantly detected in intergenic regions, where the biological relevance of alterations in DNA methylation is unclear. This study demonstrates that POPs, at environmentally relevant levels, are able to induce differential DNA methylation in human differentiating adipocytes. Copyright © 2017 Wageningen University. Published by Elsevier Ltd.. All rights reserved.

  19. Pitfalls in the analysis of ancient human mtDNA

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    The retrieval of DNA from ancient human specimens is not always successful owing to DNA deterioration and contamination although it is vital to provide new insights into the genetic structure of ancient people and to reconstruct the past history. Normally, only short DNA fragments can be retrieved from the ancient specimens. How to identify the authenticity of DNA obtained and to uncover the information it contained are difficult. We employed the ancient mtDNAs reported from Central Asia (including Xinjiang, China) as an example to discern potentially extraneous DNA contamination based on the updated mtDNA phylogeny derived from mtDNA control region, coding region, as well as complete sequence information. Our results demonstrated that many mtDNAs reported are more or less problematic. Starting from a reliable mtDNA phylogeney and combining the available modern data into analysis, one can ascertain the authenticity of the ancient DNA, distinguish the potential errors in a data set, and efficiently decipher the meager information it harbored. The reappraisal of the mtDNAs with the age of more than 2000 years from Central Asia gave support to the suggestion of extensively (pre)historical gene admixture in this region.

  20. Brown adipose tissue quantification in human neonates using water-fat separated MRI.

    Directory of Open Access Journals (Sweden)

    Jerod M Rasmussen

    Full Text Available There is a major resurgence of interest in brown adipose tissue (BAT biology, particularly regarding its determinants and consequences in newborns and infants. Reliable methods for non-invasive BAT measurement in human infants have yet to be demonstrated. The current study first validates methods for quantitative BAT imaging of rodents post mortem followed by BAT excision and re-imaging of excised tissues. Identical methods are then employed in a cohort of in vivo infants to establish the reliability of these measures and provide normative statistics for BAT depot volume and fat fraction. Using multi-echo water-fat MRI, fat- and water-based images of rodents and neonates were acquired and ratios of fat to the combined signal from fat and water (fat signal fraction were calculated. Neonatal scans (n = 22 were acquired during natural sleep to quantify BAT and WAT deposits for depot volume and fat fraction. Acquisition repeatability was assessed based on multiple scans from the same neonate. Intra- and inter-rater measures of reliability in regional BAT depot volume and fat fraction quantification were determined based on multiple segmentations by two raters. Rodent BAT was characterized as having significantly higher water content than WAT in both in situ as well as ex vivo imaging assessments. Human neonate deposits indicative of bilateral BAT in spinal, supraclavicular and axillary regions were observed. Pairwise, WAT fat fraction was significantly greater than BAT fat fraction throughout the sample (ΔWAT-BAT = 38 %, p<10(-4. Repeated scans demonstrated a high voxelwise correlation for fat fraction (Rall = 0.99. BAT depot volume and fat fraction measurements showed high intra-rater (ICCBAT,VOL = 0.93, ICCBAT,FF = 0.93 and inter-rater reliability (ICCBAT,VOL = 0.86, ICCBAT,FF = 0.93. This study demonstrates the reliability of using multi-echo water-fat MRI in human neonates for quantification throughout the torso of BAT depot volume and fat

  1. D-ribose inhibits DNA repair synthesis in human lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Zunica, G.; Marini, M.; Brunelli, M.A.; Chiricolo, M.; Franceschi, C.

    1986-07-31

    D-ribose is cytotoxic for quiescent human lymphocytes and severely inhibits their PHA-induced proliferation at concentrations (25-50 mM) at which other simple sugars are ineffective. In order to explain these effects, DNA repair synthesis was evaluated in PHA-stimulated human lymphocytes treated with hydroxyurea and irradiated. D-ribose, in contrast to other reducing sugars, did not induce repair synthesis and therefore did not apparently damage DNA in a direct way, although it markedly inhibited gamma ray-induced repair. Taking into account that lymphocytes must rejoin physiologically-formed DNA strand breaks in order to enter the cell cycle, we suggest that D-ribose exerts its cytotoxic activity by interfering with metabolic pathways critical for the repair of DNA breaks.

  2. Isolation and characterization of DNA probes for human chromosome 21.

    Science.gov (United States)

    Watkins, P C

    1990-01-01

    A coordinated effort to map and sequence the human genome has recently become a national priority. Chromosome 21, the smallest human chromosome accounting for less than 2% of the human genome, is an attractive model system for developing and evaluating genome mapping technology. Several strategies are currently being explored including the development of chromosome 21 libraries from somatic cell hybrids as reported here, the cloning of chromosome 21 in yeast artificial chromosomes (McCormick et al., 1989b), and the construction of chromosome 21 libraries using chromosome flow-sorting techniques (Fuscoe et al., 1989). This report describes the approaches used to identify DNA probes that are useful for mapping chromosome 21. Probes were successfully isolated from both phage and cosmid libraries made from two somatic cell hybrids that contain human chromosome 21 as the only human chromosome. The 15 cosmid clones from the WA17 library, reduced to cloned DNA sequences of an average size of 3 kb, total 525 kb of DNA which is approximately 1% of chromosome 21. From these clones, a set of polymorphic DNA markers that span the length of the long arm of chromosome 21 has been generated. All of the probes thus far analyzed from the WA17 libraries have been mapped to chromosome 21 both by physical and genetic mapping methods. It is therefore likely that the WA17 hybrid cell line contains human chromosome 21 as the only human component, in agreement with cytogenetic observation. The 153E7b cosmid libraries will provide an alternative source of cloned chromosome 21 DNA. Library screening techniques can be employed to obtain cloned DNA sequences from the same genetic loci of the two different chromosome 21s. Comparative analysis will allow direct estimation of DNA sequence variation for different regions of chromosome 21. Mapped DNA probes make possible the molecular analysis of chromosome 21 at a level of resolution not achievable by classical cytogenetic techniques (Graw et al

  3. Modeling and Quantification of Team Performance in Human Reliability Analysis for Probabilistic Risk Assessment

    Energy Technology Data Exchange (ETDEWEB)

    Jeffrey C. JOe; Ronald L. Boring

    2014-06-01

    Probabilistic Risk Assessment (PRA) and Human Reliability Assessment (HRA) are important technical contributors to the United States (U.S.) Nuclear Regulatory Commission’s (NRC) risk-informed and performance based approach to regulating U.S. commercial nuclear activities. Furthermore, all currently operating commercial NPPs in the U.S. are required by federal regulation to be staffed with crews of operators. Yet, aspects of team performance are underspecified in most HRA methods that are widely used in the nuclear industry. There are a variety of "emergent" team cognition and teamwork errors (e.g., communication errors) that are 1) distinct from individual human errors, and 2) important to understand from a PRA perspective. The lack of robust models or quantification of team performance is an issue that affects the accuracy and validity of HRA methods and models, leading to significant uncertainty in estimating HEPs. This paper describes research that has the objective to model and quantify team dynamics and teamwork within NPP control room crews for risk informed applications, thereby improving the technical basis of HRA, which improves the risk-informed approach the NRC uses to regulate the U.S. commercial nuclear industry.

  4. Functional interactions of DNA topoisomerases with a human replication origin.

    Science.gov (United States)

    Abdurashidova, Gulnara; Radulescu, Sorina; Sandoval, Oscar; Zahariev, Sotir; Danailov, Miltcho B; Demidovich, Alexander; Santamaria, Laura; Biamonti, Giuseppe; Riva, Silvano; Falaschi, Arturo

    2007-02-21

    The human DNA replication origin, located in the lamin B2 gene, interacts with the DNA topoisomerases I and II in a cell cycle-modulated manner. The topoisomerases interact in vivo and in vitro with precise bonds ahead of the start sites of bidirectional replication, within the pre-replicative complex region; topoisomerase I is bound in M, early G1 and G1/S border and topoisomerase II in M and the middle of G1. The Orc2 protein competes for the same sites of the origin bound by either topoisomerase in different moments of the cell cycle; furthermore, it interacts on the DNA with topoisomerase II during the assembly of the pre-replicative complex and with DNA-bound topoisomerase I at the G1/S border. Inhibition of topoisomerase I activity abolishes origin firing. Thus, the two topoisomerases are closely associated with the replicative complexes, and DNA topology plays an essential functional role in origin activation.

  5. Quantification of cortisol in human eccrine sweat by liquid chromatography - tandem mass spectrometry.

    Science.gov (United States)

    Jia, Min; Chew, Wade M; Feinstein, Yelena; Skeath, Perry; Sternberg, Esther M

    2016-03-21

    Cortisol has long been recognized as the "stress biomarker" in evaluating stress related disorders. Plasma, urine or saliva are the current source for cortisol analysis. The sampling of these biofluids is either invasive or has reliability problems that could lead to inaccurate results. Sweat has drawn increasing attention as a promising source for non-invasive stress analysis. A sensitive HPLC-MS/MS method was developed for the quantitation of cortisol ((11β)-11,17,21-trihydroxypregn-4-ene-3,20-dione) in human eccrine sweat. At least one unknown isomer that has previously not been reported and could potentially interfere with quantification was separated from cortisol with mixed mode RP HPLC. Detection of cortisol was carried out using atmospheric pressure chemical ionization (APCI) and selected reaction monitoring (SRM) in positive ion mode, using cortisol-9,11,12,12-D4 as internal standard. LOD and LOQ were estimated to be 0.04 ng ml(-1) and 0.1 ng ml(-1), respectively. Linear range of 0.10-25.00 ng ml(-1) was obtained. Intraday precision (2.5%-9.7%) and accuracy (0.5%-2.1%), interday precision (12.3%-18.7%) and accuracy (7.1%-15.1%) were achieved. This method has been successfully applied to the cortisol analysis of human eccrine sweat samples. This is the first demonstration that HPLC-MS/MS can be used for the sensitive and highly specific determination of cortisol in human eccrine sweat in the presence of at least one isomer that has similar hydrophobicity as cortisol. This study demonstrated that human eccrine sweat could be used as a promising source for non-invasive assessment of stress biomarkers such as cortisol and other steroid hormones.

  6. An in-house real-time polymerase chain reaction: standardisation and comparison with the Cobas Amplicor HBV monitor and Cobas AmpliPrep/Cobas TaqMan HBV tests for the quantification of hepatitis B virus DNA

    Science.gov (United States)

    Santos, Ana Paula de Torres; Levi, José Eduardo; Lemos, Marcilio Figueiredo; Calux, Samira Julien; Oba, Isabel Takano; Moreira, Regina Célia

    2016-01-01

    This study aimed to standardise an in-house real-time polymerase chain reaction (rtPCR) to allow quantification of hepatitis B virus (HBV) DNA in serum or plasma samples, and to compare this method with two commercial assays, the Cobas Amplicor HBV monitor and the Cobas AmpliPrep/Cobas TaqMan HBV test. Samples from 397 patients from the state of São Paulo were analysed by all three methods. Fifty-two samples were from patients who were human immunodeficiency virus and hepatitis C virus positive, but HBV negative. Genotypes were characterised, and the viral load was measure in each sample. The in-house rtPCR showed an excellent success rate compared with commercial tests; inter-assay and intra-assay coefficients correlated with commercial tests (r = 0.96 and r = 0.913, p < 0.001) and the in-house test showed no genotype-dependent differences in detection and quantification rates. The in-house assay tested in this study could be used for screening and quantifying HBV DNA in order to monitor patients during therapy. PMID:26872342

  7. Purification of human leucocyte DNA: proteinase K is not necessary.

    Science.gov (United States)

    Douglas, A M; Georgalis, A M; Benton, L R; Canavan, K L; Atchison, B A

    1992-03-01

    A rapid nontoxic method for the purification of DNA from human leucocytes is described. Preliminary experiments which tested different methods of DNA purification indicated that digestion of proteins with proteinase K was unnecessary. This led to the development of a simple procedure involving lysis of the cells in SDS followed by extraction with 6 M NaCl. The method described overcomes the requirement for lengthy incubations in the presence of expensive proteinase K and subsequent extraction with toxic chemicals.

  8. Human neuronal tau promoting the melting temperature of DNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The hyperchromic effect of ultraviolet spectroscopy shows that adding recombinant human neuronal tau to the solution of calf thymus DNA will promote the melting temperature (Tm) from 67℃ to 81℃. Similar result has been detected when adding tau to plasmid pBluescript-Ⅱ SK, by raising Tm from 75℃ to 85℃. The kinetics of thermal denaturation of DNA with tau is much slower than that of control. It suggests that tau may stabilize the double helix conformation of DNA.

  9. Detection of Streptococcus mutans Genomic DNA in Human DNA Samples Extracted from Saliva and Blood

    Science.gov (United States)

    Vieira, Alexandre R.; Deeley, Kathleen B.; Callahan, Nicholas F.; Noel, Jacqueline B.; Anjomshoaa, Ida; Carricato, Wendy M.; Schulhof, Louise P.; DeSensi, Rebecca S.; Gandhi, Pooja; Resick, Judith M.; Brandon, Carla A.; Rozhon, Christopher; Patir, Asli; Yildirim, Mine; Poletta, Fernando A.; Mereb, Juan C.; Letra, Ariadne; Menezes, Renato; Wendell, Steven; Lopez-Camelo, Jorge S.; Castilla, Eduardo E.; Orioli, Iêda M.; Seymen, Figen; Weyant, Robert J.; Crout, Richard; McNeil, Daniel W.; Modesto, Adriana; Marazita, Mary L.

    2011-01-01

    Caries is a multifactorial disease, and studies aiming to unravel the factors modulating its etiology must consider all known predisposing factors. One major factor is bacterial colonization, and Streptococcus mutans is the main microorganism associated with the initiation of the disease. In our studies, we have access to DNA samples extracted from human saliva and blood. In this report, we tested a real-time PCR assay developed to detect copies of genomic DNA from Streptococcus mutans in 1,424 DNA samples from humans. Our results suggest that we can determine the presence of genomic DNA copies of Streptococcus mutans in both DNA samples from caries-free and caries-affected individuals. However, we were not able to detect the presence of genomic DNA copies of Streptococcus mutans in any DNA samples extracted from peripheral blood, which suggests the assay may not be sensitive enough for this goal. Values of the threshold cycle of the real-time PCR reaction correlate with higher levels of caries experience in children, but this correlation could not be detected for adults. PMID:21731912

  10. Finding human promoter groups based on DNA physical properties

    Science.gov (United States)

    Zeng, Jia; Cao, Xiao-Qin; Zhao, Hongya; Yan, Hong

    2009-10-01

    DNA rigidity is an important physical property originating from the DNA three-dimensional structure. Although the general DNA rigidity patterns in human promoters have been investigated, their distinct roles in transcription are largely unknown. In this paper, we discover four highly distinct human promoter groups based on similarity of their rigidity profiles. First, we find that all promoter groups conserve relatively rigid DNAs at the canonical TATA box [a consensus TATA(A/T)A(A/T) sequence] position, which are important physical signals in binding transcription factors. Second, we find that the genes activated by each group of promoters share significant biological functions based on their gene ontology annotations. Finally, we find that these human promoter groups correlate with the tissue-specific gene expression.

  11. Finding human promoter groups based on DNA physical properties.

    Science.gov (United States)

    Zeng, Jia; Cao, Xiao-Qin; Zhao, Hongya; Yan, Hong

    2009-10-01

    DNA rigidity is an important physical property originating from the DNA three-dimensional structure. Although the general DNA rigidity patterns in human promoters have been investigated, their distinct roles in transcription are largely unknown. In this paper, we discover four highly distinct human promoter groups based on similarity of their rigidity profiles. First, we find that all promoter groups conserve relatively rigid DNAs at the canonical TATA box [a consensus TATA(A/T)A(A/T) sequence] position, which are important physical signals in binding transcription factors. Second, we find that the genes activated by each group of promoters share significant biological functions based on their gene ontology annotations. Finally, we find that these human promoter groups correlate with the tissue-specific gene expression.

  12. Quantification of low molecular weight selenium metabolites in human plasma after treatment with selenite in pharmacological doses by LC-ICP-MS

    DEFF Research Database (Denmark)

    Flouda, Konstantina; Dersch, Julie Maria; Gabel-Jensen, Charlotte

    2016-01-01

    The paper presents an analytical method for quantification of low molecular weight (LMW) selenium compounds in human plasma based on liquid chromatography inductively coupled plasma mass spectrometry (LC-ICP-MS) and post column isotope dilution-based quantification. Prior to analysis, samples were...

  13. Extracellular DNA affects NO content in human endothelial cells.

    Science.gov (United States)

    Efremova, L V; Alekseeva, A Yu; Konkova, M S; Kostyuk, S V; Ershova, E S; Smirnova, T D; Konorova, I L; Veiko, N N

    2010-08-01

    Fragments of extracellular DNA are permanently released into the blood flow due to cell apoptosis and possible de novo DNA synthesis. To find out whether extracellular DNA can affect the synthesis of nitric oxide (NO), one of key vascular tone regulators, we studied in vitro effects of three artificial DNA probes with different sequences and 10 samples of extracellular DNA (obtained from healthy people and patients with hypertension and atherosclerosis) on NO synthesis in endothelial cell culture (HUVEC). For detection of NO in live cells and culture medium, we used a NO-specific agent CuFL penetrating into the cells and forming a fluorescent product FL-NO upon interaction with NO. Human genome DNA fragments affected the content of NO in endothelial cells; this effect depended on both the base sequence and concentration of DNA fragments. Addition of artificial DNA and extracellular DNA from healthy people into the cell culture in a low concentration (5 ng/ml) increased the detected NO concentration by 4-fold at most. Cytosine-guanine (CG)-rich fragment of the transcribed sequence of ribosomal repeat was the most powerful NO-inductor. The effect of DNA fragments on NO synthesis was comparable with that of low doses of oxidizing agents, H(2)O(2) and 17β-estradiol. Extracellular DNA samples obtained from patients with hypertension and atherosclerosis decreased NO content in cells and medium by 1.3-28 times compared to the control; the effect correlated with the content of CG-rich sequences.

  14. The mutation rate of the human mtDNA deletion mtDNA4977.

    Science.gov (United States)

    Shenkar, R; Navidi, W; Tavaré, S; Dang, M H; Chomyn, A; Attardi, G; Cortopassi, G; Arnheim, N

    1996-10-01

    The human mitochondrial mutation mtDNA4977 is a 4,977-bp deletion that originates between two 13-bp direct repeats. We grew 220 colonies of cells, each from a single human cell. For each colony, we counted the number of cells and amplified the DNA by PCR to test for the presence of a deletion. To estimate the mutation fate, we used a model that describes the relationship between the mutation rate and the probability that a colony of a given size will contain no mutants, taking into account such factors as possible mitochondrial turnover and mistyping due to PCR error. We estimate that the mutation rate for mtDNA4977 in cultured human cells is 5.95 x 10(-8) per mitochondrial genome replication. This method can be applied to specific chromosomal, as well as mitochondrial, mutations.

  15. The mutation rate of the human mtDNA deletion mtDNA{sup 4977}

    Energy Technology Data Exchange (ETDEWEB)

    Shenkar, R. [Univ. of Colorado Health Science Center, Denver, CO (United States); Navidi, W. [Colorado School of Mines, Golden, CO (United States); Tavare, S. [Univ. of California, Los Angeles, CA (United States)] [and others

    1996-10-01

    The human mitochondrial mutation mtDNA{sup 4977} is a 4,977-bp deletion that originates between two 13-bp direct repeats. We grew 220 colonies of cells, each from a single human cell. For each colony, we counted the number of cells and amplified the DNA by PCR to test for the presence of a deletion. To estimate the mutation rate, we used a model that describes the relationship between the mutation rate and the probability that a colony of a given size will contain no mutants, taking into account such factors as possible mitochondrial turnover and mistyping due to PCR error. We estimate that the mutation rate for mtDNA{sup 4977} in cultured human cells is 5.95 x 10{sup {minus}8} per mitochondrial genome replication. This method can be applied to specific chromosomal, as well as mitochondrial, mutations. 17 refs., 1 fig., 1 tab.

  16. The human DNA-activated protein kinase, DNA-PK: Substrate specificity

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, C.W.; Connelly, M.A.; Zhang, H.; Sipley, J.A. [Brookhaven National Lab., Upton, NY (United States). Biology Dept.; Lees-Miller, S.P.; Lintott, L.G. [Univ. of Calgary, Alberta (Canada). Dept. of Biological Sciences; Sakaguchi, Kazuyasu; Appella, E. [National Institutes of Health, Bethesda, MD (United States). Lab. of Cell Biology

    1994-11-05

    Although much has been learned about the structure and function of p53 and the probable sequence of subsequent events that lead to cell cycle arrest, little is known about how DNA damage is detected and the nature of the signal that is generated by DNA damage. Circumstantial evidence suggests that protein kinases may be involved. In vitro, human DNA-PK phosphorylates a variety of nuclear DNA-binding, regulatory proteins including the tumor suppressor protein p53, the single-stranded DNA binding protein RPA, the heat shock protein hsp90, the large tumor antigen (TAg) of simian virus 40, a variety of transcription factors including Fos, Jun, serum response factor (SRF), Myc, Sp1, Oct-1, TFIID, E2F, the estrogen receptor, and the large subunit of RNA polymerase II (reviewed in Anderson, 1993; Jackson et al., 1993). However, for most of these proteins, the sites that are phosphorylated by DNA-PK are not known. To determine if the sites that were phosphorylated in vitro also were phosphorylated in vivo and if DNA-PK recognized a preferred protein sequence, the authors identified the sites phosphorylated by DNA-PK in several substrates by direct protein sequence analysis. Each phosphorylated serine or threonine is followed immediately by glutamine in the polypeptide chain; at no other positions are the amino acid residues obviously constrained.

  17. The human DNA-activated protein kinase, DNA-PK: Substrate specificity

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, C.W.; Connelly, M.A.; Zhang, H.; Sipley, J.A. [Brookhaven National Lab., Upton, NY (United States). Biology Dept.; Lees-Miller, S.P.; Lintott, L.G. [Univ. of Calgary, Alberta (Canada). Dept. of Biological Sciences; Sakaguchi, Kazuyasu; Appella, E. [National Institutes of Health, Bethesda, MD (United States). Lab. of Cell Biology

    1994-11-05

    Although much has been learned about the structure and function of p53 and the probable sequence of subsequent events that lead to cell cycle arrest, little is known about how DNA damage is detected and the nature of the signal that is generated by DNA damage. Circumstantial evidence suggests that protein kinases may be involved. In vitro, human DNA-PK phosphorylates a variety of nuclear DNA-binding, regulatory proteins including the tumor suppressor protein p53, the single-stranded DNA binding protein RPA, the heat shock protein hsp90, the large tumor antigen (TAg) of simian virus 40, a variety of transcription factors including Fos, Jun, serum response factor (SRF), Myc, Sp1, Oct-1, TFIID, E2F, the estrogen receptor, and the large subunit of RNA polymerase II (reviewed in Anderson, 1993; Jackson et al., 1993). However, for most of these proteins, the sites that are phosphorylated by DNA-PK are not known. To determine if the sites that were phosphorylated in vitro also were phosphorylated in vivo and if DNA-PK recognized a preferred protein sequence, the authors identified the sites phosphorylated by DNA-PK in several substrates by direct protein sequence analysis. Each phosphorylated serine or threonine is followed immediately by glutamine in the polypeptide chain; at no other positions are the amino acid residues obviously constrained.

  18. Coal tar residues produce both DNA adducts and oxidative DNA damage in human mammary epithelial cells.

    Science.gov (United States)

    Leadon, S A; Sumerel, J; Minton, T A; Tischler, A

    1995-12-01

    In the present study we compare the metabolic activation of coal tar, as measured by the production of both DNA adducts and oxidative DNA damage, with that of a single carcinogen that is a constituent of this complex mixture in human mammary epithelial cells (HMEC). We find that a significant level of DNA adducts, detected by 32P-postlabeling, are formed in HMEC following exposure to coal tar residues. This treatment also results in the generation of high levels of oxidative DNA damage, as measured by the production of one type of oxidative base modification, thymine glycols. The amounts of both DNA adducts and thymine varied considerably between the various coal tar residues and did not correlate with either the total amount of polycyclic aromatic hydrocarbons (PAH) or the amount of benzo[a]pyrene (B[a]P) present in the residue. Fractionating the residue from one of the sites by sequential extraction with organic solvents indicated that while the ability to produce both types of DNA damage was contained mostly in a hexane-soluble fraction, a benzene-soluble fraction produced high levels of reactive oxygens relative to the number of total DNA adducts. We find that the total amount of PAH or B[a]P present in the coal tars from the various sites was not a predictor of the level of total DNA damage formed.

  19. Quantification of nanoparticle uptake into hair follicles in pig ear and human forearm.

    Science.gov (United States)

    Raber, A S; Mittal, A; Schäfer, J; Bakowsky, U; Reichrath, J; Vogt, T; Schaefer, U F; Hansen, S; Lehr, C-M

    2014-04-10

    Drug delivery via the hair follicle (HF) especially with nanoparticles (NP) recently gained attention due to a depot effect and facilitated absorption conditions within the lower HF. With the prospect of transdermal drug delivery, it is of interest to optimize the follicular uptake of NP. In this study, a method was developed to quantify NP uptake into HF and applied in vitro in a pig ear model and in vivo in human volunteers. The influence of NP material on HF uptake was investigated using fluorescence-labeled NP based on poly(D,L-lactide-co-glycolide) (PLGA). All NP had similar hydrodynamic sizes (163-170 nm) but different surface modifications: (i) plain PLGA, (ii) chitosan-coated PLGA (Chit.-PLGA), and (iii) Chit.-PLGA coated with different phospholipids (PL) (DPPC (100), DPPC:Chol (85:15), and DPPC:DOTAP (92:8). Differential stripping was performed, including complete mass balance. The samples were extracted for fluorescence quantification. An effect of the PL coating on follicular uptake was observed as DPPC (100) and DPPC:DOTAP (92:8) penetrated into HF to a higher extent than the other tested NP. The effect was observed both in the pig ear model as well as in human volunteers, although it was statistically significant only in the in vitro model. An excellent in vitro-in vivo correlation (IVIVC, r(2)=0.987) between both models was demonstrated, further supporting the suitability of the pig ear model as a surrogate for the in vivo situation in humans for quantifying NP uptake into HF. These findings may help to optimize NP for targeting the HF and to improve transdermal delivery.

  20. Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits.

    Science.gov (United States)

    Demeke, Tigst; Jenkins, G Ronald

    2010-03-01

    Biotechnology-derived varieties of canola, cotton, corn and soybean are being grown in the USA, Canada and other predominantly grain exporting countries. Although the amount of farmland devoted to production of biotechnology-derived crops continues to increase, lingering concerns that unintended consequences may occur provide the EU and most grain-importing countries with justification to regulate these crops. Legislation in the EU requires traceability of grains/oilseeds, food and feed products, and labelling, when a threshold level of 0.9% w/w of genetically engineered trait is demonstrated to be present in an analytical sample. The GE content is routinely determined by quantitative PCR (qPCR) and plant genomic DNA provides the template for the initial steps in this process. A plethora of DNA extraction methods exist for qPCR applications. Implementing standardized methods for detection of genetically engineered traits is necessary to facilitate grain marketing. The International Organization for Standardization draft standard 21571 identifies detergent-based methods and commercially available kits that are widely used for DNA extraction, but also indicates that adaptations may be necessary depending upon the sample matrix. This review assesses advantages and disadvantages of various commercially available DNA extraction kits, as well as modifications to published cetyltrimethylammonium bromide methods. Inhibitors are a major obstacle for efficient amplification in qPCR. The types of PCR inhibitors and techniques to minimize inhibition are discussed. Finally, accurate quantification of DNA for applications in qPCR is not trivial. Many confounders contribute to differences in analytical measurements when a particular DNA quantification method is applied and different methods do not always provide concordant results on the same DNA sample. How these differences impact measurement uncertainty in qPCR is considered.

  1. Prospects for DNA methods to measure human heritable mutation rates

    Energy Technology Data Exchange (ETDEWEB)

    Mendelsohn, M.L.

    1985-06-14

    A workshop cosponsored by ICPEMC and the US Department of Energy was held in Alta, Utah, December 9-13, 1984 to examine the extent to which DNA-oriented methods might provide new approaches to the important but intractable problem of measuring mutation rates in control and exposed human populations. The workshop identified and analyzed six DNA methods for detection of human heritable mutation, including several created at the meeting, and concluded that none of the methods combine sufficient feasibility and efficiency to be recommended for general application. 8 refs.

  2. Distribution patterns of postmortem damage in human mitochondrial DNA

    DEFF Research Database (Denmark)

    Gilbert, M Thomas P; Willerslev, Eske; Hansen, Anders J

    2002-01-01

    The distribution of postmortem damage in mitochondrial DNA retrieved from 37 ancient human DNA samples was analyzed by cloning and was compared with a selection of published animal data. A relative rate of damage (rho(v)) was calculated for nucleotide positions within the human hypervariable region......, such as MT5, have lower in vivo mutation rates and lower postmortem-damage rates. The postmortem data also identify a possible functional subregion of the HVR1, termed "low-diversity 1," through the lack of sequence damage. The amount of postmortem damage observed in mitochondrial coding regions...

  3. Recombinational DNA repair and human disease

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, Larry H.; Schild, David

    2002-11-30

    We review the genes and proteins related to the homologous recombinational repair (HRR) pathway that are implicated in cancer through either genetic disorders that predispose to cancer through chromosome instability or the occurrence of somatic mutations that contribute to carcinogenesis. Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and an ataxia-like disorder (ATLD), are chromosome instability disorders that are defective in the ataxia telangiectasia mutated (ATM), NBS, and Mre11 genes, respectively. These genes are critical in maintaining cellular resistance to ionizing radiation (IR), which kills largely by the production of double-strand breaks (DSBs). Bloom syndrome involves a defect in the BLM helicase, which seems to play a role in restarting DNA replication forks that are blocked at lesions, thereby promoting chromosome stability. The Werner syndrome gene (WRN) helicase, another member of the RecQ family like BLM, has very recently been found to help mediate homologous recombination. Fanconi anemia (FA) is a genetically complex chromosomal instability disorder involving seven or more genes, one of which is BRCA2. FA may be at least partially caused by the aberrant production of reactive oxidative species. The breast cancer-associated BRCA1 and BRCA2 proteins are strongly implicated in HRR; BRCA2 associates with Rad51 and appears to regulate its activity. We discuss in detail the phenotypes of the various mutant cell lines and the signaling pathways mediated by the ATM kinase. ATM's phosphorylation targets can be grouped into oxidative stress-mediated transcriptional changes, cell cycle checkpoints, and recombinational repair. We present the DNA damage response pathways by using the DSB as the prototype lesion, whose incorrect repair can initiate and augment karyotypic abnormalities.

  4. Inaccurate DNA synthesis in cell extracts of yeast producing active human DNA polymerase iota.

    Directory of Open Access Journals (Sweden)

    Alena V Makarova

    Full Text Available Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn(2+ ions, can bypass some DNA lesions and misincorporates "G" opposite template "T" more frequently than incorporates the correct "A." We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of "G" versus "A" method of Gening, abbreviated as "misGvA". We provide unambiguous proof of the "misGvA" approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The "misGvA" activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts.

  5. A sandwich ELISA for the conformation-specific quantification of the activated form of human Bax.

    Science.gov (United States)

    Teijido, Oscar; Ganesan, Yogesh Tengarai; Llanos, Raul; Peton, Ashley; Urtecho, Jean-Baptiste; Soprani, Adauri; Villamayor, Aimee; Antonsson, Bruno; Manon, Stéphen; Dejean, Laurent

    2016-03-15

    Bcl-2 family proteins are critical regulators of mitochondrial outer membrane permeabilization (MOMP), which represents the point of no return of apoptotic cell death. The exposure of the Bax N-terminus at the mitochondria reflects Bax activation; and this activated configuration of the Bax protein is associated with MOMP. N-terminal exposure can be detected using specific monoclonal and/or polyclonal antibodies, and the onset of activated Bax has extensively been used as an early marker of apoptosis. The protocols of immunoprecipitation and/or immunocytochemistry commonly used to detect activated Bax are long and tedious, and allow semiquantification of the antigen at best. The sandwich ELISA protocol we developed has a 5 ng/mL detection limit and is highly specific for the activated conformation of Bax. This ELISA allows a rapid quantification of activated human Bax in whole cells and isolated mitochondria protein extracts. These properties grant this assay the potential to further clarify the prognostic and diagnostic value of activated Bax in disorders associated with deregulated apoptotic pathways such as degenerative diseases or cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Sensitive quantification of omeprazole and its metabolites in human plasma by liquid chromatography-mass spectrometry.

    Science.gov (United States)

    Hofmann, Ute; Schwab, Matthias; Treiber, Gerd; Klotz, Ulrich

    2006-02-02

    A sensitive method was developed for the simultaneous determination of omeprazole and its major metabolites 5-hydroxyomeprazole and omeprazole sulfone in human plasma by HPLC-electrospray mass spectrometry. Following liquid-liquid extraction HPLC separation was achieved on a ProntoSil AQ, C18 column using a gradient with 10 mM ammonium acetate in water (pH 7.25) and acetonitrile. The mass spectrometer was operated in the selected ion monitoring mode using the respective MH(+) ions, m/z 346 for omeprazole, m/z 362 for 5-hydroxy-omeprazole and omeprazol-sulfone and m/z 300 for the internal standard (2-{[(3,5-dimethylpyridine-2-yl)methyl]thio}-1H-benzimidazole-5-yl)methanol. The limit of quantification (LOQ) achieved with this method was 5 ng/ml for 5-hydroxyomeprazole and 10 ng/ml for omeprazole and omeprazole-sulfone using 0.25 ml of plasma. Intra- and inter-assay variability was below 11% over the whole concentration range from 5 to 250 ng/ml for 5-hydroxyomeprazol and from 10 to 750 ng/ml for omeprazole and omeprazole-sulfone. The method was successfully applied to the determination of pharmacokinetic parameters of esomeprazole and the two major metabolites after a single dose and under steady state conditions.

  7. Mechanism of Ribonucleotide Incorporation by Human DNA Polymerase η.

    Science.gov (United States)

    Su, Yan; Egli, Martin; Guengerich, F Peter

    2016-02-19

    Ribonucleotides and 2'-deoxyribonucleotides are the basic units for RNA and DNA, respectively, and the only difference is the extra 2'-OH group on the ribonucleotide sugar. Cellular rNTP concentrations are much higher than those of dNTP. When copying DNA, DNA polymerases not only select the base of the incoming dNTP to form a Watson-Crick pair with the template base but also distinguish the sugar moiety. Some DNA polymerases use a steric gate residue to prevent rNTP incorporation by creating a clash with the 2'-OH group. Y-family human DNA polymerase η (hpol η) is of interest because of its spacious active site (especially in the major groove) and tolerance of DNA lesions. Here, we show that hpol η maintains base selectivity when incorporating rNTPs opposite undamaged DNA and the DNA lesions 7,8-dihydro-8-oxo-2'-deoxyguanosine and cyclobutane pyrimidine dimer but with rates that are 10(3)-fold lower than for inserting the corresponding dNTPs. X-ray crystal structures show that the hpol η scaffolds the incoming rNTP to pair with the template base (dG) or 7,8-dihydro-8-oxo-2'-deoxyguanosine with a significant propeller twist. As a result, the 2'-OH group avoids a clash with the steric gate, Phe-18, but the distance between primer end and Pα of the incoming rNTP increases by 1 Å, elevating the energy barrier and slowing polymerization compared with dNTP. In addition, Tyr-92 was identified as a second line of defense to maintain the position of Phe-18. This is the first crystal structure of a DNA polymerase with an incoming rNTP opposite a DNA lesion.

  8. Retrieval of human DNA from rodent-human genomic libraries by a recombination process.

    Science.gov (United States)

    Neve, R L; Bruns, G A; Dryja, T P; Kurnit, D M

    1983-09-01

    Human Alu repeat ("BLUR") sequences have been cloned into the mini-plasmid vector piVX. The resulting piBLUR clones have been used to rescue selectively, by recombination, bacteriophage carrying human DNA sequences from genomic libraries constructed using DNA from rodent-human somatic cell hybrids. piBLUR clones are able to retrieve human clones from such libraries because at least one Alu family repeat is present on most 15 to 20 kb fragments of human DNA and because of the relative species-specificity of the sequences comprising the Alu family. The rapid, selective plaque purification achieved results in the construction of a collection of recombinant phage carrying diverse human DNA inserts from a specific subset of the human karyotype. Subfragments of two recombinants rescued from a mouse-human somatic cell hybrid containing human chromosomes X, 10, 13, and 22 were mapped to human chromosomes X and 13, respectively, demonstrating the utility of this protocol for the isolation of human chromosome-specific DNA sequences from appropriate somatic cell hybrids.

  9. PET quantification of the norepinephrine transporter in human brain with (S,S)-18F-FMeNER-D2.

    Science.gov (United States)

    Moriguchi, Sho; Kimura, Yasuyuki; Ichise, Masanori; Arakawa, Ryosuke; Takano, Harumasa; Seki, Chie; Ikoma, Yoko; Takahata, Keisuke; Nagashima, Tomohisa; Yamada, Makiko; Mimura, Masaru; Suhara, Tetsuya

    2016-12-15

    Norepinephrine transporter (NET) in the brain plays important roles in human cognition and the pathophysiology of psychiatric disorders. Two radioligands, (S,S)-(11)C-MRB and (S,S)-(18)F-FMeNER-D2, have been used for imaging NETs in the thalamus and midbrain (including locus coeruleus) using positron emission topography (PET) in humans. However, NET density in the equally important cerebral cortex has not been well quantified because of unfavorable kinetics with (S,S)-(11)C-MRB and defluorination with (S,S)-(18)F-FMeNER-D2, which can complicate NET quantification in the cerebral cortex adjacent to the skull containing defluorinated (18)F radioactivity. In this study, we have established analysis methods of quantification of NET density in the brain including cerebral cortex using (S,S)-(18)F-FMeNER-D2 PET.

  10. Assessment of Human DNA Repair (NER) Capacity With DNA Repair Rate (DRR) by Comet Assay

    Institute of Scientific and Technical Information of China (English)

    WEI ZHENG; JI-LIANG HE; LI-FEN JIN; JIAN-LIN LOU; BAO-HONG WANG

    2005-01-01

    Objective Alkaline comet assay was used to evaluate DNA repair (nucleotide excision repair, NER) capacity of human fresh lymphocytes from 12 young healthy non-smokers (6 males and 6 females). Methods Lymphocytes were exposed to UV-C (254 nm) at the dose rate of 1.5 J/m2/sec. Novobiocin (NOV) and aphidicolin (APC), DNA repair inhibitors, were utilized to imitate the deficiency of DNA repair capacity at the incision and ligation steps of NER. Lymphocytes from each donor were divided into three grougs: UVC group, UVC plus NOV group, and UVC plus APC group. DNA single strand breaks were detected in UVC irradiated cells incubated for 0, 30, 60, 90, 120, 180, and 240 min after UVC irradiation. DNA repair rate (DRR) served as an indicator of DNA repair capacity. Results The results indicated that the maximum DNA damage (i.e. maximum tail length) in the UVC group mainly appeared at 90 min. The ranges of DRRs in the UVC group were 62.84%-98.71%. Average DRR value was 81.84%. The DRR difference between males and females was not significant (P<0.05). However, the average DRR value in the UVC plus NOV group and the UVC plus APC group was 52.98% and 39.57% respectively, which were significantly lower than that in the UVC group (P<0.01). Conclusion The comet assay is a rapid, simple and sensitive screening test to assess individual DNA repair (NER) capacity. It is suggested that the time to detect DNA single strand breaks in comet assay should include 0 (before UV irradiation), 90 and 240 min after exposure to 1.5 J·m-2 UVC at least. The DRR, as an indicator, can represent the individual DNA repair capacity in comet assay.

  11. The DNA methylome of human peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Li, Yingrui; Zhu, Jingde; Tian, Geng

    2010-01-01

    DNA methylation plays an important role in biological processes in human health and disease. Recent technological advances allow unbiased whole-genome DNA methylation (methylome) analysis to be carried out on human cells. Using whole-genome bisulfite sequencing at 24.7-fold coverage (12.3-fold per...... strand), we report a comprehensive (92.62%) methylome and analysis of the unique sequences in human peripheral blood mononuclear cells (PBMC) from the same Asian individual whose genome was deciphered in the YH project. PBMC constitute an important source for clinical blood tests world-wide. We found...... that 68.4% of CpG sites and 80% displayed allele-specific expression (ASE). These data demonstrate that ASM is a recurrent phenomenon and is highly correlated with ASE in human PBMCs. Together with recently reported similar studies, our study provides a comprehensive resource for future epigenomic...

  12. Recovery of human DNA profiles from poached deer remains: a feasibility study.

    Science.gov (United States)

    Tobe, Shanan S; Govan, James; Welch, Lindsey A

    2011-12-01

    Poaching is a crime that occurs worldwide and can be extremely difficult to investigate and prosecute due to the nature of the evidence available. If a species is protected by international legislation such as the Convention on International Trade in Endangered Species of Wild Fauna and Flora then simply possessing any part of that species is illegal. Previous studies have focused on the identification of endangered species in cases of potential poaching. Difficulties arise if the poached animal is not endangered. Species such as deer have hunting seasons whereby they can legally be hunted however poaching is the illegal take of deer, irrespective of season. Therefore, identification of deer alone has little probative value as samples could have originated from legal hunting activities in season. After a deer is hunted it is usual to remove the innards, head and lower limbs. The limbs are removed through manual force and represent a potential source of human touch DNA. We investigate the potential to recover and profile human autosomal DNA from poached deer remains. Samples from the legs of ten culled deer were obtained (40 in total) using minitapes. DNA from samples was extracted, quantified and amplified to determine if it would be possible to recover human STR profiles. Low quantification data led to the use of an extended PCR cycling protocol of 34 cycles. Samples from seven deer amplified, however some samples were excluded from further analysis due to 'drop in' alleles or the low level of successfully amplified loci. Samples from five deer could be further analysed and gave match probabilities ranging from 6.37×10(-3) to 9.53×10(-11). This study demonstrates the potential of recovering human touch DNA from poached animal remains. There is the potential for this test to be used in relation to other species of poached remains or other types of wildlife crimes. This is the first time, to our knowledge, that human STR profiling has been successfully applied to

  13. Genome-Wide Prediction of DNA Methylation Using DNA Composition and Sequence Complexity in Human

    Science.gov (United States)

    Wu, Chengchao; Yao, Shixin; Li, Xinghao; Chen, Chujia; Hu, Xuehai

    2017-01-01

    DNA methylation plays a significant role in transcriptional regulation by repressing activity. Change of the DNA methylation level is an important factor affecting the expression of target genes and downstream phenotypes. Because current experimental technologies can only assay a small proportion of CpG sites in the human genome, it is urgent to develop reliable computational models for predicting genome-wide DNA methylation. Here, we proposed a novel algorithm that accurately extracted sequence complexity features (seven features) and developed a support-vector-machine-based prediction model with integration of the reported DNA composition features (trinucleotide frequency and GC content, 65 features) by utilizing the methylation profiles of embryonic stem cells in human. The prediction results from 22 human chromosomes with size-varied windows showed that the 600-bp window achieved the best average accuracy of 94.7%. Moreover, comparisons with two existing methods further showed the superiority of our model, and cross-species predictions on mouse data also demonstrated that our model has certain generalization ability. Finally, a statistical test of the experimental data and the predicted data on functional regions annotated by ChromHMM found that six out of 10 regions were consistent, which implies reliable prediction of unassayed CpG sites. Accordingly, we believe that our novel model will be useful and reliable in predicting DNA methylation. PMID:28212312

  14. Protein-associated intercalator-induced DNA scission is enhanced by estrogen stimulation in human breast cancer cells.

    Science.gov (United States)

    Zwelling, L A; Kerrigan, D; Lippman, M E

    1983-01-01

    Estrogen-responsive human breast cancer cells (MCF-7) displayed a higher frequency of intercalator-induced protein-associated DNA scission after treatment with 17 beta-estradiol (E2) than did cells that had not received estrogen treatment. This effect was dependent on estrogen concentration (maximum enhancement at approximately equal to 1 nM E2) and time (maximum effect seen approximately equal to 24 hr after E2 addition). Human breast cancer cells lacking estrogen receptors did not display the enhanced response. Antiestrogens produced a slight decrease in intercalator-induced DNA scission, whereas insulin produced an enhanced effect. The DNA breaks produced by the intercalators 5-iminodaunorubicin and 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) in these cells were undetectable without enzymatic deproteinization of cell lysates prior to quantification by alkaline elution. Intercalator-induced DNA-protein crosslinking also was enhanced in E2-treated MCF-7 cells. Studies with m-[14C]AMSA revealed no estrogen-associated increases in drug uptake. The data suggest that E2 treatment, either by specifically and directly increasing active transcription in chromatin or through secondary effects on DNA that accompany alterations in cell growth or cell cycle distribution, alters the susceptibility of DNA to intercalator-induced protein-associated DNA scission. If this enhanced protein-associated scission is selectively localized to transcriptionally active chromatin, the adsorption of the DNA-bound proteins to membrane filters (DNA-protein crosslinking) may allow identification and isolation of estrogen-regulated gene sequences. PMID:6353411

  15. Involvement of DNA ligase III and ribonuclease H1 in mitochondrial DNA replication in cultured human cells.

    Science.gov (United States)

    Ruhanen, Heini; Ushakov, Kathy; Yasukawa, Takehiro

    2011-12-01

    Recent evidence suggests that coupled leading and lagging strand DNA synthesis operates in mammalian mitochondrial DNA (mtDNA) replication, but the factors involved in lagging strand synthesis are largely uncharacterised. We investigated the effect of knockdown of the candidate proteins in cultured human cells under conditions where mtDNA appears to replicate chiefly via coupled leading and lagging strand DNA synthesis to restore the copy number of mtDNA to normal levels after transient mtDNA depletion. DNA ligase III knockdown attenuated the recovery of mtDNA copy number and appeared to cause single strand nicks in replicating mtDNA molecules, suggesting the involvement of DNA ligase III in Okazaki fragment ligation in human mitochondria. Knockdown of ribonuclease (RNase) H1 completely prevented the mtDNA copy number restoration, and replication intermediates with increased single strand nicks were readily observed. On the other hand, knockdown of neither flap endonuclease 1 (FEN1) nor DNA2 affected mtDNA replication. These findings imply that RNase H1 is indispensable for the progression of mtDNA synthesis through removing RNA primers from Okazaki fragments. In the nucleus, Okazaki fragments are ligated by DNA ligase I, and the RNase H2 is involved in Okazaki fragment processing. This study thus proposes that the mitochondrial replication system utilises distinct proteins, DNA ligase III and RNase H1, for Okazaki fragment maturation.

  16. Sulforaphane induces DNA single strand breaks in cultured human cells

    Energy Technology Data Exchange (ETDEWEB)

    Sestili, Piero, E-mail: piero.sestili@uniurb.it [Dipartimento di Scienze Biomolecolari, Via Maggetti, 21, Universita degli Studi di Urbino ' Carlo Bo' , 61029 Urbino, PU (Italy); Paolillo, Marco [Dipartimento di Scienze Biomolecolari, Via Maggetti, 21, Universita degli Studi di Urbino ' Carlo Bo' , 61029 Urbino, PU (Italy); Lenzi, Monia [Dipartimento di Farmacologia, Universita degli Studi di Bologna, Via Irnerio 48, 40126 Bologna (Italy); Colombo, Evelin; Vallorani, Luciana; Casadei, Lucia; Martinelli, Chiara [Dipartimento di Scienze Biomolecolari, Via Maggetti, 21, Universita degli Studi di Urbino ' Carlo Bo' , 61029 Urbino, PU (Italy); Fimognari, Carmela [Dipartimento di Farmacologia, Universita degli Studi di Bologna, Via Irnerio 48, 40126 Bologna (Italy)

    2010-07-07

    Sulforaphane (SFR), an isothiocyanate from cruciferous vegetables, possesses growth-inhibiting and apoptosis-inducing activities in cancer cell lines. Recently, SFR has been shown to promote the mitochondrial formation of reactive oxygen species (ROS) in human cancer cell lines. The present study was undertaken to see whether SFR-derived ROS might cause DNA damage in cultured human cells, namely T limphoblastoid Jurkat and human umbilical vein endothelial cells (HUVEC). 1-3 h treatments with 10-30 {mu}M SFR elicited intracellular ROS formation (as assayed with dihydrorhodamine, DHR, oxidation) as well as DNA breakage (as assessed with fast halo assay, FHA). These effects lacked cell-type specificity, since could be observed in both Jurkat and HUVEC. Differential-pH FHA analysis of damaged DNA showed that SFR causes frank DNA single strand breaks (SSBs); no DNA double strand breaks (DSBs) were found within the considered treatment times (up to 3 h). SFR-derived ROS were formed at the mitochondrial respiratory chain (MRC) level: indeed rotenone or myxothiazol (MRC Complex I and III inhibitors, respectively) abrogated ROS formation. Furthermore ROS were not formed in Jurkat cells pharmacologically depleted of respiring mitochondria (MRC-/Jurkat). Formation of ROS was causally linked to the induction of SSBs: indeed all the experimental conditions capable of preventing ROS formation also prevented the damage of nuclear DNA from SFR-intoxicated cells. As to the toxicological relevance of SSBs, we found that their prevention slightly but significantly attenuated SFR cytotoxicity, suggesting that high-dose SFR toxicity is the result of a complex series of events among which GSH depletion seems to play a pivotal role. In conclusion, the present study identifies a novel mechanism contributing to SFR toxicity which - since DNA damage is a prominent mechanism underlying the cytotoxic activity of established antineoplastic agents - might help to exploit the therapeutic value

  17. Destabilization of the human epigenome: consequences of foreign DNA insertions.

    Science.gov (United States)

    Weber, Stefanie; Hofmann, Andrea; Herms, Stefan; Hoffmann, Per; Doerfler, Walter

    2015-08-01

    We previously reported changes of DNA methylation and transcription patterns in mammalian cells that carry integrated foreign DNA. Experiments were now designed to assess the epigenetic consequences of inserting a 5.6 kbp plasmid into the human genome. Differential transcription and CpG methylation patterns were compared between transgenomic and nontransgenomic cell clones by using gene chip microarray systems. In 4.7% of the 28.869 gene segments analyzed, transcriptional activities were up- or downregulated in the transgenomic cell clones. Genome-wide profiling revealed differential methylation in 3791 of > 480,000 CpG's examined in transgenomic versus nontransgenomic clones. The data document genome-wide effects of foreign DNA insertions on the epigenetic stability of human cells. Many fields in experimental biology and medicine employ transgenomic or otherwise genome-manipulated cells or organisms without considering the epigenetic consequences for the recipient genomes.

  18. The DNA-damage response in human biology and disease

    DEFF Research Database (Denmark)

    Jackson, Stephen P; Bartek, Jiri

    2009-01-01

    , signal its presence and mediate its repair. Such responses, which have an impact on a wide range of cellular events, are biologically significant because they prevent diverse human diseases. Our improving understanding of DNA-damage responses is providing new avenues for disease management....

  19. False-positive Human Papillomavirus DNA tests in cervical screening

    DEFF Research Database (Denmark)

    Rebolj, Matejka; Pribac, Igor; Lynge, Elsebeth

    2011-01-01

    Based on data from randomised controlled trials (RCT) on primary cervical screening, it has been reported that the problem of more frequent false-positive tests in Human Papillomavirus (HPV) DNA screening compared to cytology could be overcome. However, these reports predominantly operated...

  20. Ancient DNA in human bone remains from Pompeii archaeological site.

    Science.gov (United States)

    Cipollaro, M; Di Bernardo, G; Galano, G; Galderisi, U; Guarino, F; Angelini, F; Cascino, A

    1998-06-29

    aDNA extraction and amplification procedures have been optimized for Pompeian human bone remains whose diagenesis has been determined by histological analysis. Single copy genes amplification (X and Y amelogenin loci and Y specific alphoid repeat sequences) have been performed and compared with anthropometric data on sexing.

  1. Translesion synthesis past acrolein-derived DNA adducts by human mitochondrial DNA polymerase γ.

    Science.gov (United States)

    Kasiviswanathan, Rajesh; Minko, Irina G; Lloyd, R Stephen; Copeland, William C

    2013-05-17

    Acrolein, a mutagenic aldehyde, is produced endogenously by lipid peroxidation and exogenously by combustion of organic materials, including tobacco products. Acrolein reacts with DNA bases forming exocyclic DNA adducts, such as γ-hydroxy-1,N(2)-propano-2'-deoxyguanosine (γ-HOPdG) and γ-hydroxy-1,N(6)-propano-2'-deoxyadenosine (γ-HOPdA). The bulky γ-HOPdG adduct blocks DNA synthesis by replicative polymerases but can be bypassed by translesion synthesis polymerases in the nucleus. Although acrolein-induced adducts are likely to be formed and persist in mitochondrial DNA, animal cell mitochondria lack specialized translesion DNA synthesis polymerases to tolerate these lesions. Thus, it is important to understand how pol γ, the sole mitochondrial DNA polymerase in human cells, acts on acrolein-adducted DNA. To address this question, we investigated the ability of pol γ to bypass the minor groove γ-HOPdG and major groove γ-HOPdA adducts using single nucleotide incorporation and primer extension analyses. The efficiency of pol γ-catalyzed bypass of γ-HOPdG was low, and surprisingly, pol γ preferred to incorporate purine nucleotides opposite the adduct. Pol γ also exhibited ∼2-fold lower rates of excision of the misincorporated purine nucleotides opposite γ-HOPdG compared with the corresponding nucleotides opposite dG. Extension of primers from the termini opposite γ-HOPdG was accomplished only following error-prone purine nucleotide incorporation. However, pol γ preferentially incorporated dT opposite the γ-HOPdA adduct and efficiently extended primers from the correctly paired terminus, indicating that γ-HOPdA is probably nonmutagenic. In summary, our data suggest that acrolein-induced exocyclic DNA lesions can be bypassed by mitochondrial DNA polymerase but, in the case of the minor groove γ-HOPdG adduct, at the cost of unprecedented high mutation rates.

  2. Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR.

    Science.gov (United States)

    Tatti, Enrico; McKew, Boyd A; Whitby, Corrine; Smith, Cindy J

    2016-06-11

    Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with 'end-point' PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

  3. Traumatic stress and human DNA methylation: a critical review.

    Science.gov (United States)

    Vinkers, Christiaan H; Kalafateli, Aimilia Lydia; Rutten, Bart P F; Kas, Martien J; Kaminsky, Zachary; Turner, Jonathan D; Boks, Marco P M

    2015-01-01

    Animal studies have identified persistent and functional effects of traumatic stress on the epigenome. This review discusses the clinical evidence for trauma-induced changes in DNA methylation across the life span in humans. Studies are reviewed based on reports of trauma exposure during the prenatal period (13 studies), early life (20 studies), and adulthood (ten studies). Even though it is apparent that traumatic stress influences the human epigenome, there are significant drawbacks in the existing human literature. These include a lack of longitudinal studies, methodological heterogeneity, selection of tissue type, and the influence of developmental stage and trauma type on methylation outcomes. These issues are discussed in order to present a way in which future studies can gain more insight into the functional relevance of trauma-related DNA methylation changes. Epigenetic studies investigating the detrimental effects of traumatic stress have great potential for an improved detection and treatment of trauma-related psychiatric disorders.

  4. DNA integrity of human leukocytes after magnetic resonance imaging.

    Science.gov (United States)

    Szerencsi, Ágnes; Kubinyi, Györgyi; Váliczkó, Éva; Juhász, Péter; Rudas, Gábor; Mester, Ádám; Jánossy, Gábor; Bakos, József; Thuróczy, György

    2013-10-01

    This study focuses on the effects of high-field (3T) magnetic resonance imaging (MRI) scans on the DNA integrity of human leukocytes in vitro in order to validate the study where genotoxic effects were obtained and published by Lee et al. The scanning protocol and exposure situation were the same as those used under routine clinical brain MRI scan. Peripheral blood samples from healthy non-smoking male donors were exposed to electromagnetic fields (EMF) produced by 3T magnetic resonance imaging equipment for 0, 22, 45, 67, and 89 min during the scanning procedure. Samples of positive control were exposed to ionizing radiation (4 Gy of (60)Co-γ). Single breaks of DNA in leukocytes were detected by single-cell gel electrophoresis (Comet assay). Chromosome breakage, chromosome loss and micronuclei formations were detected by a micronucleus test (MN). Three independent experiments were performed. The data of comet tail DNA%, olive tail moment and micronucleus frequency showed no DNA damages due to MRI exposure. The results of the Comet assay and the micronucleus test indicate that the applied exposure of MRI does not appear to produce breaks in the DNA and has no significant effect on DNA integrity.

  5. Collective behavior quantification on human odor effects against female Aedes aegypti mosquitoes-Open source development.

    Science.gov (United States)

    Poh, Abdul Halim; Moghavvemi, Mahmoud; Leong, Cherng Shii; Lau, Yee Ling; Safdari Ghandari, Alireza; Apau, Alexlee; Mahamd Adikan, Faisal Rafiq

    2017-01-01

    Classifying and quantifying mosquito activity includes a plethora of categories, ranging from measuring flight speeds, repellency, feeding rates, and specific behaviors such as home entry, swooping and resting, among others. Entomologists have been progressing more toward using machine vision for efficiency for this endeavor. Digital methods have been used to study the behavior of insects in labs, for instance via three-dimensional tracking with specialized cameras to observe the reaction of mosquitoes towards human odor, heat and CO2, although virtually none was reported for several important fields, such as repellency studies which have a significant need for a proper response quantification. However, tracking mosquitoes individually is a challenge and only limited number of specimens can be studied. Although tracking large numbers of individual insects is hailed as one of the characteristics of an ideal automated image-based tracking system especially in 3D, it also is a costly method, often requiring specialized hardware and limited access to the algorithms used for mapping the specimens. The method proposed contributes towards (a) unlimited open source use, (b) a low-cost setup, (c) complete guide for any entomologist to adapt in terms of hardware and software, (d) simple to use, and (e) a lightweight data output for collective behavior analysis of mosquitoes. The setup is demonstrated by testing a simple response of mosquitoes in the presence of human odor versus control, one session with continuous human presence as a stimuli and the other with periodic presence. A group of female Aedes aegypti (Linnaeus) mosquitoes are released into a white-background chamber with a transparent acrylic panel on one side. The video feed of the mosquitoes are processed using filtered contours in a threshold-adjustable video. The mosquitoes in the chamber are mapped on the raster where the coordinates of each mosquito are recorded with the corresponding timestamp. The average

  6. Collective behavior quantification on human odor effects against female Aedes aegypti mosquitoes—Open source development

    Science.gov (United States)

    Leong, Cherng Shii; Lau, Yee Ling; Safdari Ghandari, Alireza; Apau, Alexlee; Mahamd Adikan, Faisal Rafiq

    2017-01-01

    Classifying and quantifying mosquito activity includes a plethora of categories, ranging from measuring flight speeds, repellency, feeding rates, and specific behaviors such as home entry, swooping and resting, among others. Entomologists have been progressing more toward using machine vision for efficiency for this endeavor. Digital methods have been used to study the behavior of insects in labs, for instance via three-dimensional tracking with specialized cameras to observe the reaction of mosquitoes towards human odor, heat and CO2, although virtually none was reported for several important fields, such as repellency studies which have a significant need for a proper response quantification. However, tracking mosquitoes individually is a challenge and only limited number of specimens can be studied. Although tracking large numbers of individual insects is hailed as one of the characteristics of an ideal automated image-based tracking system especially in 3D, it also is a costly method, often requiring specialized hardware and limited access to the algorithms used for mapping the specimens. The method proposed contributes towards (a) unlimited open source use, (b) a low-cost setup, (c) complete guide for any entomologist to adapt in terms of hardware and software, (d) simple to use, and (e) a lightweight data output for collective behavior analysis of mosquitoes. The setup is demonstrated by testing a simple response of mosquitoes in the presence of human odor versus control, one session with continuous human presence as a stimuli and the other with periodic presence. A group of female Aedes aegypti (Linnaeus) mosquitoes are released into a white-background chamber with a transparent acrylic panel on one side. The video feed of the mosquitoes are processed using filtered contours in a threshold-adjustable video. The mosquitoes in the chamber are mapped on the raster where the coordinates of each mosquito are recorded with the corresponding timestamp. The average

  7. Design and performance testing of a DNA extraction assay for sensitive and reliable quantification of acetic acid bacteria directly in red wine using real time PCR

    Directory of Open Access Journals (Sweden)

    Cédric eLONGIN

    2016-06-01

    Full Text Available Although strategies exist to prevent AAB contamination, the increased interest for wines with low sulfite addition leads to greater AAB spoilage. Hence there is a real need for a rapid, specific, sensitive and reliable method for detecting these spoilage bacteria. All these requirements are met by real time Polymerase Chain Reaction (or quantitative PCR; qPCR. Here, we compare existing methods of isolating DNA and their adaptation to a red wine matrix. Two different protocols for isolating DNA and three PCR mix compositions were tested to select the best method. The addition of insoluble polyvinylpolypyrrolidone (PVPP at 1% (v/v during DNA extraction using a protocol succeeded in eliminating PCR inhibitors from red wine. We developed a bacterial internal control which was efficient in avoiding false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The specificity, linearity, repeatability and reproducibility of the method were evaluated. A standard curve was established for the enumeration of AAB inoculated into red wines. The limit of quantification in red wine was 3.7 log AAB/mL and about 2.8 log AAB/mL when the volume of the samples was increased from 1 mL to 10 mL. Thus the DNA extraction method developed in this paper allows sensitive and reliable AAB quantification without underestimation thanks to the presence of an internal control. Moreover, monitoring of both the AAB population and the amount of acetic acid in ethanol medium and red wine highlighted that a minimum about 6.0 log cells/mL of AAB is needed to significantly increase the production of acetic acid leading to spoilage.

  8. Design and Performance Testing of a DNA Extraction Assay for Sensitive and Reliable Quantification of Acetic Acid Bacteria Directly in Red Wine Using Real Time PCR.

    Science.gov (United States)

    Longin, Cédric; Guilloux-Benatier, Michèle; Alexandre, Hervé

    2016-01-01

    Although strategies exist to prevent AAB contamination, the increased interest for wines with low sulfite addition leads to greater AAB spoilage. Hence, there is a real need for a rapid, specific, sensitive, and reliable method for detecting these spoilage bacteria. All these requirements are met by real time Polymerase Chain Reaction (or quantitative PCR; qPCR). Here, we compare existing methods of isolating DNA and their adaptation to a red wine matrix. Two different protocols for isolating DNA and three PCR mix compositions were tested to select the best method. The addition of insoluble polyvinylpolypyrrolidone (PVPP) at 1% (v/v) during DNA extraction using a protocol succeeded in eliminating PCR inhibitors from red wine. We developed a bacterial internal control which was efficient in avoiding false negative results due to decreases in the efficiency of DNA isolation and/or amplification. The specificity, linearity, repeatability, and reproducibility of the method were evaluated. A standard curve was established for the enumeration of AAB inoculated into red wines. The limit of quantification in red wine was 3.7 log AAB/mL and about 2.8 log AAB/mL when the volume of the samples was increased from 1 to 10 mL. Thus, the DNA extraction method developed in this paper allows sensitive and reliable AAB quantification without underestimation thanks to the presence of an internal control. Moreover, monitoring of both the AAB population and the amount of acetic acid in ethanol medium and red wine highlighted that a minimum about 6.0 log cells/mL of AAB is needed to significantly increase the production of acetic acid leading to spoilage.

  9. Assessment of okadaic acid effects on cytotoxicity, DNA damage and DNA repair in human cells.

    Science.gov (United States)

    Valdiglesias, Vanessa; Méndez, Josefina; Pásaro, Eduardo; Cemeli, Eduardo; Anderson, Diana; Laffon, Blanca

    2010-07-07

    Okadaic acid (OA) is a phycotoxin produced by several types of dinoflagellates causing diarrheic shellfish poisoning (DSP) in humans. Symptoms induced by DSP toxins are mainly gastrointestinal, but the intoxication does not appear to be fatal. Despite this, this toxin presents a potential threat to human health even at concentrations too low to induce acute toxicity, since previous animal studies have shown that OA has very potent tumour promoting activity. However, its concrete action mechanism has not been described yet and the results reported with regard to OA cytotoxicity and genotoxicity are often contradictory. In the present study, the genotoxic and cytotoxic effects of OA on three different types of human cells (peripheral blood leukocytes, HepG2 hepatoma cells, and SHSY5Y neuroblastoma cells) were evaluated. Cells were treated with a range of OA concentrations in the presence and absence of S9 fraction, and MTT test and Comet assay were performed in order to evaluate cytotoxicity and genotoxicity, respectively. The possible effects of OA on DNA repair were also studied by means of the DNA repair competence assay, using bleomycin as DNA damage inductor. Treatment with OA in absence of S9 fraction induced not statistically significant decrease in cell viability and significant increase in DNA damage in all cell types at the highest concentrations investigated. However, only SHSY5Y cells showed OA induced genotoxic and cytotoxic effects in presence of S9 fraction. Furthermore, we found that OA can induce modulations in DNA repair processes when exposure was performed prior to BLM treatment, in co-exposure, or during the subsequent DNA repair process. Copyright 2010 Elsevier B.V. All rights reserved.

  10. Identification and quantification of metabolites of the fungicide tebuconazole in human urine.

    Science.gov (United States)

    Mercadante, R; Polledri, E; Scurati, S; Moretto, A; Fustinoni, S

    2014-11-17

    Tebuconazole (TEB) is a fungicide used in agriculture; the objective of this work was to identify and quantify TEB metabolites in human urine. Samples from seven vineyard workers exposed to TEB were submitted to liquid chromatography interfaced with a triple quadrupole mass spectrometer, equipped with an electron spray source, and a linear ion trap to gain a profile of candidate metabolites. Based on the presence of the ion m/z 70 in the MS/MS spectra, which corresponds to protonated triazole (a specific moiety of TEB), and the isotopic pattern of the molecular ions, typical of molecules with one chlorine atom, hydroxyl and carboxyl derivatives of TEB, that is, TEB-OH and TEB-COOH, were identified as major metabolites, both as free molecules and as glucuronide (Glc) conjugates. The mean molar fractions were 0.67, 0.13, 0.13, and 0.07 for TEB-O-Glc, TEB-OH, TEB-COO-Glc, and TEB-COOH. Urine samples were submitted to hydrolysis with β-glucuronidase, and the free compounds were quantified in the presence of deuterated TEB (TEB-d6) as the internal standard (IS), by multiple reaction monitoring (MRM) mode. The assay was linear in the ranges of 0.2-600 μg/L and 0.1-240 μg/L for TEB-OH and TEB-COOH, respectively; precision, accuracy, and the limit of quantification (LOQ) were <3.1%, 98-103%, and 0.3 μg/L for both analytes. An evaluation of matrix effects showed that the use of TEB-d6 controlled these sources of bias. The urinary levels of TEB-OH and TEB-COOH in specimens collected from farmers exposed to TEB ranged from 10 to 473 and from 3 to 159 μg/L, respectively.

  11. In vivo quantification of {sup 18}F-Fdg uptake in human placenta during early pregnancy

    Energy Technology Data Exchange (ETDEWEB)

    Zanotti-Fregonara, P.; Jan, S.; Trebossen, R.; Maroy, R. [CEA, DSV, I2BM, SHFJ, F-91401 Orsay (France); Champion, C. [Univ Paul Verlaine Metz, Lab Phys Mol et Collis, Inst Phys, Metz (France); Hindie, E. [Hop St Antoine, AP-HP, F-75571 Paris (France); Hindie, E. [Univ Paris 07, IMDCT, IUH, Ecole Doctorale B2T, F-75221 Paris (France)

    2008-07-01

    {sup 18}F-FDG is the most widely used PET radiopharmaceutical. Nevertheless, no data for {sup 18}F-FDG uptake in the human placenta have been reported. We recently reported on embryo dosimetry in a woman who underwent an {sup 18}F-FDG PET/CT scan during early pregnancy. In the present work we attempt an in vivo quantification of the {sup 18}F-FDG uptake by the placenta. The 27-y-old woman received 320 MBq of {sup 18}F-FDG for a follow-up study for Hodgkin's lymphoma and was later discovered to be pregnant (embryo age 8 wk). Imaging started 1 h after injection. The maximum placental tissue uptake (SUVmax) was 2.5. This value was conservatively attributed to the entire placental volume, i.e., 45 mL, a value representative of the average dimensions of a normal placenta at 8 wk. On the basis of these measurements, placenta {sup 18}F-FDG uptake in our patient was 0.19% of the injected activity. A Monte Carlo simulation was used to derive the photon dose to the embryo from the placenta (0.022 * 10{sup -2} mGy per MBq of injected {sup 18}F-FDG) and from the surrounding amniotic fluid (0.017 * 10{sup -2} mGy MBq{sup -1}). This increases our previously calculated dose (3.3 * 10{sup -2} mGy MBq{sup -1}) by only a small fraction (1.18%), which does not justify modifying the previous estimate given the overall uncertainties. (authors)

  12. Detection and quantification of human adenovirus genomes in Acanthamoeba isolated from swimming pools

    Directory of Open Access Journals (Sweden)

    RODRIGO STAGGEMEIER

    2016-01-01

    Full Text Available ABSTRACT Acanthamoeba is the most common free-living environmental amoeba, it may serve as an important vehicle for various microorganisms living in the same environment, such as viruses, being pathogenic to humans. This study aimed to detect and quantify human adenoviruses (HAdV in Acanthamoebas isolated from water samples collected from swimming pools in the city of Porto Alegre, Southern Brazil. Free-living amoebae of the genus Acanthamoeba were isolated from water samples, and isolates (n=16 were used to investigate the occurrence of HAdVs. HAdV detection was performed by quantitative real-time polymerase chain reaction (qPCR. HAdVs were detected in 62.5% (10/16 of Acanthamoeba isolates, ranging from 3.24x103 to 5.14x105 DNA copies per milliliter of isolate. HAdV viral loads found in this study are not negligible, especially because HAdV infections are associated with several human diseases, including gastroenteritis, respiratory distress, and ocular diseases. These findings reinforce the concept that Acanthamoeba may act as a reservoir and promote HAdV transmission through water.

  13. Comprehensive Assessment of Oxidatively Induced Modifications of DNA in a Rat Model of Human Wilson's Disease.

    Science.gov (United States)

    Yu, Yang; Guerrero, Candace R; Liu, Shuo; Amato, Nicholas J; Sharma, Yogeshwar; Gupta, Sanjeev; Wang, Yinsheng

    2016-03-01

    Defective copper excretion from hepatocytes in Wilson's disease causes accumulation of copper ions with increased generation of reactive oxygen species via the Fenton-type reaction. Here we developed a nanoflow liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry coupled with the isotope-dilution method for the simultaneous quantification of oxidatively induced DNA modifications. This method enabled measurement, in microgram quantities of DNA, of four oxidative stress-induced lesions, including direct ROS-induced purine cyclonucleosides (cPus) and two exocyclic adducts induced by byproducts of lipid peroxidation, i.e. 1,N(6)-etheno-2'-deoxyadenosine (εdA) and 1,N(2)-etheno-2'-deoxyguanosine (εdG). Analysis of liver tissues of Long-Evans Cinnamon rats, which constitute an animal model of human Wilson's disease, and their healthy counterparts [i.e. Long-Evans Agouti rats] showed significantly higher levels of all four DNA lesions in Long-Evans Cinnamon than Long-Evans Agouti rats. Moreover, cPus were present at much higher levels than εdA and εdG lesions. In contrast, the level of 5-hydroxymethyl-2'-deoxycytidine (5-HmdC), an oxidation product of 5-methyl-2'-deoxycytidine (5-mdC), was markedly lower in the liver tissues of Long-Evans Cinnamon than Long-Evans Agouti rats, though no differences were observed for the levels of 5-mdC. In vitro biochemical assay showed that Cu(2+) ions could directly inhibit the activity of Tet enzymes. Together, these results suggest that aberrant copper accumulation may perturb genomic stability by elevating oxidatively induced DNA lesions, and by altering epigenetic pathways of gene regulation.

  14. Detecting multiple DNA human profile from a mosquito blood meal.

    Science.gov (United States)

    Rabêlo, K C N; Albuquerque, C M R; Tavares, V B; Santos, S M; Souza, C A; Oliveira, T C; Moura, R R; Brandão, L A C; Crovella, S

    2016-08-26

    Criminal traces commonly found at crime scenes may present mixtures from two or more individuals. The scene of the crime is important for the collection of various types of traces in order to find the perpetrator of the crime. Thus, we propose that hematophagous mosquitoes found at crime scenes can be used to perform genetic testing of human blood and aid in suspect investigation. The aim of the study was to obtain a single Aedes aegypti mosquito profile from a human DNA mixture containing genetic materials of four individuals. We also determined the effect of blood acquisition time by setting time intervals of 24, 48, and 72 h after the blood meal. STR loci and amelogenin were analyzed, and the results showed that human DNA profiles could be obtained from hematophagous mosquitos at 24 h following the blood meal. It is possible that hematophagous mosquitoes can be used as biological remains at the scene of the crime, and can be used to detect human DNA profiles of up to four individuals.

  15. DNA-duplex linker for AFM-SELEX of DNA aptamer against human serum albumin.

    Science.gov (United States)

    Takenaka, Musashi; Okumura, Yuzo; Amino, Tomokazu; Miyachi, Yusuke; Ogino, Chiaki; Kondo, Akihiko

    2017-02-15

    DNA-duplex interactions in thymines and adenins are used as a linker for the novel methodology of Atomic Force Microscope-Systematic Evolution of Ligands by EXpotential enrichment (AFM-SELEX). This study used the hydrogen bonds in 10 mer of both thymines (T10) and adenines (A10). Initially, the interactive force in T10-A10 was measured by AFM, which returned an average interactive force of approximately 350pN. Based on this result, DNA aptamers against human serum albumin could be selected in the 4th round, and 15 different clones could be sequenced. The lowest dissociation constant of the selected aptamer was identified via surface plasmon resonance, and it proved to be identical to that of the commercial aptamer. Therefore, specific hydrogen bonds in DNA can be useful linkers for AFM-SELEX. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Analysis of Human Accelerated DNA Regions Using Archaic Hominin Genomes

    Science.gov (United States)

    Burbano, Hernán A.; Green, Richard E.; Maricic, Tomislav; Lalueza-Fox, Carles; de la Rasilla, Marco; Rosas, Antonio; Kelso, Janet; Pollard, Katherine S.; Lachmann, Michael; Pääbo, Svante

    2012-01-01

    Several previous comparisons of the human genome with other primate and vertebrate genomes identified genomic regions that are highly conserved in vertebrate evolution but fast-evolving on the human lineage. These human accelerated regions (HARs) may be regions of past adaptive evolution in humans. Alternatively, they may be the result of non-adaptive processes, such as biased gene conversion. We captured and sequenced DNA from a collection of previously published HARs using DNA from an Iberian Neandertal. Combining these new data with shotgun sequence from the Neandertal and Denisova draft genomes, we determine at least one archaic hominin allele for 84% of all positions within HARs. We find that 8% of HAR substitutions are not observed in the archaic hominins and are thus recent in the sense that the derived allele had not come to fixation in the common ancestor of modern humans and archaic hominins. Further, we find that recent substitutions in HARs tend to have come to fixation faster than substitutions elsewhere in the genome and that substitutions in HARs tend to cluster in time, consistent with an episodic rather than a clock-like process underlying HAR evolution. Our catalog of sequence changes in HARs will help prioritize them for functional studies of genomic elements potentially responsible for modern human adaptations. PMID:22412940

  17. Orthographic Structuring of Human Speech and Texts Linguistic Application of Recurrence Quantification Analysis

    CERN Document Server

    Orsucci, F; Giuliani, A; Webber, C L; Zbilut, J P

    1997-01-01

    A methodology based upon recurrence quantification analysis is proposed for the study of orthographic structure of written texts. Five different orthographic data sets (20th century Italian poems, 20th century American poems, contemporary Swedish poems with their corresponding Italian translations, Italian speech samples, and American speech samples) were subjected to recurrence quantification analysis, a procedure which has been found to be diagnostically useful in the quantitative assessment of ordered series in fields such as physics, molecular dynamics, physiology, and general signal processing. Recurrence quantification was developed from recurrence plots as applied to the analysis of nonlinear, complex systems in the physical sciences, and is based on the computation of a distance matrix of the elements of an ordered series (in this case the letters consituting selected speech and poetic texts). From a strictly mathematical view, the results show the possibility of demonstrating invariance between diffe...

  18. Androgen receptor function links human sexual dimorphism to DNA methylation.

    Directory of Open Access Journals (Sweden)

    Ole Ammerpohl

    Full Text Available Sex differences are well known to be determinants of development, health and disease. Epigenetic mechanisms are also known to differ between men and women through X-inactivation in females. We hypothesized that epigenetic sex differences may also result from sex hormone functions, in particular from long-lasting androgen programming. We aimed at investigating whether inactivation of the androgen receptor, the key regulator of normal male sex development, is associated with differences of the patterns of DNA methylation marks in genital tissues. To this end, we performed large scale array-based analysis of gene methylation profiles on genomic DNA from labioscrotal skin fibroblasts of 8 males and 26 individuals with androgen insensitivity syndrome (AIS due to inactivating androgen receptor gene mutations. By this approach we identified differential methylation of 167 CpG loci representing 162 unique human genes. These were significantly enriched for androgen target genes and low CpG content promoter genes. Additional 75 genes showed a significant increase of heterogeneity of methylation in AIS compared to a high homogeneity in normal male controls. Our data show that normal and aberrant androgen receptor function is associated with distinct patterns of DNA-methylation marks in genital tissues. These findings support the concept that transcription factor binding to the DNA has an impact on the shape of the DNA methylome. These data which derived from a rare human model suggest that androgen programming of methylation marks contributes to sexual dimorphism in the human which might have considerable impact on the manifestation of sex-associated phenotypes and diseases.

  19. mtDNA-Server: next-generation sequencing data analysis of human mitochondrial DNA in the cloud

    Science.gov (United States)

    Weissensteiner, Hansi; Forer, Lukas; Fuchsberger, Christian; Schöpf, Bernd; Kloss-Brandstätter, Anita; Specht, Günther; Kronenberg, Florian; Schönherr, Sebastian

    2016-01-01

    Next generation sequencing (NGS) allows investigating mitochondrial DNA (mtDNA) characteristics such as heteroplasmy (i.e. intra-individual sequence variation) to a higher level of detail. While several pipelines for analyzing heteroplasmies exist, issues in usability, accuracy of results and interpreting final data limit their usage. Here we present mtDNA-Server, a scalable web server for the analysis of mtDNA studies of any size with a special focus on usability as well as reliable identification and quantification of heteroplasmic variants. The mtDNA-Server workflow includes parallel read alignment, heteroplasmy detection, artefact or contamination identification, variant annotation as well as several quality control metrics, often neglected in current mtDNA NGS studies. All computational steps are parallelized with Hadoop MapReduce and executed graphically with Cloudgene. We validated the underlying heteroplasmy and contamination detection model by generating four artificial sample mix-ups on two different NGS devices. Our evaluation data shows that mtDNA-Server detects heteroplasmies and artificial recombinations down to the 1% level with perfect specificity and outperforms existing approaches regarding sensitivity. mtDNA-Server is currently able to analyze the 1000G Phase 3 data (n = 2,504) in less than 5 h and is freely accessible at https://mtdna-server.uibk.ac.at. PMID:27084948

  20. mtDNA-Server: next-generation sequencing data analysis of human mitochondrial DNA in the cloud.

    Science.gov (United States)

    Weissensteiner, Hansi; Forer, Lukas; Fuchsberger, Christian; Schöpf, Bernd; Kloss-Brandstätter, Anita; Specht, Günther; Kronenberg, Florian; Schönherr, Sebastian

    2016-07-08

    Next generation sequencing (NGS) allows investigating mitochondrial DNA (mtDNA) characteristics such as heteroplasmy (i.e. intra-individual sequence variation) to a higher level of detail. While several pipelines for analyzing heteroplasmies exist, issues in usability, accuracy of results and interpreting final data limit their usage. Here we present mtDNA-Server, a scalable web server for the analysis of mtDNA studies of any size with a special focus on usability as well as reliable identification and quantification of heteroplasmic variants. The mtDNA-Server workflow includes parallel read alignment, heteroplasmy detection, artefact or contamination identification, variant annotation as well as several quality control metrics, often neglected in current mtDNA NGS studies. All computational steps are parallelized with Hadoop MapReduce and executed graphically with Cloudgene. We validated the underlying heteroplasmy and contamination detection model by generating four artificial sample mix-ups on two different NGS devices. Our evaluation data shows that mtDNA-Server detects heteroplasmies and artificial recombinations down to the 1% level with perfect specificity and outperforms existing approaches regarding sensitivity. mtDNA-Server is currently able to analyze the 1000G Phase 3 data (n = 2,504) in less than 5 h and is freely accessible at https://mtdna-server.uibk.ac.at.

  1. A promoter DNA demethylation landscape of human hematopoietic differentiation.

    Science.gov (United States)

    Calvanese, Vincenzo; Fernández, Agustín F; Urdinguio, Rocío G; Suárez-Alvarez, Beatriz; Mangas, Cristina; Pérez-García, Vicente; Bueno, Clara; Montes, Rosa; Ramos-Mejía, Verónica; Martínez-Camblor, Pablo; Ferrero, Cecilia; Assenov, Yassen; Bock, Christoph; Menendez, Pablo; Carrera, Ana Clara; Lopez-Larrea, Carlos; Fraga, Mario F

    2012-01-01

    Global mechanisms defining the gene expression programs specific for hematopoiesis are still not fully understood. Here, we show that promoter DNA demethylation is associated with the activation of hematopoietic-specific genes. Using genome-wide promoter methylation arrays, we identified 694 hematopoietic-specific genes repressed by promoter DNA methylation in human embryonic stem cells and whose loss of methylation in hematopoietic can be associated with gene expression. The association between promoter methylation and gene expression was studied for many hematopoietic-specific genes including CD45, CD34, CD28, CD19, the T cell receptor (TCR), the MHC class II gene HLA-DR, perforin 1 and the phosphoinositide 3-kinase (PI3K) and results indicated that DNA demethylation was not always sufficient for gene activation. Promoter demethylation occurred either early during embryonic development or later on during hematopoietic differentiation. Analysis of the genome-wide promoter methylation status of induced pluripotent stem cells (iPSCs) generated from somatic CD34(+) HSPCs and differentiated derivatives from CD34(+) HSPCs confirmed the role of DNA methylation in regulating the expression of genes of the hemato-immune system, and indicated that promoter methylation of these genes may be associated to stemness. Together, these data suggest that promoter DNA demethylation might play a role in the tissue/cell-specific genome-wide gene regulation within the hematopoietic compartment.

  2. Recovery of latent fingerprints and DNA on human skin.

    Science.gov (United States)

    Färber, Doris; Seul, Andrea; Weisser, Hans-Joachim; Bohnert, Michael

    2010-11-01

    The project "Latent Fingerprints and DNA on Human Skin" was the first systematic research in Europe dealing with detection of fingerprints and DNA left by offenders on the skin of corpses. One thousand samples gave results that allow general statements on the materials and methods used. The tests were carried out according to a uniform trial structure. Fingerprints were deposited by natural donors on corpses. The latent fingerprints were treated with magnetic powder or black fingerprint powder. Afterward, they were lifted with silicone casting material (Isomark(®)) or gelatine foil. All lifts were swabbed to recover DNA. It was possible to visualize comparable and identifiable fingerprints on the skin of corpses (16%). In the same categories, magnetic powder (18.4%) yielded better results than black fingerprint powder (13.6%). The number of comparable and identifiable fingerprints decreased on the lifts (12.7%). Isomark(®) (14.9%) was the better lifting material in comparison with gelatine foil (10.1%). In one-third of the samples, DNA could be extracted from the powdered and lifted latents. Black fingerprint powder delivered the better result with a rate of 2.2% for full DNA profiles and profiles useful for exclusion in comparison with 1.8% for the magnetic powder traces. Isomark(®) (3.1%) yielded better results than gelatine foil (0.6%).

  3. Nuclear responses to depletion of mitochondrial DNA in human cells.

    Science.gov (United States)

    Li, K; Neufer, P D; Williams, R S

    1995-11-01

    The derivation of human cell lines devoid of mitochondrial (mt) DNA (rho 0) provides an opportunity to study nuclear responses to a chronic impairment of mitochondrial oxidative phosphorylation. Expression of several nuclear genes is induced in human rho 0 cells, including those encoding integral proteins of the mitochondrial inner membrane, intermediate filaments, and ribosomes. In contrast to conditions in which mitochondrial respiration is altered acutely, expression of heat shock proteins and immediate early genes is not induced. Mitochondria from rho 0 cells maintain a transmembrane electrochemical potential and are distributed within the cytoplasm of these cells in a manner indistinguishable from that of wild-type cells. We conclude that a chronic deficiency of mitochondrial oxidative phosphorylation produced by elimination of mtDNA is associated with a different pattern of gene induction than that provoked by other acute or subacute conditions that impair mitochondrial respiration or create energy demands in excess of mitochondrial respiratory capacity.

  4. DNA-binding specificities of human transcription factors.

    Science.gov (United States)

    Jolma, Arttu; Yan, Jian; Whitington, Thomas; Toivonen, Jarkko; Nitta, Kazuhiro R; Rastas, Pasi; Morgunova, Ekaterina; Enge, Martin; Taipale, Mikko; Wei, Gonghong; Palin, Kimmo; Vaquerizas, Juan M; Vincentelli, Renaud; Luscombe, Nicholas M; Hughes, Timothy R; Lemaire, Patrick; Ukkonen, Esko; Kivioja, Teemu; Taipale, Jussi

    2013-01-17

    Although the proteins that read the gene regulatory code, transcription factors (TFs), have been largely identified, it is not well known which sequences TFs can recognize. We have analyzed the sequence-specific binding of human TFs using high-throughput SELEX and ChIP sequencing. A total of 830 binding profiles were obtained, describing 239 distinctly different binding specificities. The models represent the majority of human TFs, approximately doubling the coverage compared to existing systematic studies. Our results reveal additional specificity determinants for a large number of factors for which a partial specificity was known, including a commonly observed A- or T-rich stretch that flanks the core motifs. Global analysis of the data revealed that homodimer orientation and spacing preferences, and base-stacking interactions, have a larger role in TF-DNA binding than previously appreciated. We further describe a binding model incorporating these features that is required to understand binding of TFs to DNA.

  5. DNA barcoding of fungi causing infections in humans and animals.

    Science.gov (United States)

    Irinyi, Laszlo; Lackner, Michaela; de Hoog, G Sybren; Meyer, Wieland

    2016-02-01

    Correct species identification is becoming increasingly important in clinical diagnostics. Till now, many mycological laboratories rely on conventional phenotypic identification. But this is slow and strongly operator-dependent. Therefore, to improve the quality of pathogen identification, rapid, reliable, and objective identification methods are essential. One of the most encouraging approaches is molecular barcoding using the internal transcribed spacer (ITS) of the rDNA, which is rapid, easily achievable, accurate, and applicable directly from clinical specimens. It relies on the comparison of a single ITS sequence with a curated reference database. The International Society for Human and Animal Mycology (ISHAM) working group for DNA barcoding has recently established such a database, focusing on the majority of human and animal pathogenic fungi (ISHAM-ITS, freely accessible at http://www.isham.org/ or directly from http://its.mycologylab.org). For some fungi the use of secondary barcodes may be necessary.

  6. Recurrent DNA inversion rearrangements in the human genome

    DEFF Research Database (Denmark)

    Flores, Margarita; Morales, Lucía; Gonzaga-Jauregui, Claudia

    2007-01-01

    Several lines of evidence suggest that reiterated sequences in the human genome are targets for nonallelic homologous recombination (NAHR), which facilitates genomic rearrangements. We have used a PCR-based approach to identify breakpoint regions of rearranged structures in the human genome...... on chromosomes 3, 15, and 19, were analyzed. The relative proportion of wild-type to rearranged structures was determined in DNA samples from blood obtained from different, unrelated individuals. The results obtained indicate that recurrent genomic rearrangements occur at relatively high frequency in somatic...... cells. Interestingly, the rearrangements studied were significantly more abundant in adults than in newborn individuals, suggesting that such DNA rearrangements might start to appear during embryogenesis or fetal life and continue to accumulate after birth. The relevance of our results in regard...

  7. Simultaneous determination and validated quantification of human insulin and its synthetic analogues in human blood serum by immunoaffinity purification and liquid chromatography-mass spectrometry.

    Science.gov (United States)

    Hess, Cornelius; Thomas, Andreas; Thevis, Mario; Stratmann, Bernd; Quester, Wulf; Tschoepe, Diethelm; Madea, Burkhard; Musshoff, Frank

    2012-10-01

    Possible fatal complications of human insulin and its synthetic analogues like hypoglycemia require precise classification and quantitative determination of these drugs both for clinical purposes as well as for forensic toxicologists. A procedure was developed for the identification and quantification of human insulin and different long-acting as well as short-acting synthetic insulins in human blood serum specimens. After an immunoaffinity purification step and separation by liquid chromatography, the insulins were characterized by their five- or sixfold protonated molecule ions and diagnostic product ions. Clinical samples of 207 diabetic and 50 non-diabetic patients after the administration of human insulin or oral antidiabetics and forensic samples were analyzed for human/synthetic insulin concentrations. The method was validated according to international guidelines. Limits of detection of the insulins ranged between 1.3 and 2.8 μU/ml. Recoveries ranged between 33.2 % and 51.7 %. Precision data was in accordance with international guidelines. Clinical samples showed concentrations of human insulin lower than 301 μU/ml. Our liquid chromatography tandem mass spectrometry procedure allows unambiguous identification and quantification of the intact human insulin and its intact synthetic analogues Humalog®, Novolog®, Apidra®, Lantus®, and Levemir® in human blood serum in clinical and overdose cases. The assay could be successfully tested in patients with diabetes mellitus on therapy with insulins or oral antidiabetics.

  8. Detection of extracellular genomic DNA scaffold in human thrombus

    DEFF Research Database (Denmark)

    Oklu, Rahmi; Albadawi, Hassan; Watkins, Michael T

    2012-01-01

    PURPOSE: Mechanisms underlying transition of a thrombus susceptible to tissue plasminogen activator (TPA) fibrinolysis to one that is resistant is unclear. Demonstration of a new possible thrombus scaffold may open new avenues of research in thrombolysis and may provide mechanistic insight...... thrombi. CONCLUSIONS: Extensive detection of genomic DNA associated with histones in the extracellular matrix of human and mouse thrombi suggest the presence of a new thrombus-associated scaffold....

  9. Quantification of Human and Animal Viruses to Differentiate the Origin of the Fecal Contamination Present in Environmental Samples

    Directory of Open Access Journals (Sweden)

    Sílvia Bofill-Mas

    2013-01-01

    Full Text Available Many different viruses are excreted by humans and animals and are frequently detected in fecal contaminated waters causing public health concerns. Classical bacterial indicator such as E. coli and enterococci could fail to predict the risk for waterborne pathogens such as viruses. Moreover, the presence and levels of bacterial indicators do not always correlate with the presence and concentration of viruses, especially when these indicators are present in low concentrations. Our research group has proposed new viral indicators and methodologies for determining the presence of fecal pollution in environmental samples as well as for tracing the origin of this fecal contamination (microbial source tracking. In this paper, we examine to what extent have these indicators been applied by the scientific community. Recently, quantitative assays for quantification of poultry and ovine viruses have also been described. Overall, quantification by qPCR of human adenoviruses and human polyomavirus JC, porcine adenoviruses, bovine polyomaviruses, chicken/turkey parvoviruses, and ovine polyomaviruses is suggested as a toolbox for the identification of human, porcine, bovine, poultry, and ovine fecal pollution in environmental samples.

  10. DNA typing of Calliphorids collected from human corpses in Malaysia.

    Science.gov (United States)

    Kavitha, R; Tan, T C; Lee, H L; Nazni, W A; Sofian-Azirun, M

    2013-03-01

    Estimation of post-mortem interval (PMI) is crucial for time of death determination. The advent of DNA-based identification techniques forensic entomology saw the beginning of a proliferation of molecular studies into forensically important Calliphoridae (Diptera). The use of DNA to characterise morphologically indistinguishable immature calliphorids was recognised as a valuable molecular tool with enormous practical utility. The local entomofauna in most cases is important for the examination of entomological evidences. The survey of the local entomofauna has become a fundamental first step in forensic entomological studies, because different geographical distributions, seasonal and environmental factors may influence the decomposition process and the occurrence of different insect species on corpses. In this study, calliphorids were collected from 13 human corpses recovered from indoors, outdoors and aquatic conditions during the post-mortem examination by pathologists from the government hospitals in Malaysia. Only two species, Chrysomya megacephala and Chrysomya rufifacies were recovered from human corpses. DNA sequencing was performed to study the mitochondrial encoded COI gene and to evaluate the suitability of the 1300 base pairs of COI fragments for identification of blow fly species collected from real crime scene. The COI gene from blow fly specimens were sequenced and deposited in GenBank to expand local databases. The sequenced COI gene was useful in identifying calliphorids retrieved from human corpses.

  11. Defining functional DNA elements in the human genome.

    Science.gov (United States)

    Kellis, Manolis; Wold, Barbara; Snyder, Michael P; Bernstein, Bradley E; Kundaje, Anshul; Marinov, Georgi K; Ward, Lucas D; Birney, Ewan; Crawford, Gregory E; Dekker, Job; Dunham, Ian; Elnitski, Laura L; Farnham, Peggy J; Feingold, Elise A; Gerstein, Mark; Giddings, Morgan C; Gilbert, David M; Gingeras, Thomas R; Green, Eric D; Guigo, Roderic; Hubbard, Tim; Kent, Jim; Lieb, Jason D; Myers, Richard M; Pazin, Michael J; Ren, Bing; Stamatoyannopoulos, John A; Weng, Zhiping; White, Kevin P; Hardison, Ross C

    2014-04-29

    With the completion of the human genome sequence, attention turned to identifying and annotating its functional DNA elements. As a complement to genetic and comparative genomics approaches, the Encyclopedia of DNA Elements Project was launched to contribute maps of RNA transcripts, transcriptional regulator binding sites, and chromatin states in many cell types. The resulting genome-wide data reveal sites of biochemical activity with high positional resolution and cell type specificity that facilitate studies of gene regulation and interpretation of noncoding variants associated with human disease. However, the biochemically active regions cover a much larger fraction of the genome than do evolutionarily conserved regions, raising the question of whether nonconserved but biochemically active regions are truly functional. Here, we review the strengths and limitations of biochemical, evolutionary, and genetic approaches for defining functional DNA segments, potential sources for the observed differences in estimated genomic coverage, and the biological implications of these discrepancies. We also analyze the relationship between signal intensity, genomic coverage, and evolutionary conservation. Our results reinforce the principle that each approach provides complementary information and that we need to use combinations of all three to elucidate genome function in human biology and disease.

  12. A strategy with label-free quantification of the targeted peptides for quantitative peptidome analysis of human serum

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    Peptidomics draws more and more attention in discovering useful biomarkers for early diagnosis of disease. However, there is lack of efficient quantification strategy in peptidome analysis. In this study, a strategy with label-free quantification of the targeted endogenous peptides based on peak intensity using μUPLC-Q-TOF-MS/MS was developed for quantitative peptidome analysis of human serum. Different amounts of standard BSA tryptic digesting peptides were added into the same serum extracts for evaluation of the developed strategy, and it was observed that the average relative error of the targeted peptides was 6.42%, which was superior to the result obtained directly by commercially available software PLGS. It was also demonstrated that this quantification strategy could obviously increase the detection sensitivity of the peptide by DDA analysis. Then, this strategy was applied to comparatively analyze the peptides extracted from the serum of HCC or breast cancer patients and healthy individuals, respectively. Peptides with charge states up to 5 and molecular weight over 4000 can be reliably identified and quantified. This quantitative analysis method based on μUPLC-Q-TOF-MS/MS exhibited superior sensitivity than that by MALDI-TOF-MS commonly used in peptidome analysis. Finally, some interesting endogenous peptides related to corresponding diseases were successfully obtained.

  13. Structural elucidation and quantification of phenolic conjugates present in human urine after tea intake

    NARCIS (Netherlands)

    Hooft, van der J.J.J.; Vos, de R.C.H.; Mihaleva, V.; Bino, R.J.; Ridder, L.O.; Roo, de N.; Jacobs, D.M.; Duynhoven, van J.P.M.; Vervoort, J.J.M.

    2012-01-01

    In dietary polyphenol exposure studies, annotation and identification of urinary metabolites present at low (micromolar) concentrations are major obstacles. In order to determine the biological activity of specific components, it is necessary to have the correct structures and the quantification of

  14. Trapping DNA replication origins from the human genome.

    Science.gov (United States)

    Eki, Toshihiko; Murakami, Yasufumi; Hanaoka, Fumio

    2013-04-17

    Synthesis of chromosomal DNA is initiated from multiple origins of replication in higher eukaryotes; however, little is known about these origins' structures. We isolated the origin-derived nascent DNAs from a human repair-deficient cell line by blocking the replication forks near the origins using two different origin-trapping methods (i.e., UV- or chemical crosslinker-treatment and cell synchronization in early S phase using DNA replication inhibitors). Single-stranded DNAs (of 0.5-3 kb) that accumulated after such treatments were labeled with bromodeoxyuridine (BrdU). BrdU-labeled DNA was immunopurified after fractionation by alkaline sucrose density gradient centrifugation and cloned by complementary-strand synthesis and PCR amplification. Competitive PCR revealed an increased abundance of DNA derived from known replication origins (c-myc and lamin B2 genes) in the nascent DNA fractions from the UV-treated or crosslinked cells. Nucleotide sequences of 85 and 208 kb were obtained from the two libraries (I and II) prepared from the UV-treated log-phase cells and early S phase arrested cells, respectively. The libraries differed from each other in their G+C composition and replication-related motif contents, suggesting that differences existed between the origin fragments isolated by the two different origin-trapping methods. The replication activities for seven out of 12 putative origin loci from the early-S phase cells were shown by competitive PCR. We mapped 117 (library I) and 172 (library II) putative origin loci to the human genome; approximately 60% and 50% of these loci were assigned to the G-band and intragenic regions, respectively. Analyses of the flanking sequences of the mapped loci suggested that the putative origin loci tended to associate with genes (including conserved sites) and DNase I hypersensitive sites; however, poor correlations were found between such loci and the CpG islands, transcription start sites, and K27-acetylated histone H3 peaks.

  15. The Cell Cycle Timing of Human Papillomavirus DNA Replication.

    Science.gov (United States)

    Reinson, Tormi; Henno, Liisi; Toots, Mart; Ustav, Mart; Ustav, Mart

    2015-01-01

    Viruses manipulate the cell cycle of the host cell to optimize conditions for more efficient viral genome replication. One strategy utilized by DNA viruses is to replicate their genomes non-concurrently with the host genome; in this case, the viral genome is amplified outside S phase. This phenomenon has also been described for human papillomavirus (HPV) vegetative genome replication, which occurs in G2-arrested cells; however, the precise timing of viral DNA replication during initial and stable replication phases has not been studied. We developed a new method to quantitate newly synthesized DNA levels and used this method in combination with cell cycle synchronization to show that viral DNA replication is initiated during S phase and is extended to G2 during initial amplification but follows the replication pattern of cellular DNA during S phase in the stable maintenance phase. E1 and E2 protein overexpression changes the replication time from S only to both the S and G2 phases in cells that stably maintain viral episomes. These data demonstrate that the active synthesis and replication of the HPV genome are extended into the G2 phase to amplify its copy number and the duration of HPV genome replication is controlled by the level of the viral replication proteins E1 and E2. Using the G2 phase for genome amplification may be an important adaptation that allows exploitation of changing cellular conditions during cell cycle progression. We also describe a new method to quantify newly synthesized viral DNA levels and discuss its benefits for HPV research.

  16. Quantification of Cell-Free DNA in Red Blood Cell Units in Different Whole Blood Processing Methods

    Directory of Open Access Journals (Sweden)

    Andrew W. Shih

    2016-01-01

    Full Text Available Background. Whole blood donations in Canada are processed by either the red cell filtration (RCF or whole blood filtration (WBF methods, where leukoreduction is potentially delayed in WBF. Fresh WBF red blood cells (RBCs have been associated with increased in-hospital mortality after transfusion. Cell-free DNA (cfDNA is released by neutrophils prior to leukoreduction, degraded during RBC storage, and is associated with adverse patient outcomes. We explored cfDNA levels in RBCs prepared by RCF and WBF and different storage durations. Methods. Equal numbers of fresh (stored ≤14 days and older RBCs were sampled. cfDNA was quantified by spectrophotometry and PicoGreen. Separate regression models determined the association with processing method and storage duration and their interaction on cfDNA. Results. cfDNA in 120 RBC units (73 RCF, 47 WBF were measured. Using PicoGreen, WBF units overall had higher cfDNA than RCF units (p=0.0010; fresh WBF units had higher cfDNA than fresh RCF units (p=0.0093. Using spectrophotometry, fresh RBC units overall had higher cfDNA than older units (p=0.0031; fresh WBF RBCs had higher cfDNA than older RCF RBCs (p=0.024. Conclusion. Higher cfDNA in fresh WBF was observed compared to older RCF blood. Further study is required for association with patient outcomes.

  17. High-performance liquid chromatography using UV detection for the simultaneous quantification of ropivacaine and bupivacaine in human plasma.

    Science.gov (United States)

    Gaudreault, François; Drolet, Pierre; Varin, France

    2009-12-01

    A specific high-performance liquid chromatography assay coupled with UV detection has been developed and validated for the simultaneous determination of ropivacaine and bupivacaine in human plasma. A liquid-liquid back extraction procedure was used to increase specificity, and very good and consistent recoveries were obtained: 93%-95% for ropivacaine and 90%-96% for bupivacaine. The lowest limit of quantification was 4 and 8 ng/mL for ropivacaine and bupivacaine, respectively. The method was sensitive, reproducible (coefficient of variation

  18. Thermodynamics of the DNA damage repair steps of human 8-oxoguanine DNA glycosylase.

    Directory of Open Access Journals (Sweden)

    Nikita A Kuznetsov

    Full Text Available Human 8-oxoguanine DNA glycosylase (hOGG1 is a key enzyme responsible for initiating the base excision repair of 7,8-dihydro-8-oxoguanosine (oxoG. In this study a thermodynamic analysis of the interaction of hOGG1 with specific and non-specific DNA-substrates is performed based on stopped-flow kinetic data. The standard Gibbs energies, enthalpies and entropies of specific stages of the repair process were determined via kinetic measurements over a temperature range using the van't Hoff approach. The three steps which are accompanied with changes in the DNA conformations were detected via 2-aminopurine fluorescence in the process of binding and recognition of damaged oxoG base by hOGG1. The thermodynamic analysis has demonstrated that the initial step of the DNA substrates binding is mainly governed by energy due to favorable interactions in the process of formation of the recognition contacts, which results in negative enthalpy change, as well as due to partial desolvation of the surface between the DNA and enzyme, which results in positive entropy change. Discrimination of non-specific G base versus specific oxoG base is occurring in the second step of the oxoG-substrate binding. This step requires energy consumption which is compensated by the positive entropy contribution. The third binding step is the final adjustment of the enzyme/substrate complex to achieve the catalytically competent state which is characterized by large endothermicity compensated by a significant increase of entropy originated from the dehydration of the DNA grooves.

  19. Quantitative PCR - new diagnostic tool for quantifying specific mRNA and DNA molecules: HER2/neu DNA quantification with LightCycler real-time PCR in comparison with immunohistochemistry and fluorescence in situhybridization

    DEFF Research Database (Denmark)

    Schlemmer, B.O.; Sørensen, B. S.; Overgaard, J.

    2004-01-01

    , and the treatment is considered to be justified if the tumor displays an increased amount of HER2. For this reason there is a need for techniques suitable for HER2 measurements. A LightCycler real-time PCR method used for HER2/neu DNA quantification was evaluated and the results compared with those obtained...... significant (p PCR compares with FISH and IHC. The data show...... that results obtained for amplification of HER2 by real-time PCR on the LightCycler instrument are comparable to results obtained by IHC and FISH....

  20. The DNA methylome of human peripheral blood mononuclear cells.

    Directory of Open Access Journals (Sweden)

    Yingrui Li

    Full Text Available DNA methylation plays an important role in biological processes in human health and disease. Recent technological advances allow unbiased whole-genome DNA methylation (methylome analysis to be carried out on human cells. Using whole-genome bisulfite sequencing at 24.7-fold coverage (12.3-fold per strand, we report a comprehensive (92.62% methylome and analysis of the unique sequences in human peripheral blood mononuclear cells (PBMC from the same Asian individual whose genome was deciphered in the YH project. PBMC constitute an important source for clinical blood tests world-wide. We found that 68.4% of CpG sites and 80% displayed allele-specific expression (ASE. These data demonstrate that ASM is a recurrent phenomenon and is highly correlated with ASE in human PBMCs. Together with recently reported similar studies, our study provides a comprehensive resource for future epigenomic research and confirms new sequencing technology as a paradigm for large-scale epigenomics studies.

  1. Holes influence the mutation spectrum of human mitochondrial DNA

    Science.gov (United States)

    Villagran, Martha; Miller, John

    Mutations drive evolution and disease, showing highly non-random patterns of variant frequency vs. nucleotide position. We use computational DNA hole spectroscopy [M.Y. Suarez-Villagran & J.H. Miller, Sci. Rep. 5, 13571 (2015)] to reveal sites of enhanced hole probability in selected regions of human mitochondrial DNA. A hole is a mobile site of positive charge created when an electron is removed, for example by radiation or contact with a mutagenic agent. The hole spectra are quantum mechanically computed using a two-stranded tight binding model of DNA. We observe significant correlation between spectra of hole probabilities and of genetic variation frequencies from the MITOMAP database. These results suggest that hole-enhanced mutation mechanisms exert a substantial, perhaps dominant, influence on mutation patterns in DNA. One example is where a trapped hole induces a hydrogen bond shift, known as tautomerization, which then triggers a base-pair mismatch during replication. Our results deepen overall understanding of sequence specific mutation rates, encompassing both hotspots and cold spots, which drive molecular evolution.

  2. Environmental car exhaust pollution damages human sperm chromatin and DNA.

    Science.gov (United States)

    Calogero, A E; La Vignera, S; Condorelli, R A; Perdichizzi, A; Valenti, D; Asero, P; Carbone, U; Boggia, B; De Rosa, N; Lombardi, G; D'Agata, R; Vicari, L O; Vicari, E; De Rosa, M

    2011-06-01

    The adverse role of traffic pollutants on male fertility is well known. Aim of this study was to evaluate their effects on sperm chromatin/DNA integrity. To accomplish this, 36 men working at motorway tollgates and 32 unexposed healthy men (controls) were enrolled. All of them were interviewed about their lifestyle. Hormone, semen samples, and environmental and biological markers of pollution were evaluated. Sperm chromatin and DNA integrity were evaluated by flow cytometry following propidium iodide staining and TUNEL assay, respectively. LH, FSH, and testosterone serum levels were within the normal range in tollgate workers. Sperm concentration, total sperm count, total and progressive motility, and normal forms were significantly lower in these men compared with controls. Motorway tollgate workers had a significantly higher percentage of spermatozoa with damaged chromatin and DNA fragmentation, a late sign of apoptosis, compared with controls. A significant direct correlation was found between spermatozoa with damaged chromatin or fragmented DNA and the length of occupational exposure, suggesting a time-dependent relationship. This study showed that car exhaust exposure has a genotoxic effect on human spermatozoa. This may be of relevant importance not only for the reproductive performance of the men exposed, but also for the offspring health.

  3. Genome-Wide Analysis of DNA Methylation in Human Amnion

    Directory of Open Access Journals (Sweden)

    Jinsil Kim

    2013-01-01

    Full Text Available The amnion is a specialized tissue in contact with the amniotic fluid, which is in a constantly changing state. To investigate the importance of epigenetic events in this tissue in the physiology and pathophysiology of pregnancy, we performed genome-wide DNA methylation profiling of human amnion from term (with and without labor and preterm deliveries. Using the Illumina Infinium HumanMethylation27 BeadChip, we identified genes exhibiting differential methylation associated with normal labor and preterm birth. Functional analysis of the differentially methylated genes revealed biologically relevant enriched gene sets. Bisulfite sequencing analysis of the promoter region of the oxytocin receptor (OXTR gene detected two CpG dinucleotides showing significant methylation differences among the three groups of samples. Hypermethylation of the CpG island of the solute carrier family 30 member 3 (SLC30A3 gene in preterm amnion was confirmed by methylation-specific PCR. This work provides preliminary evidence that DNA methylation changes in the amnion may be at least partially involved in the physiological process of labor and the etiology of preterm birth and suggests that DNA methylation profiles, in combination with other biological data, may provide valuable insight into the mechanisms underlying normal and pathological pregnancies.

  4. Genome-Wide Analysis of DNA Methylation in Human Amnion

    Science.gov (United States)

    Kim, Jinsil; Pitlick, Mitchell M.; Christine, Paul J.; Schaefer, Amanda R.; Saleme, Cesar; Comas, Belén; Cosentino, Viviana; Gadow, Enrique; Murray, Jeffrey C.

    2013-01-01

    The amnion is a specialized tissue in contact with the amniotic fluid, which is in a constantly changing state. To investigate the importance of epigenetic events in this tissue in the physiology and pathophysiology of pregnancy, we performed genome-wide DNA methylation profiling of human amnion from term (with and without labor) and preterm deliveries. Using the Illumina Infinium HumanMethylation27 BeadChip, we identified genes exhibiting differential methylation associated with normal labor and preterm birth. Functional analysis of the differentially methylated genes revealed biologically relevant enriched gene sets. Bisulfite sequencing analysis of the promoter region of the oxytocin receptor (OXTR) gene detected two CpG dinucleotides showing significant methylation differences among the three groups of samples. Hypermethylation of the CpG island of the solute carrier family 30 member 3 (SLC30A3) gene in preterm amnion was confirmed by methylation-specific PCR. This work provides preliminary evidence that DNA methylation changes in the amnion may be at least partially involved in the physiological process of labor and the etiology of preterm birth and suggests that DNA methylation profiles, in combination with other biological data, may provide valuable insight into the mechanisms underlying normal and pathological pregnancies. PMID:23533356

  5. Exploring the utility of human DNA methylation arrays for profiling mouse genomic DNA.

    Science.gov (United States)

    Wong, Nicholas C; Ng, Jane; Hall, Nathan E; Lunke, Sebastian; Salmanidis, Marika; Brumatti, Gabriela; Ekert, Paul G; Craig, Jeffrey M; Saffery, Richard

    2013-07-01

    Illumina Infinium Human Methylation (HM) BeadChips are widely used for measuring genome-scale DNA methylation, particularly in relation to epigenome-wide association studies (EWAS) studies. The methylation profile of human samples can be assessed accurately and reproducibly using the HM27 BeadChip (27,578 CpG sites) or its successor, the HM450 BeadChip (482,421 CpG sites). To date no mouse equivalent has been developed, greatly hindering the application of this methodology to the wide range of valuable murine models of disease and development currently in existence. We found 1308 and 13,715 probes from HM27 and HM450 BeadChip respectively, uniquely matched the bisulfite converted reference mouse genome (mm9). We demonstrate reproducible measurements of DNA methylation at these probes in a range of mouse tissue samples and in a murine cell line model of acute myeloid leukaemia. In the absence of a mouse counterpart, the Infinium Human Methylation BeadChip arrays have utility for methylation profiling in non-human species.

  6. Human Rad51 mediated DNA unwinding is facilitated by conditions that favour Rad51-dsDNA aggregation

    Directory of Open Access Journals (Sweden)

    Kulkarni Anagha

    2009-01-01

    Full Text Available Abstract Background Human Rad51 (RAD51, analogous to its bacterial homolog, RecA, binds and unwinds double stranded DNA (dsDNA in the presence of certain nucleotide cofactors. ATP hydrolysis is not required for this process, because even ATP non hydrolysable analogs like AMP-PNP and ATPγS, support DNA unwinding. Even ADP, the product of ATP hydrolysis, feebly supports DNA unwinding. Results We find that human Rad52 (RAD52 stimulates RAD51 mediated DNA unwinding in the presence of all Adenine nucleotide cofactors, (except in AMP and no nucleotide conditions that intrinsically fail to support unwinding reaction while enhancing aggregation of RAD51-dsDNA complexes in parallel. Interestingly, salt at low concentration can substitute the role of RAD52, in facilitating aggregation of RAD51-dsDNA complexes, that concomitantly also leads to better unwinding. Conclusion RAD52 itself being a highly aggregated protein perhaps acts as scaffold to bring together RAD51 and DNA molecules into large co-aggregates of RAD52-RAD51-DNA complexes to promote RAD51 mediated DNA unwinding reaction, when appropriate nucleotide cofactors are available, presumably through macromolecular crowding effects. Our work highlights the functional link between aggregation of protein-DNA complexes and DNA unwinding in RAD51 system.

  7. Evolutionarily different alphoid repeat DNA on homologous chromosomes in human and chimpanzee.

    OpenAIRE

    Jørgensen, A L; Laursen, H B; Jones, C; Bak, A L

    1992-01-01

    Centromeric alphoid DNA in primates represents a class of evolving repeat DNA. In humans, chromosomes 13 and 21 share one subfamily of alphoid DNA while chromosomes 14 and 22 share another subfamily. We show that similar pairwise homogenizations occur in the chimpanzee (Pan troglodytes), where chromosomes 14 and 22, homologous to human chromosomes 13 and 21, share one partially homogenized alphoid DNA subfamily and chromosomes 15 and 23, homologous to human chromosomes 14 and 22, share anothe...

  8. [Detection of DNA human cytomegalovirus of a molecular methods: hybrid capture DNA CMV by immunocompromised].

    Science.gov (United States)

    Mhiri, Leila; Arrouji, Zakia; Slim, Amine; Ben Redjeb, Saida

    2006-10-01

    Human cytomegalovirus (HCMV), a member of the beta-virus herpes family, is a ubiquitous human pathogen. After a primary infection, HCMV establishes life latency. HCMV rarely causes symptomatic disease in an immunocompetent host, however, it is a major cause of infectious morbidity and mortality in immunocompromised individuals and developing fetuses. The HCMV genome consists of 240 kbp of double stranded DNA. Early diagnosis molecular of CMV infection is important. The objective of this study was to develop a molecular methods: Quantitative Hybrid capture for the detection of DNA CMV. We present results for 200 immunocompromised collected from 1999 to 2003 (122 men and 78 women, whom mean age was 35 years). Our results showed that 25% of women and 36% of men were positif for hybrid capture DNA CMV. This simple test (cold probe) provide quantitative and fast results. Also the efficacity of anti-CMV therapy can be followed. More over, in contrary with pp65-antigenemia assay and CMV PCR, this test can be managed on biopsy sample.

  9. The use of dimorphic Alu insertions in human DNA fingerprinting

    Energy Technology Data Exchange (ETDEWEB)

    Novick, G.E.; Gonzalez, T.; Garrison, J.; Novick, C.C.; Herrera, R.J. [Florida International Univ., Miami, FL (United States). Dept. of Biological Sciences; Batzer, M.A. [Lawrence Livermore National Lab., CA (United States); Deininger, P.L. [Louisiana State Univ., New Orleans, LA (United States). Medical Center

    1992-12-04

    We have characterized certain Human Specific Alu Insertions as either dimorphic (TPA25, PV92, APO), sightly dimorphic (C2N4 and C4N4) or monomorphic (C3N1, C4N6, C4N2, C4N5, C4N8), based on studies of Caucasian, Asian, American Black and African Black populations. Our approach is based upon: (1) PCR amplification using primers directed to the sequences that flank the site of insertion of the different Alu elements studied; (2) gel electrophoresis and scoring according to the presence or absence of an Alu insertion in one or both homologous chromosomes; (3) allelic frequencies calculated and compared according to Hardy-Weinberg equilibrium. Our DNA fingerprinting procedure using PCR amplification of dimorphic Human Specific Alu insertions, is stable enough to be used not only as a tool for genetic mapping but also to characterize populations, study migrational patterns and track the inheritance of human genetic disorders.

  10. DNA structure in human RNA polymerase II promoters

    DEFF Research Database (Denmark)

    Pedersen, Anders Gorm; Baldi, Pierre; Chauvin, Yves

    1998-01-01

    the high-bendability regions position nucleosomes at the downstream end of the transcriptional start point, and consider the possibility of interaction between histone-like TAFs and this area. We also propose the use of this structural signature in computational promoter-finding algorithms.......The fact that DNA three-dimensional structure is important for transcriptional regulation begs the question of whether eukaryotic promoters contain general structural features independently of what genes they control. We present an analysis of a large set of human RNA polymerase II promoters...... with a very low level of sequence similarity. The sequences, which include both TATA-containing and TATA-less promoters, are aligned by hidden Markov models. Using three different models of sequence-derived DNA bendability, the aligned promoters display a common structural profile with bendability being low...

  11. Towards standardisation of cell-free DNA measurement in plasma: controls for extraction efficiency, fragment size bias and quantification.

    Science.gov (United States)

    Devonshire, Alison S; Whale, Alexandra S; Gutteridge, Alice; Jones, Gerwyn; Cowen, Simon; Foy, Carole A; Huggett, Jim F

    2014-10-01

    Circulating cell-free DNA (cfDNA) is becoming an important clinical analyte for prenatal testing, cancer diagnosis and cancer monitoring. The extraction stage is critical in ensuring clinical sensitivity of analytical methods measuring minority nucleic acid fractions, such as foetal-derived sequences in predominantly maternal cfDNA. Consequently, quality controls are required for measurement of extraction efficiency, fragment size bias and yield for validation of cfDNA methods. We evaluated the utility of an external DNA spike for monitoring these parameters in a study comparing three specific cfDNA extraction methods [QIAamp circulating nucleic acid (CNA) kit, NucleoSpin Plasma XS (NS) kit and FitAmp plasma/serum DNA isolation (FA) kit] with the commonly used QIAamp DNA blood mini (DBM) kit. We found that the extraction efficiencies of the kits ranked in the order CNA kit > DBM kit > NS kit > FA kit, and the CNA and NS kits gave a better representation of smaller DNA fragments in the extract than the DBM kit. We investigated means of improved reporting of cfDNA yield by comparing quantitative PCR measurements of seven different reference gene assays in plasma samples and validating these with digital PCR. We noted that the cfDNA quantities based on measurement of some target genes (e.g. TERT) were, on average, more than twofold higher than those of other assays (e.g. ERV3). We conclude that analysis and averaging of multiple reference genes using a GeNorm approach gives a more reliable estimate of total cfDNA quantity.

  12. Antibody-free quantification of seven tau peptides in human CSF using targeted mass spectrometry

    Directory of Open Access Journals (Sweden)

    Pauline eBros

    2015-09-01

    Full Text Available Tau protein concentration in cerebrospinal fluid (CSF is currently used as a sensitive and specific biomarker for Alzheimer's disease. Its detection currently relies on ELISA but the perspective of using mass spectrometry (MS to detect its different proteoforms represents an interesting alternative. This is however an analytical challenge because of its low concentration in the CSF, a biological fluid collected in small volume by lumbar puncture, and with a high structural heterogeneity. To overcome these issues, instead of using immunocapture as previously done, we rather relied on an original two steps pre-fractionation technique of CSF: perchloric acid followed by micro solid phase extraction (µSPE. We could then measure seven tau trypsic peptides by Multiple Reaction Monitoring (MRM on a triple quadrupole mass spectrometer. Quantification was performed using isotopically labelled 15N- recombinant Tau protein as internal standard and validated using CSF pools with low, medium or high tau concentrations. Repeatability, intermediate precision, linearity, limit of quantification and recovery were calculated for the different peptides. This new MRM assay, which allowed for the first time CSF tau protein quantification without immunocapture, has important potential application to follow tau metabolism in both diagnostic and therapeutic research.

  13. Antibody-free quantification of seven tau peptides in human CSF using targeted mass spectrometry.

    Science.gov (United States)

    Bros, Pauline; Vialaret, Jérôme; Barthelemy, Nicolas; Delatour, Vincent; Gabelle, Audrey; Lehmann, Sylvain; Hirtz, Christophe

    2015-01-01

    Tau protein concentration in cerebrospinal fluid (CSF) is currently used as a sensitive and specific biomarker for Alzheimer's disease. Its detection currently relies on ELISA but the perspective of using mass spectrometry (MS) to detect its different proteoforms represents an interesting alternative. This is however an analytical challenge because of its low concentration in the CSF, a biological fluid collected in small volume by lumbar puncture, and with a high structural heterogeneity. To overcome these issues, instead of using immunocapture as previously done, we rather relied on an original two steps pre-fractionation technique of CSF: perchloric acid (PCA) followed by micro solid phase extraction (μSPE). We could then measure seven tau trypsic peptides by Multiple Reaction Monitoring (MRM) on a triple quadrupole mass spectrometer. Quantification was performed using isotopically labeled (15)N- recombinant tau protein as internal standard and validated using CSF pools with low, medium, or high tau concentrations (HTCs). Repeatability, intermediate precision, linearity, limit of quantification (LOQ), and recovery were calculated for the different peptides. This new MRM assay, which allowed for the first time CSF tau protein quantification without immunocapture, has important potential application to follow tau metabolism in both diagnostic and therapeutic research.

  14. Quantification of DNA strand breaks and abasic sites by oxime derivatization and accelerator mass spectrometry: application to gamma-radiation and peroxynitrite.

    Science.gov (United States)

    Zhou, Xinfeng; Liberman, Rosa G; Skipper, Paul L; Margolin, Yelena; Tannenbaum, Steven R; Dedon, Peter C

    2005-08-01

    We report a highly sensitive method to quantify abasic sites and deoxyribose oxidation products arising in damaged DNA. The method exploits the reaction of aldehyde- and ketone-containing deoxyribose oxidation products and abasic sites with [(14)C]methoxyamine to form stable oxime derivatives, as originally described by Talpaert-Borle and Liuzzi [Reaction of apurinic/apyrimidinic sites with [(14)C]methoxyamine. A method for the quantitative assay of AP sites in DNA, Biochim. Biophys. Acta 740 (1983) 410-416]. The sensitivity of the method was dramatically improved by the application of accelerator mass spectrometry to quantify the (14)C, with a limit of detection of 1 lesion in 10(6) nucleotides in 1 microg of DNA. The method was validated using DNA containing a defined quantity of abasic sites, with a >0.95 correlation between the quantities of abasic sites and those of methoxyamine labels. The original applications of this and similar oxyamine derivatization methods have assumed that abasic sites are the only aldehyde-containing DNA damage products. However, deoxyribose oxidation produces strand breaks and abasic sites containing a variety of degradation products with aldehyde and ketone moieties. To assess the utility of methoxyamine labeling for quantifying strand breaks and abasic sites, the method was applied to plasmid DNA treated with gamma-radiation and peroxynitrite. For gamma-radiation, there was a 0.99 correlation between the quantity of methoxyamine labels and the quantity of strand breaks and abasic sites determined by a plasmid nicking assay; the abasic sites comprised less than 10% of the radiation-induced DNA damage. Studies with peroxynitrite demonstrate that the method, in conjunction with DNA repair enzymes that remove damaged bases to produce aldehydic sugar residues or abasic sites, is also applicable to quantifying nucleobase lesions in addition to strand break products. Compared to other abasic site quantification techniques, the modified

  15. Quantitative PCR - new diagnostic tool for quantifying specific mRNA and DNA molecules: HER2/neu DNA quantification with LightCycler real-time PCR in comparison with immunohistochemistry and fluorescence in situhybridization

    DEFF Research Database (Denmark)

    Schlemmer, B.O.; Sørensen, B. S.; Overgaard, J.

    2004-01-01

    Previously, polymerase chain reaction (PCR) technology has been hampered by its inability to generate quantitative results, a drawback inherent to the high degree of amplification taking place in the reaction. Recently, PCR techniques have been described with the potential of quantifying the amount...... of mRNA or DNA in biological samples. In this study quantitative PCR was used to investigate the role of the EGF (epidermal growth factor) system in cancer both for measurements of mRNA concentrations and for measurements of the number of copies of specific genes. It is shown that the mRNA expression......, and the treatment is considered to be justified if the tumor displays an increased amount of HER2. For this reason there is a need for techniques suitable for HER2 measurements. A LightCycler real-time PCR method used for HER2/neu DNA quantification was evaluated and the results compared with those obtained...

  16. Robotics for recombinant DNA and human genetics research

    Energy Technology Data Exchange (ETDEWEB)

    Beugelsdijk, T.J.

    1990-01-01

    In October of 1989, molecular biologists throughout the world formally embarked on ultimately determining the set of genetic instructions for a human being. Called by some the Manhattan Project'' a molecular biology, pursuit of this goal is projected to require approximately 3000 man years of effort over a 15-year period. The Humane Genome Initiative is a worldwide research effort that has the goal of analyzing the structure of human deoxyribonucleic acid (DNA) and determining the location of all human genes. The Department of Energy (DOE) has designated three of its national laboratories as centers for the Human Genome Project. These are Los Alamos National Laboratory (LANL), Lawrence Livermore National Laboratory (LLNL), and Lawrence Berkeley Laboratory (LBL). These laboratories are currently working on different, but complementary technology development areas in support of the Human Genome Project. The robotics group at LANL is currently working at developing the technologies that address the problems associated with physical mapping. This article describes some of these problems and discusses some of the robotics approaches and engineering tolls applicable to their solution. 7 refs., 8 figs., 1 tab.

  17. Development and evaluation of a sandwich ELISA for quantification of the 20S proteasome in human plasma

    DEFF Research Database (Denmark)

    Dutaud, Dominique; Aubry, Laurent; Henry, Laurent

    2002-01-01

    Because quantification of the 20S proteasome by functional activity measurements is difficult and inaccurate, we have developed an indirect sandwich enzyme-linked immunosorbent assays (ELISA) for quantification of the 20S proteasome in human plasma. This sandwich ELISA uses a combination...... of a monoclonal antibody (mcp 20) recognizing the C2-beta subunit of human 20S proteasome (Mr˜30,000) and a polyclonal rabbit anti-20S antibody which labels different subunits of the complex. The detection limit of the assay was established as 10 ng/ml (n=10, mean of zero standard+2 S.D.) and the recovery rate...... ranged from 96% to 104%. The within-run and between-run coefficients of variation (CV) ranges were 2.8–3.3 and 3.0–3.4, respectively. Using serial dilutions of plasma to which various amounts of purified 20S proteasome were added, a linear dose–response was observed between 102 and 2050 ng...

  18. Detection and validated quantification of 21 benzodiazepines and 3 "z-drugs" in human hair by LC-MS/MS.

    Science.gov (United States)

    Rust, Kristina Y; Baumgartner, Markus R; Meggiolaro, Natascha; Kraemer, Thomas

    2012-02-10

    A method for detection and quantification of 21 benzodiazepines and the pharmacologically related "z-drugs" in human hair samples was developed and fully validated using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). After methanolic and methanolic/aqueous extraction, the analytes were separated using two different LC-MS systems (AB Sciex 3200 QTRAP and AB Sciex 5500 QTRAP). Separation columns, mobile phases and MS modes for both systems were: Phenomenex Kinetex, 2.6 μm, 50/2.1; 5mM ammonium formate buffer pH 3.5/methanol, total flow 0.75 mL/min; electrospray ionization (ESI), multiple reaction monitoring (MRM), information dependent acquisition (IDA), enhanced product ion scan (EPI). The assays were found to be selective for the tested compounds (alprazolam, 7-aminoclonazepam, 7-aminoflunitrazepam, bromazepam, chlordiazepoxide, clonazepam, N-desalkylflurazepam, diazepam, flunitrazepam, flurazepam, alpha-hydroxymidazolam, lorazepam, lormetazepam, midazolam, nitrazepam, nordazepam, oxazepam, phenazepam, prazepam, temazepam, triazolam, zaleplon, zolpidem and zopiclone), all validation criteria were in the required ranges according to international guidelines, except for bromazepam. Matrix effects, and process efficiencies were in the acceptable ranges evaluated using the post-extraction addition approach. Lower limits of quantification were between 0.6 and 16 pg/mg of hair. The LC-MS/MS assay has proven to be applicable for determination of the studied analytes in human hair in numerous authentic cases (n=175). Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  19. Quantification of 5-HT{sub 1A} receptors in human brain using p-MPPF kinetic modelling and PET

    Energy Technology Data Exchange (ETDEWEB)

    Sanabria-Bohorquez, S.M.; Veraart, C. [Neural Rehabilitation Engineering Lab., Univ. Catholique de Louvain, Brussels (Belgium); Biver, F.; Damhaut, P.; Wikler, D.; Goldman, S. [PET/Biomedical Cyclotron Unit, Univ. Libre de Bruxelles (Belgium)

    2002-01-01

    Serotonin-1A (5-HT{sub 1A}) receptors are implicated in neurochemical mechanisms underlying anxiety and depression and their treatment. Animal studies have suggested that 4-(2'-methoxyphenyl)-1-[2'-[N-(2''-pyridinyl)-p-[{sup 18}F]fluorobenzamido] ethyl] piperazine (p-MPPF) may be a suitable positron emission tomography (PET) tracer of 5-HT{sub 1A} receptors. To test p-MPPF in humans, we performed 60-min dynamic PET scans in 13 healthy volunteers after single bolus injection. Metabolite quantification revealed a fast decrease in tracer plasma concentration, such that at 5 min post injection about 25% of the total radioactivity in plasma corresponded to p-MPPF. Radioactivity concentration was highest in hippocampus, intermediate in neocortex and lowest in basal ganglia and cerebellum. The interactions between p-MPPF and 5-HT{sub 1A} receptors were described using linear compartmental models with plasma input and reference tissue approaches. The two quantification methods provided similar results which are in agreement with previous reports on 5-HT{sub 1A} receptor brain distribution. In conclusion, our results show that p-MPPF is a suitable PET radioligand for 5-HT{sub 1A} receptor human studies. (orig.)

  20. Hydroxytyrosol Protects against Oxidative DNA Damage in Human Breast Cells

    Directory of Open Access Journals (Sweden)

    José J. Gaforio

    2011-10-01

    Full Text Available Over recent years, several studies have related olive oil ingestion to a low incidence of several diseases, including breast cancer. Hydroxytyrosol and tyrosol are two of the major phenols present in virgin olive oils. Despite the fact that they have been linked to cancer prevention, there is no evidence that clarifies their effect in human breast tumor and non-tumor cells. In the present work, we present hydroxytyrosol and tyrosol’s effects in human breast cell lines. Our results show that hydroxytyrosol acts as a more efficient free radical scavenger than tyrosol, but both fail to affect cell proliferation rates, cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A or breast cancer cells (MDA-MB-231 and MCF7. We found that hydroxytyrosol decreases the intracellular reactive oxygen species (ROS level in MCF10A cells but not in MCF7 or MDA-MB-231 cells while very high amounts of tyrosol is needed to decrease the ROS level in MCF10A cells. Interestingly, hydroxytyrosol prevents oxidative DNA damage in the three breast cell lines. Therefore, our data suggest that simple phenol hydroxytyrosol could contribute to a lower incidence of breast cancer in populations that consume virgin olive oil due to its antioxidant activity and its protection against oxidative DNA damage in mammary cells.

  1. Hydroxytyrosol protects against oxidative DNA damage in human breast cells.

    Science.gov (United States)

    Warleta, Fernando; Quesada, Cristina Sánchez; Campos, María; Allouche, Yosra; Beltrán, Gabriel; Gaforio, José J

    2011-10-01

    Over recent years, several studies have related olive oil ingestion to a low incidence of several diseases, including breast cancer. Hydroxytyrosol and tyrosol are two of the major phenols present in virgin olive oils. Despite the fact that they have been linked to cancer prevention, there is no evidence that clarifies their effect in human breast tumor and non-tumor cells. In the present work, we present hydroxytyrosol and tyrosol's effects in human breast cell lines. Our results show that hydroxytyrosol acts as a more efficient free radical scavenger than tyrosol, but both fail to affect cell proliferation rates, cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A) or breast cancer cells (MDA-MB-231 and MCF7). We found that hydroxytyrosol decreases the intracellular reactive oxygen species (ROS) level in MCF10A cells but not in MCF7 or MDA-MB-231 cells while very high amounts of tyrosol is needed to decrease the ROS level in MCF10A cells. Interestingly, hydroxytyrosol prevents oxidative DNA damage in the three breast cell lines. Therefore, our data suggest that simple phenol hydroxytyrosol could contribute to a lower incidence of breast cancer in populations that consume virgin olive oil due to its antioxidant activity and its protection against oxidative DNA damage in mammary cells.

  2. Comparison of methods for accurate quantification of DNA mass concentration with traceability to the international system of units.

    Science.gov (United States)

    Bhat, Somanath; Curach, Natalie; Mostyn, Thomas; Bains, Gursharan Singh; Griffiths, Kate R; Emslie, Kerry R

    2010-09-01

    Accurate estimation of total DNA concentration (mass concentration, e.g., ng/muL) that is traceable to the International System of Units (SI) is a crucial starting point for improving reproducible measurements in many applications involving nucleic acid testing and requires a DNA reference material which has been certified for its total DNA concentration. In this study, the concentrations of six different lambda DNA preparations were determined using different measurement platforms: UV Absorbance at 260 nm (A(260)) with and without prior sodium hydroxide (NaOH) treatment of the DNA, PicoGreen assay, and digital polymerase chain reaction (dPCR). DNA concentration estimates by A(260) with and without prior NaOH treatment were significantly different for five of the six samples tested. There were no significant differences in concentration estimates based on A(260) with prior NaOH treatment, PicoGreen analysis, and dPCR for two of the three samples tested using dPCR. Since the measurand in dPCR is amount (copy number) concentration (copies/muL), the results suggest that accurate estimation of DNA mass concentration based on copy number concentration is achievable provided the DNA is fully characterized and in the double-stranded form or amplification is designed to be initiated from only one of the two complementary strands.

  3. 基于数字PCR的单分子DNA定量技术研究进展%Progress of Digital PCR for Single DNA Quantification

    Institute of Scientific and Technical Information of China (English)

    李亮; 隋志伟; 王晶; 臧超; 余笑波

    2012-01-01

    数字PCR是一项针对单分子目标DNA的绝对定量技术.该技术是将含有DNA模板的反应溶液分配到大量独立的反应室中并且发生扩增反应,通过统计反应室中的阳性信号来定量DNA的拷贝数.DNA样品在反应室中随机和独立分布是单分子成功扩增和准确定量DNA拷贝数的关键因素.本文综述了数字PCR的发展历史、数字PCR与实时荧光定量PCR的区别,以及数字PCR在临床诊断、转基因成分定量、单细胞基因表达、环境微生物检测和下一代测序等方面的最新进展,并展望了该技术的应用前景.%Digital PCR is an absolute DNA quantification technique to determine the copy number of target DNA. PCR reaction solution is divided into aliquot throughout numerous partitions and amplifies independently. The copy number of target DNA is estimated by the statistical analysis of positive signals. Random and independent distribution of the target DNA molecules throughout the partitions of the digital panel and successful amplification from single molecules are critical to the validity of this approach for estimating target DNA copy number. In this review, we discuss the advances in development of digital PCR, the differences between digital PCR and real time quantitative PCR. We further show the advances in application of digital PCR in the clinical diagnosis, GMO analysis, single cell expression, environmental microbiology and next generation sequencing. The prospects for the applications of digital PCR are discussed.

  4. Molecular diagnosis of canine visceral leishmaniasis: identification of Leishmania species by PCR-RFLP and quantification of parasite DNA by real-time PCR.

    Science.gov (United States)

    Quaresma, Patrícia Flávia; Murta, Silvane Maria Fonseca; Ferreira, Eduardo de Castro; da Rocha-Lima, Ana Cristina Vianna Mariano; Xavier, Ana Amélia Prates; Gontijo, Célia Maria Ferreira

    2009-09-01

    The efficacies of polymerase chain reaction (PCR) procedures for the diagnosis of canine visceral leishmaniasis (CVL), and of PCR-restriction fragment length polymorphism (RFLP) analysis for the identification of Leishmania species, have been assessed. Quantitative real-time PCR employing a SYBR Green dye-based system was standardised for the quantification of Leishmania kDNA minicircles. Skin, peripheral blood and bone marrow samples collected from 217 dogs, asymptomatic or symptomatic for CVL, were analysed. The PCR method, which was based on the amplification of a 120 bp kDNA fragment conserved across Leishmania species, was able to detect the presence in clinical samples of protozoan parasite DNA in amounts as low as 0.1 fg. Bone marrow and skin samples proved to be more suitable than peripheral blood for the detection of Leishmania by PCR and presented positive indices of 84.9% and 80.2%, respectively. PCR-RFLP analysis indicated that 192 of the PCR-positive dogs were infected with Leishmania infantum chagasi, whilst L. braziliensis was identified in two other animals. Quantitative PCR revealed that bone marrow samples from dogs presenting positive conventional tests contained a higher number of copies of Leishmania kDNA than peripheral blood, although no significant differences were detected between symptomatic and asymptomatic dogs in terms of parasite load. This study demonstrates that PCR can be used for the detection of Leishmania in clinical samples derived from naturally infected dogs, and that PCR-RFLP represents a rapid and sensitive tool for the identification of Leishmania species. Additionally, qPCR is effective in quantifying Leishmania DNA load in clinical samples.

  5. Quantification of vitamin B6 vitamers in human cerebrospinal fluid by ultra performance liquid chromatography-tandem mass spectrometry

    Energy Technology Data Exchange (ETDEWEB)

    Ham, M. van der, E-mail: M.vanderHam-3@umcutrecht.nl [Department of Metabolic Diseases and Netherlands Metabolomics Center, University Medical Center (UMC) Utrecht, Huispost KC02.069.1, Lundlaan 6, 3584 EA Utrecht (Netherlands); Albersen, M., E-mail: M.Albersen@umcutrecht.nl [Department of Metabolic Diseases and Netherlands Metabolomics Center, University Medical Center (UMC) Utrecht, Huispost KC02.069.1, Lundlaan 6, 3584 EA Utrecht (Netherlands); Koning, T.J. de, E-mail: T.deKoning@umcutrecht.nl [Department of Pediatric Metabolic Diseases, Wilhelmina Children' s Hospital, University Medical Center (UMC) Utrecht, Huispost KC03.063.0, Lundlaan 6, 3584 EA Utrecht (Netherlands); Visser, G., E-mail: G.Visser-4@umcutrecht.nl [Department of Pediatric Metabolic Diseases, Wilhelmina Children' s Hospital, University Medical Center (UMC) Utrecht, Huispost KC03.063.0, Lundlaan 6, 3584 EA Utrecht (Netherlands); Middendorp, A., E-mail: Alfred_Middendorp@waters.com [Waters Chromatography B.V., Florijnstraat 19, Postbus 379, 4870 AJ Etten-Leur (Netherlands); Bosma, M., E-mail: M.Bosma@umcutrecht.nl [Department of Metabolic Diseases and Netherlands Metabolomics Center, University Medical Center (UMC) Utrecht, Huispost KC02.069.1, Lundlaan 6, 3584 EA Utrecht (Netherlands); Verhoeven-Duif, N.M., E-mail: N.Verhoeven@umcutrecht.nl [Department of Metabolic Diseases and Netherlands Metabolomics Center, University Medical Center (UMC) Utrecht, Huispost KC02.069.1, Lundlaan 6, 3584 EA Utrecht (Netherlands); Sain-van der Velden, M.G.M. de, E-mail: M.G.deSain@umcutrecht.nl [Department of Metabolic Diseases and Netherlands Metabolomics Center, University Medical Center (UMC) Utrecht, Huispost KC02.069.1, Lundlaan 6, 3584 EA Utrecht (Netherlands)

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer We present a sensitive UPLC-MS/MS method for quantification of B6 vitamers in human CSF. Black-Right-Pointing-Pointer Our method is very accurate since stable isotope labeled internal standards are used. Black-Right-Pointing-Pointer We present data on light sensitivity, temperature dependence and rostrocaudal gradient. Black-Right-Pointing-Pointer With PN supplementation, concentrations of PL, PM, PN and PA in CSF are increased. Black-Right-Pointing-Pointer Our fully validated method is suitable for implementation in a diagnostic setting. - Abstract: Since vitamin B6 is essential for normal functioning of the central nervous system, there is growing need for sensitive analysis of B6 vitamers in cerebrospinal fluid (CSF). This manuscript describes the development and validation of a rapid, sensitive and accurate method for quantification of the vitamin B6 vitamers pyridoxal (PL), pyridoxamine (PM), pyridoxine (PN), pyridoxic acid (PA), pyridoxal 5 Prime -phosphate (PLP), pyridoxamine 5 Prime -phosphate (PMP) and pyridoxine 5 Prime -phosphate (PNP) in human CSF. The method is based on ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with a simple sample preparation procedure of protein precipitation using 50 g L{sup -1} trichloroacetic acid containing stable isotope labeled internal standards: PL-D{sub 3} for PL and PM, PN-{sup 13}C{sub 4} for PN, PA-D{sub 2} for PA and PLP-D{sub 3} for the phosphorylated vitamers. B6 vitamers were separated (Acquity HSS-T3 UPLC column) with a buffer containing acetic acid, heptafluorobutyric acid and acetonitrile. Positive electrospray ionization was used to monitor transitions m/z 168.1 {yields} 150.1 (PL), 169.1 {yields} 134.1 (PM), 170.1 {yields} 134.1 (PN), 184.1 {yields} 148.1 (PA), 248.1 {yields} 150.1 (PLP), 249.1 {yields} 232.1 (PMP) and 250.1 {yields} 134.1 (PNP). The method was validated at three concentration levels for each B6 vitamer in CSF

  6. Performance Evaluation of the Real-Q Cytomegalovirus (CMV) Quantification Kit Using Two Real-Time PCR Systems for Quantifying CMV DNA in Whole Blood.

    Science.gov (United States)

    Park, Jong Eun; Kim, Ji Youn; Yun, Sun Ae; Lee, Myoung Keun; Huh, Hee Jae; Kim, Jong Won; Ki, Chang Seok

    2016-11-01

    Standardized cytomegalovirus (CMV) DNA quantification is important for managing CMV disease. We evaluated the performance of the Real-Q CMV Quantification Kit (Real-Q assay; BioSewoom, Korea) using whole blood (WB), with nucleic acid extraction using MagNA Pure 96 (Roche Diagnostics, Germany). Real-time PCR was performed on two platforms: the 7500 Fast real-time PCR (7500 Fast; Applied Biosystems, USA) and CFX96 real-time PCR detection (CFX96; Bio-Rad, USA) systems. The WHO international standard, diluted with CMV-negative WB, was used to validate the analytical performance. We used 90 WB clinical samples for comparison with the artus CMV RG PCR kit (artus assay; Qiagen, Germany). Limits of detections (LODs) in 7500 Fast and CFX96 were 367 and 479 IU/mL, respectively. The assay was linear from the LOD to 10⁶ IU/mL (R² ≥0.9886). The conversion factors from copies to IU in 7500 Fast and CFX96 were 0.95 and 1.06, respectively. Compared with the artus assay, for values 1,000 copies/mL, 73.3% and 80.6% of samples in 7500 Fast and CFX96, respectively, had real-time PCR platforms.

  7. Base composition at mtDNA boundaries suggests a DNA triple helix model for human mitochondrial DNA large-scale rearrangements.

    Science.gov (United States)

    Rocher, Christophe; Letellier, Thierry; Copeland, William C; Lestienne, Patrick

    2002-06-01

    Different mechanisms have been proposed to account for mitochondrial DNA (mtDNA) instability based on the presence of short homologous sequences (direct repeats, DR) at the potential boundaries of mtDNA rearrangements. Among them, slippage-mispairing of the replication complex during the asymmetric replication cycle of the mammalian mitochondrial DNA has been proposed to account for the preferential localization of deletions. This mechanism involves a transfer of the replication complex from the first neo-synthesized heavy (H) strand of the DR1, to the DR2, thus bypassing the intervening sequence and producing a deleted molecule. Nevertheless, the nature of the bonds between the DNA strands remains unknown as the forward sequence of DR2, beyond the replication complex, stays double-stranded. Here, we have analyzed the base composition of the DR at the boundaries of mtDNA deletions and duplications and found a skewed pyrimidine content of about 75% in the light-strand DNA template. This suggests the possible building of a DNA triple helix between the G-rich neo-synthesized DR1 and the base-paired homologous G.C-rich DR2. In vitro experiments with the purified human DNA polymerase gamma subunits enabled us to show that the third DNA strand may be used as a primer for DNA replication, using a template with the direct repeat forming a hairpin, with which the primer could initiate DNA replication. These data suggest a novel molecular basis for mitochondrial DNA rearrangements through the distributive nature of the DNA polymerase gamma, at the level of the direct repeats. A general model accounting for large-scale mitochondrial DNA deletion and duplication is proposed. These experiments extend to a DNA polymerase from an eucaryote source the use of a DNA triple helix strand as a primer, like other DNA polymerases from phage and bacterial origins.

  8. The effect of chronic alcohol consumption on mitochondrial DNA mutagenesis in human blood.

    Science.gov (United States)

    von Wurmb-Schwark, N; Ringleb, A; Schwark, T; Broese, T; Weirich, S; Schlaefke, D; Wegener, R; Oehmichen, M

    2008-01-01

    The 4977bp deletion of mitochondrial DNA (mtDNA) is known to accumulate with increasing age in post mitotic tissues. Recently, studies came out detecting this specific alteration also in fast replicating cells, e.g. in blood or skin tissue, often in correlation to specific diseases or -- specifically in skin -- external stressors such as UV radiation. In this study, we investigated mitochondrial mutagenesis in 69 patients with a chronic alcoholic disease and 46 age matched controls with a moderate drinking behavior. Two different fragments, specific for total and for deleted mtDNA (dmtDNA) were amplified in a duplex-PCR. A subsequent fragment analysis was performed and for relative quantification, the quotient of the peak areas of amplification products specific for deleted and total mtDNA was determined. Additionally, a real time PCR was performed to quantify mtDNA copy number. The relative amount of 4977bp deleted mtDNA in alcoholics was significantly increased compared to controls. On the other hand, no difference regarding the mtDNA/nuclear DNA ratio in both investigated groups was detected. Additionally, no age dependence could be found nor in alcoholics, neither in the control group. These findings indicate that mtDNA mutagenesis in blood can be influenced by stressors such as alcohol. Ethanol seems to be a significant factor to alter mitochondrial DNA in blood and might be an additional contributor for the cellular aging process.

  9. The effect of chronic alcohol consumption on mitochondrial DNA mutagenesis in human blood

    Energy Technology Data Exchange (ETDEWEB)

    Wurmb-Schwark, N. von [Institute of Legal Medicine, Christian Albrecht University of Kiel, Arnold-Heller-Str. 12, 24105 Kiel (Germany)], E-mail: nvonwurmb@rechtsmedizin.uni-kiel.de; Ringleb, A.; Schwark, T. [Institute of Legal Medicine, Christian Albrecht University of Kiel, Arnold-Heller-Str. 12, 24105 Kiel (Germany); Broese, T.; Weirich, S.; Schlaefke, D. [Clinic of Psychiatry and Psychotherapy, University of Rostock, Gehlsheimer Str. 20, Rostock (Germany); Wegener, R. [Institute of Legal Medicine, St-Georg-Str. 108, University of Rostock, 18055 Rostock (Germany); Oehmichen, M. [Institute of Legal Medicine, Christian Albrecht University of Kiel, Arnold-Heller-Str. 12, 24105 Kiel (Germany)

    2008-01-01

    The 4977 bp deletion of mitochondrial DNA (mtDNA) is known to accumulate with increasing age in post mitotic tissues. Recently, studies came out detecting this specific alteration also in fast replicating cells, e.g. in blood or skin tissue, often in correlation to specific diseases or - specifically in skin - external stressors such as UV radiation. In this study, we investigated mitochondrial mutagenesis in 69 patients with a chronic alcoholic disease and 46 age matched controls with a moderate drinking behavior. Two different fragments, specific for total and for deleted mtDNA (dmtDNA) were amplified in a duplex-PCR. A subsequent fragment analysis was performed and for relative quantification, the quotient of the peak areas of amplification products specific for deleted and total mtDNA was determined. Additionally, a real time PCR was performed to quantify mtDNA copy number. The relative amount of 4977 bp deleted mtDNA in alcoholics was significantly increased compared to controls. On the other hand, no difference regarding the mtDNA/nuclear DNA ratio in both investigated groups was detected. Additionally, no age dependence could be found nor in alcoholics, neither in the control group. These findings indicate that mtDNA mutagenesis in blood can be influenced by stressors such as alcohol. Ethanol seems to be a significant factor to alter mitochondrial DNA in blood and might be an additional contributor for the cellular aging process.

  10. DNA Sequences Proximal to Human Mitochondrial DNA Deletion Breakpoints Prevalent in Human Disease Form G-quadruplexes, a Class of DNA Structures Inefficiently Unwound by the Mitochondrial Replicative Twinkle Helicase

    NARCIS (Netherlands)

    Bharti, S.K.; Sommers, J.A.; Zhou, J.; Kaplan, D.L.; Spelbrink, J.N.; Mergny, J.L.; Brosh, R.M., Jr.

    2014-01-01

    Mitochondrial DNA deletions are prominent in human genetic disorders, cancer, and aging. It is thought that stalling of the mitochondrial replication machinery during DNA synthesis is a prominent source of mitochondrial genome instability; however, the precise molecular determinants of defective

  11. Analytical Performance of a Multiplex Real-Time PCR Assay Using TaqMan Probes for Quantification of Trypanosoma cruzi Satellite DNA in Blood Samples

    Science.gov (United States)

    Abate, Teresa; Cayo, Nelly M.; Parrado, Rudy; Bello, Zoraida Diaz; Velazquez, Elsa; Muñoz-Calderon, Arturo; Juiz, Natalia A.; Basile, Joaquín; Garcia, Lineth; Riarte, Adelina; Nasser, Julio R.; Ocampo, Susana B.; Yadon, Zaida E.; Torrico, Faustino; de Noya, Belkisyole Alarcón; Ribeiro, Isabela; Schijman, Alejandro G.

    2013-01-01

    Background The analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy. Methods/Principal Findings We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR) based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC) in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD) of 0.70 parasite equivalents/mL and a limit of quantification (LOQ) of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CL-Brener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject. Conclusions/Significance The performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment. PMID:23350002

  12. Analytical performance of a multiplex Real-Time PCR assay using TaqMan probes for quantification of Trypanosoma cruzi satellite DNA in blood samples.

    Directory of Open Access Journals (Sweden)

    Tomas Duffy

    Full Text Available BACKGROUND: The analytical validation of sensitive, accurate and standardized Real-Time PCR methods for Trypanosoma cruzi quantification is crucial to provide a reliable laboratory tool for diagnosis of recent infections as well as for monitoring treatment efficacy. METHODS/PRINCIPAL FINDINGS: We have standardized and validated a multiplex Real-Time quantitative PCR assay (qPCR based on TaqMan technology, aiming to quantify T. cruzi satellite DNA as well as an internal amplification control (IAC in a single-tube reaction. IAC amplification allows rule out false negative PCR results due to inhibitory substances or loss of DNA during sample processing. The assay has a limit of detection (LOD of 0.70 parasite equivalents/mL and a limit of quantification (LOQ of 1.53 parasite equivalents/mL starting from non-boiled Guanidine EDTA blood spiked with T. cruzi CL-Brener stock. The method was evaluated with blood samples collected from Chagas disease patients experiencing different clinical stages and epidemiological scenarios: 1- Sixteen Venezuelan patients from an outbreak of oral transmission, 2- Sixty three Bolivian patients suffering chronic Chagas disease, 3- Thirty four Argentinean cases with chronic Chagas disease, 4- Twenty seven newborns to seropositive mothers, 5- A seronegative receptor who got infected after transplantation with a cadaveric kidney explanted from an infected subject. CONCLUSIONS/SIGNIFICANCE: The performing parameters of this assay encourage its application to early assessment of T. cruzi infection in cases in which serological methods are not informative, such as recent infections by oral contamination or congenital transmission or after transplantation with organs from seropositive donors, as well as for monitoring Chagas disease patients under etiological treatment.

  13. Quantification of resting myocardial blood flow velocity in normal humans using real-time contrast echocardiography. A feasibility study

    Directory of Open Access Journals (Sweden)

    Slordahl Stig

    2005-06-01

    Full Text Available Abstract Background Real-time myocardial contrast echocardiography (MCE is a novel method for assessing myocardial perfusion. The aim of this study was to evaluate the feasibility of a very low-power real-time MCE for quantification of regional resting myocardial blood flow (MBF velocity in normal human myocardium. Methods Twenty study subjects with normal left ventricular (LV wall motion and normal coronary arteries, underwent low-power real-time MCE based on color-coded pulse inversion Doppler. Standard apical LV views were acquired during constant IV. infusion of SonoVue®. Following transient microbubble destruction, the contrast replenishment rate (β, reflecting MBF velocity, was derived by plotting signal intensity vs. time and fitting data to the exponential function; y (t =A (1-e-β(t-t0 + C. Results Quantification was feasible in 82%, 49% and 63% of four-chamber, two-chamber and apical long-axis view segments, respectively. The LAD (left anterior descending artery and RCA (right coronary artery territories could potentially be evaluated in most, but contrast detection in the LCx (left circumflex artery bed was poor. Depending on localisation and which frames to be analysed, mean values of were 0.21–0.69 s-1, with higher values in medial than lateral, and in basal compared to apical regions of scan plane (p = 0.03 and p Conclusion Low-power real-time MCE did have the potential to give contrast enhancement for quantification of resting regional MBF velocity. However, the technique is difficult and subjected to several limitations. Significant variability in β suggests that this parameter is best suited for with-in patient changes, comparing values of stress studies to baseline.

  14. Droplet digital PCR for simultaneous quantification of general and human-associated fecal indicators for water quality assessment.

    Science.gov (United States)

    Cao, Yiping; Raith, Meredith R; Griffith, John F

    2015-03-01

    Despite wide application to beach water monitoring and microbial source identification, results produced by quantitative PCR (qPCR) methods are subject to bias introduced by reliance on quantitative standards. Digital PCR technology provides direct, standards-free quantification and may potentially alleviate or greatly reduce other qPCR limitations such as difficulty in multiplexing and susceptibility to PCR inhibition. This study examined the efficacy of employing a duplex droplet digital PCR (ddPCR) assay that simultaneously quantifies Enterococcus spp. and the human fecal-associated HF183 marker for water quality assessment. Duplex ddPCR performance was evaluated side-by-side with qPCR and simplex ddPCR using reference material and 131 fecal and water samples. Results for fecal and water samples were highly correlated between ddPCR and simplex qPCR (coefficients > 0.93, p competition and resulted in non-detection or underestimation of the target with low concentration relative to the other, while results produced by simplex and duplex ddPCR were consistent and often indistinguishable from one another. ddPCR showed greater tolerance for inhibition, with no discernable effect on quantification at inhibitor concentrations one to two orders of magnitude higher than that tolerated by qPCR. Overall, ddPCR also exhibited improved precision, higher run-to-run repeatability, similar diagnostic sensitivity and specificity on the HF183 marker, but a lower upper limit of quantification than qPCR. Digital PCR has the potential to become a reliable and economical alternative to qPCR for recreational water monitoring and fecal source identification. Findings from this study may also be of interest to other aspects of water research such as detection of pathogens and antibiotic resistance genes.

  15. An integrated encyclopedia of DNA elements in the human genome.

    Science.gov (United States)

    2012-09-01

    The human genome encodes the blueprint of life, but the function of the vast majority of its nearly three billion bases is unknown. The Encyclopedia of DNA Elements (ENCODE) project has systematically mapped regions of transcription, transcription factor association, chromatin structure and histone modification. These data enabled us to assign biochemical functions for 80% of the genome, in particular outside of the well-studied protein-coding regions. Many discovered candidate regulatory elements are physically associated with one another and with expressed genes, providing new insights into the mechanisms of gene regulation. The newly identified elements also show a statistical correspondence to sequence variants linked to human disease, and can thereby guide interpretation of this variation. Overall, the project provides new insights into the organization and regulation of our genes and genome, and is an expansive resource of functional annotations for biomedical research.

  16. The Three Genetics (Nuclear DNA, Mitochondrial DNA, and Gut Microbiome) of Longevity in Humans Considered as Metaorganisms

    Science.gov (United States)

    Candela, Marco; Brigidi, Patrizia; Luiselli, Donata; Bacalini, Maria Giulia; Salvioli, Stefano; Capri, Miriam; Collino, Sebastiano; Franceschi, Claudio

    2014-01-01

    Usually the genetics of human longevity is restricted to the nuclear genome (nDNA). However it is well known that the nDNA interacts with a physically and functionally separated genome, the mitochondrial DNA (mtDNA) that, even if limited in length and number of genes encoded, plays a major role in the ageing process. The complex interplay between nDNA/mtDNA and the environment is most likely involved in phenomena such as ageing and longevity. To this scenario we have to add another level of complexity represented by the microbiota, that is, the whole set of bacteria present in the different part of our body with their whole set of genes. In particular, several studies investigated the role of gut microbiota (GM) modifications in ageing and longevity and an age-related GM signature was found. In this view, human being must be considered as “metaorganism” and a more holistic approach is necessary to grasp the complex dynamics of the interaction between the environment and nDNA-mtDNA-GM of the host during ageing. In this review, the relationship between the three genetics and human longevity is addressed to point out that a comprehensive view will allow the researchers to properly address the complex interactions that occur during human lifespan. PMID:24868529

  17. Gypenosides causes DNA damage and inhibits expression of DNA repair genes of human oral cancer SAS cells.

    Science.gov (United States)

    Lu, Kung-Wen; Chen, Jung-Chou; Lai, Tung-Yuan; Yang, Jai-Sing; Weng, Shu-Wen; Ma, Yi-Shih; Tang, Nou-Ying; Lu, Pei-Jung; Weng, Jing-Ru; Chung, Jing-Gung

    2010-01-01

    Gypenosides (Gyp) are the major components of Gynostemma pentaphyllum Makino, a Chinese medical plant. Recently, Gyp has been shown to induce cell cycle arrest and apoptosis in many human cancer cell lines. However, there is no available information to address the effects of Gyp on DNA damage and DNA repair-associated gene expression in human oral cancer cells. Therefore, we investigated whether Gyp induced DNA damage and DNA repair gene expression in human oral cancer SAS cells. The results from flow cytometric assay indicated that Gyp-induced cytotoxic effects led to a decrease in the percentage of viable SAS cells. The results from comet assay revealed that the incubation of SAS cells with Gyp led to a longer DNA migration smear (comet tail) when compared with control and this effect was dose-dependent. The results from real-time PCR analysis indicated that treatment of SAS cells with 180 mug/ml of Gyp for 24 h led to a decrease in 14-3-3sigma, DNA-dependent serine/threonine protein kinase (DNAPK), p53, ataxia telangiectasia mutated (ATM), ataxia-telangiectasia and Rad3-related (ATR) and breast cancer gene 1 (BRCA1) mRNA expression. These observations may explain the cell death caused by Gyp in SAS cells. Taken together, Gyp induced DNA damage and inhibited DNA repair-associated gene expressions in human oral cancer SAS cells in vitro.

  18. The human Bloom syndrome gene suppresses the DNA replication and repair defects of yeast dna2 mutants.

    Science.gov (United States)

    Imamura, Osamu; Campbell, Judith L

    2003-07-08

    Bloom syndrome is a disorder of profound and early cancer predisposition in which cells become hypermutable, exhibit high frequency of sister chromatid exchanges, and show increased micronuclei. BLM, the gene mutated in Bloom syndrome, has been cloned previously, and the BLM protein is a member of the RecQ family of DNA helicases. Many lines of evidence suggest that BLM is involved either directly in DNA replication or in surveillance during DNA replication, but its specific roles remain unknown. Here we show that hBLM can suppress both the temperature-sensitive growth defect and the DNA damage sensitivity of the yeast DNA replication mutant dna2-1. The dna2-1 mutant is defective in a helicase-nuclease that is required either to coordinate with the crucial Saccharomyces cerevisiae (sc) FEN1 nuclease in Okazaki fragment maturation or to compensate for scFEN1 when its activity is impaired. We show that human BLM interacts with both scDna2 and scFEN1 by using coimmunoprecipitation from yeast extracts, suggesting that human BLM participates in the same steps of DNA replication or repair as scFEN1 and scDna2.

  19. Sensitive and simultaneous quantification of zinc pyrithione and climbazole deposition from anti-dandruff shampoos onto human scalp.

    Science.gov (United States)

    Chen, Guoqiang; Miao, Miao; Hoptroff, Michael; Fei, Xiaoqing; Collins, Luisa Z; Jones, Andrew; Janssen, Hans-Gerd

    2015-10-15

    A sensitive ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed and validated for simultaneous quantification of zinc pyrithione (ZPT) and climbazole (CBZ) deposited onto human scalp from anti-dandruff (AD) shampoos. Scrubbing with a buffer solution was used as the sampling method for the extraction of ZPT and CBZ from scalp. Derivatization of ZPT was carried out prior to UHPLC-MS/MS analysis. The identification of ZPT and CBZ was performed by examining ratios of selected multiple reaction monitoring (MRM) transitions in combination with UHPLC retention times. The limit of detection for ZPT and CBZ was established to be 1 and 2ng/mL, respectively. This sensitivity enables the quantification of ZPT and CBZ at deposition levels in the low ng/cm(2) range. The method was successfully applied for the analysis of scalp buffer scrub samples from an in vivo study. The levels of ZPT and CBZ deposited on the scalp at different time points after application of the AD shampoo were measured. The results revealed that dual-active AD shampoo delivered more ZPT onto the scalp in a single wash than single active shampoo did. The amount of ZPT and CBZ retained on the scalp after AD shampoo application declined over 72h. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Power Analysis for Real-Time PCR Quantification of Genes in Activated Sludge and Analysis of the Variability Introduced by DNA Extraction

    Science.gov (United States)

    Dionisi, Hebe M.; Harms, Gerda; Layton, Alice C.; Gregory, Igrid R.; Parker, Jack; Hawkins, Shawn A.; Robinson, Kevin G.; Sayler, Gary S.

    2003-01-01

    The aims of this study were to determine the power of discrimination of the real-time PCR assay for monitoring fluctuations in microbial populations within activated sludge and to identify sample processing points where methodological changes are needed to minimize the variability in target quantification. DNA was extracted using a commercially available kit from mixed liquor samples taken from the aeration tank of four bench-scale activated-sludge reactors operating at 2-, 5-, 10-, and 20-day solid retention times, with mixed-liquor volatile suspended solid (MLVSS) values ranging from 260 to 2,610 mg/liter. Real-time PCR assays for bacterial and Nitrospira 16S rRNA genes were chosen because they represent, respectively, a highly abundant and a less-abundant bacterial target subject to clustering within the activated sludge matrix. The mean coefficient of variation in DNA yields (measured as microgram of DNA per milligram of MLVSS) in triplicate extractions of 12 different samples was 12.2%. Based on power analyses, the variability associated with DNA extraction had a small impact on the overall variability of the real-time PCR assay. Instead, a larger variability was associated with the PCR assay. The less-abundant target (Nitrospira 16S rRNA gene) had more variability than the highly abundant target (bacterial 16S rRNA gene), and samples from the lower-biomass reactors had more variability than samples from the higher-biomass reactors. Power analysis of real-time PCR assays indicated that three to five samples were necessary to detect a twofold increase in bacterial 16S rRNA genes, whereas three to five samples were required to detect a fivefold increase in Nitrospira 16S rRNA genes. PMID:14602618

  1. A novel method for the quantification of adeno-associated virus vectors for RNA interference applications using quantitative polymerase chain reaction and purified genomic adeno-associated virus DNA as a standard.

    Science.gov (United States)

    Wagner, Anke; Röhrs, Viola; Kedzierski, Radoslaw; Fechner, Henry; Kurreck, Jens

    2013-12-01

    Recombinant adeno-associated virus (rAAV) vectors are promising tools in gene therapy, but accurate quantification of the vector dose remains a critical issue for their successful application. We therefore aimed at the precise determination of the titer of self-complementary AAV (scAAV) vectors to improve the reliability of RNA interference (RNAi)-mediated knockdown approaches. Vector titers were initially determined by quantitative polymerase chain reaction (qPCR) using four primer sets targeting different regions within the AAV vector genome (VG) and either coiled or linearized plasmid standards. Despite very low variability between replicates in each assay, these quantification experiments revealed up to 20-fold variation in vector titers. Therefore, we developed a novel approach for the reproducible determination of titers of scAAV vectors based on the use of purified genomic vector DNA as a standard (scAAVStd). Consistent results were obtained in qPCR assays using the four primer sets mentioned above. RNAi-mediated silencing of human cyclophilin B (hCycB) by short hairpin RNA-expressing scAAV vectors was investigated in HeLa cells using two independent vector preparations. We found that the required vector titers for efficient knockdown differed by a factor of 3.5 between both preparations. Hence, we also investigated the number of internalized scAAV vectors, termed transduction units (TUs). TUs were determined by qPCR applying the scAAVStd. Very similar values for 80% hCycB knockdown were obtained for the two AAV vector preparations. Thus, only the determination of TUs, rather than vector concentration, allows for reproducible results in functional analyses using AAV vectors.

  2. Spectroscopic quantification of 5-hydroxymethylcytosine in genomic DNA using boric acid-functionalized nano-microsphere fluorescent probes.

    Science.gov (United States)

    Chen, Hua-Yan; Wei, Jing-Ru; Pan, Jiong-Xiu; Zhang, Wei; Dang, Fu-Quan; Zhang, Zhi-Qi; Zhang, Jing

    2017-05-15

    5-hydroxymethylcytosine (5hmC) is the sixth base of DNA. It is involved in active DNA demethylation and can be a marker of diseases such as cancer. In this study, we developed a simple and sensitive 2-(4-boronophenyl)quinoline-4-carboxylic acid modified poly (glycidyl methacrylate (PBAQA-PGMA) fluorescent probe to detect the 5hmC content of genomic DNA based on T4 β-glucosyltransferase-catalyzed glucosylation of 5hmC. The fluorescence-enhanced intensity recorded from the DNA sample was proportional to its 5-hydroxymethylcytosine content and could be quantified by fluorescence spectrophotometry. The developed probe showed good detection sensitivity and selectivity and a good linear relationship between the fluorescence intensity and the concentration of 5 hmC within a 0-100nM range. Compared with other fluorescence detection methods, this method not only could determine trace amounts of 5 hmC from genomic DNA but also could eliminate the interference of fluorescent dyes and the need for purification. It also could avoid multiple labeling. Because the PBAQA-PGMA probe could enrich the content of glycosyl-5-hydroxymethyl-2-deoxycytidine from a complex ground substance, it will broaden the linear detection range and improve sensitivity. The limit of detection was calculated to be 0.167nM after enrichment. Furthermore, the method was successfully used to detect 5-hydroxymethylcytosine from mouse tissues. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. The single-strand DNA binding activity of human PC4 preventsmutagenesis and killing by oxidative DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jen-Yeu; Sarker, Altaf Hossain; Cooper, Priscilla K.; Volkert, Michael R.

    2004-02-01

    Human positive cofactor 4 (PC4) is a transcriptional coactivator with a highly conserved single-strand DNA (ssDNA) binding domain of unknown function. We identified PC4 as a suppressor of the oxidative mutator phenotype of the Escherichia coli fpg mutY mutant and demonstrate that this suppression requires its ssDNA binding activity. Yeast mutants lacking their PC4 ortholog Sub1 are sensitive to hydrogen peroxide and exhibit spontaneous and peroxide induced hypermutability. PC4 expression suppresses the peroxide sensitivity of the yeast sub l{Delta} mutant, suggesting that the human protein has a similar function. A role for yeast and human proteins in DNA repair is suggested by the demonstration that Sub1 acts in a peroxide-resistance pathway involving Rad2 and by the physical interaction of PC4 with the human Rad2 homolog XPG. We show XPG recruits PC4 to a bubble-containing DNA substrate with resulting displacement of XPG and formation of a PC4-DNA complex. We discuss the possible requirement for PC4 in either global or transcription-coupled repair of oxidative DNA damage to mediate the release of XPG bound to its substrate.

  4. ISFG: Recommendations regarding the use of non-human (animal) DNA in forensic genetic investigations

    DEFF Research Database (Denmark)

    Linacre, A.; Gusmão, L.; Hecht, W.;

    2010-01-01

    The use of non-human DNA typing in forensic science investigations, and specifically that from animal DNA, is ever increasing. The term animal DNA in this document refers to animal species encountered in a forensic science examination but does not include human DNA. Non-human DNA may either be......: the trade and possession of a species, or products derived from a species, which is contrary to legislation; as evidence where the crime is against a person or property; instances of animal cruelty; or where the animal is the offender. The first instance is addressed by determining the species present......, and the other scenarios can often be addressed by assigning a DNA sample to a particular individual organism. Currently there is little standardization of methodologies used in the forensic analysis of animal DNA or in reporting styles. The recommendations in this document relate specifically to animal DNA...

  5. A preliminary analysis of the DNA and diet of the extinct Beothuk: a systematic approach to ancient human DNA

    DEFF Research Database (Denmark)

    Kuch, Melanie; Gröcke, Darren R; Knyf, Martin C

    2007-01-01

    We have used a systematic protocol for extracting, quantitating, sexing and validating ancient human mitochondrial and nuclear DNA of one male and one female Beothuk, a Native American population from Newfoundland, which became extinct approximately 180 years ago. They carried mtDNA haplotypes......, and that their water sources were pooled or stored water. Both mtDNA sequence data and Y SNP data hint at possible gene flow or a common ancestral population for both the Beothuk and the current day Mikmaq, but more importantly the data do not lend credence to the proposed idea that the Beothuk (specifically......, Nonosabasut) were of admixed (European-Native American) descent. We also analyzed patterns of DNA damage in the clones of authentic mtDNA sequences; there is no tendency for DNA damage to occur preferentially at previously defined mutational hotspots, suggesting that such mutational hotspots...

  6. The Cloning of the Human Tumor Supressor Gene INGI: DNA Cloning into Plasmid Vector and DNA Analysis by Restriction Enzymes

    Directory of Open Access Journals (Sweden)

    Elza Ibrahim Auerkari

    2015-11-01

    Full Text Available DNA cloning is one of the most important techniques In the field of molecular biology, with a critical role in analyzing the structure and function of genes and their adjacent regulatory regions. DNA cloning is helpful in learning fundamental molecular biological techniques, since DNA cloning involves a series of them, such as polymerase chain reaction (PCR, DNA ligation, bacterial transformation, bacterial culture, plasmid DNA extraction, DNA digestion with restriction enzymes and agarose gel electrophoresis. In this paper the cloning of the human tumor suppressor gene INGI has been used to illustrate the methodology. The gene was amplified by PCR, cloned into a TA-cloning vectore, and restriction enzyme mapping was used to distinguish the sense INGI construct from the antisense INGI construct.

  7. Direct visual detection of DNA based on the light scattering of silica nanoparticles on a human papillomavirus DNA chip.

    Science.gov (United States)

    Piao, Jing Yu; Park, Eun Hee; Choi, Kihwan; Quan, Bo; Kang, Dong Ho; Park, Pan Yun; Kim, Dai Sik; Chung, Doo Soo

    2009-12-15

    A detection system for a human papillomavirus (HPV) DNA chip based on the light scattering of aggregated silica nanoparticle probes is presented. In the assay, a target HPV DNA is sandwiched between the capture DNA immobilized on the chip and the probe DNA immobilized on the plain silica nanoparticle. The spot where the sandwich reaction occurs appears bright white and is readily distinguishable to the naked eye. Scanning electron microscopy images clearly show the aggregation of the silica nanoparticle probes. When three different sized (55 nm, 137 nm, 286 nm) plain silica nanoparticles were compared, probes of the larger silica nanoparticles showed a higher scattering intensity. Using 286-nm silica nanoparticles, the spots obtained with 200 pM of target DNA were visually detectable. The demonstrated capability to detect a disease related target DNA with direct visualization without using a complex detection instrument provides the prerequisite for the development of portable testing kits for genotyping.

  8. Quantification of age-related changes in the structure model type and trabecular thickness of human tibial cancellous

    DEFF Research Database (Denmark)

    Ding, Ming; Hvid, I

    2000-01-01

    quantification of structure model type, such as plate, rod objects, or mixture of plates or rods. Trabecular thickness is calculated directly from 3-D images, which is especially important for an a priori unknown or changing structure. Furthermore, 2-D trabecular thickness was also calculated based on the plate......Structure model type and trabecular thickness are important characteristics in describing cancellous bone architecture. It has been qualitatively observed that a radical change of trabeculae from plate-like to rod-like occurs in aging, bone remodeling, and osteoporosis. Thickness of trabeculae has......) cancellous bone. In this study, 160 human proximal tibial cancellous bone specimens from 40 normal donors, aged 16 to 85 years, were collected. These specimens were micro-computed tomography (micro-CT) scanned, then the micro-CT images were segmented using optimal thresholds. From accurate 3-D data sets...

  9. Quantification of Lumefantrine in Human Plasma Using LC-MS/MS and Its Application to a Bioequivalence Study.

    Science.gov (United States)

    Pingale, Satish G; Mangaonkar, Kiran V

    2013-01-01

    An analytical method based on protein precipitation has been developed and validated for analysis of lumefantrine in human plasma. Artesunate was used as an internal standard for lumefantrine. Inertsil ODS column provided chromatographic separation of analytes followed by detection with mass spectrometry. The method involves simple isocratic chromatographic condition and mass spectrometric detection in the positive ionization mode using an API-3000 system. The total run time was 2.5 minutes. The proposed method has been validated with linear range of 200-20000 ng/mL for lumefantrine. The intrarun and interrun precision values are within 6.66% and 5.56%, respectively, for lumefantrine at the lower limit of quantification level. The overall recovery for lumefantrine and artesunate was 93.16% and 91.05%, respectively. This validated method was used successfully for analysis of plasma samples from a bioequivalence study.

  10. Quantification of Lumefantrine in Human Plasma Using LC-MS/MS and Its Application to a Bioequivalence Study

    Directory of Open Access Journals (Sweden)

    Satish G. Pingale

    2013-01-01

    Full Text Available An analytical method based on protein precipitation has been developed and validated for analysis of lumefantrine in human plasma. Artesunate was used as an internal standard for lumefantrine. Inertsil ODS column provided chromatographic separation of analytes followed by detection with mass spectrometry. The method involves simple isocratic chromatographic condition and mass spectrometric detection in the positive ionization mode using an API-3000 system. The total run time was 2.5 minutes. The proposed method has been validated with linear range of 200–20000 ng/mL for lumefantrine. The intrarun and interrun precision values are within 6.66% and 5.56%, respectively, for lumefantrine at the lower limit of quantification level. The overall recovery for lumefantrine and artesunate was 93.16% and 91.05%, respectively. This validated method was used successfully for analysis of plasma samples from a bioequivalence study.

  11. Laser Capture Microdissection of Cervical Human Papillomavirus Infections: Copy Number of the Virus in Cancerous and Normal Tissue and Heterogeneous DNA Methylation

    Science.gov (United States)

    Kalantari, Mina; Garcia-Carranca, Alejandro; Morales-Vazquez, Claudia Dalia; Zuna, Rosemary; Montiel, Delia Perez; Calleja-Macias, Itzel E.; Johansson, Bo; Andersson, Sonia; Bernard, Hans-Ulrich

    2009-01-01

    Research on the pathogenicity of human papillomaviruses (HPVs) during cervical carcinogenesis often relies on the study of homogenized tissue or cultured cells. This approach does not detect molecular heterogeneities within the infected tissue. It is desirable to understand molecular properties in specific histological contexts. We asked whether Laser Capture Microdissection (LCM) of archival cervical tumors in combination with real-time polymerase chain reaction and bisulfite sequencing permits (i) sensitive DNA diagnosis of small clusters of formalin fixed cells, (ii) quantification of HPV DNA in neoplastic and normal cells, and (iii) analysis of HPV DNA methylation, a marker of tumor progression. We analyzed 26 tumors containing HPV-16 or 18. We prepared DNA from LCM dissected thin sections of 100 to 2000 cells, and analyzed aliquots corresponding to between nine and 70 cells. We detected nine to 630 HPV-16 genome copies and one to 111 HPV-18 genome copies per tumor cell, respectively. In 17 of the 26 samples, HPV DNA existed in histologically normal cells distant from the margins of the tumors, but at much lower concentrations than in the tumor, suggesting that HPVs can infect at low levels without pathogenic changes. Methylation of HPV DNA, a biomarker of integration of the virus into cellular DNA, could be measured only in few samples due to limited sensitivity, and indicated heterogeneous methylation patterns in small clusters of cancerous and normal cells. LCM is powerful to study molecular parameters of cervical HPV infections like copy number, latency and epigenetics. PMID:19497607

  12. Enhancing the Sensitivity of DNA Microarray Using Dye-Doped Silica Nanoparticles: Detection of Human Papilloma Virus

    Science.gov (United States)

    Enrichi, F.; Riccò, R.; Meneghello, A.; Pierobon, R.; Canton, G.; Cretaio, E.

    2010-10-01

    DNA microarray is a high-throughput technology used for detection and quantification of nucleic acid molecules and others of biological interest. The analysis is based on the specific hybridization between probe sequences deposited in array and a target ss-DNA amplified by PCR and functionalized by a fluorescent dye. Organic labels have well known disadvantages like photobleaching and low signal intensities, which put a limitation to the lower amount of DNA material that can be detected. Therefore for trace analysis the development of more efficient biomarkers is required. With this aim we present in this paper the synthesis and application of alternative hybrid nanosystems obtained by incorporating standard fluorescent molecules into monodisperse silica nanoparticles. Efficient application to the detection of Human Papilloma Virus is demonstrated. This virus is associated to the formation of cervical cancer, a leading cause of death by cancer for women worldwide. It is shown that the use of the novel biomarkers increases the optical signal of about one order of magnitude with respect to the free dyes or quantum dots in conventional instruments. This is due to the high number of molecules that can be accommodated into each nanoparticle, to the reduced photobleaching and to the improved environmental protection of the dyes when encapsulated in the silica matrix. The cheap and easy synthesis of these luminescent particles, the stability in water, the surface functionalizability and bio-compatibility make them very promising for present and future bio-labeling and bio-imaging applications.

  13. HaloJ: an ImageJ program for semiautomatic quantification of DNA damage at single-cell level.

    Science.gov (United States)

    Maurya, Dharmendra Kumar

    2014-01-01

    Although Halo assay is a fast and more economic technique, it is not popular compared to comet assay for the measurement of DNA damage. One of the reasons behind this was nonavailability of suitable user-friendly program. Currently, most of the researchers were analyzing halo images manually using image analysis software (Scion Image or ImageJ). To address this problem, I have developed a semiautomatic halo analysis ImageJ program, HaloJ, and applied in the assessment of DNA damage at the single-cell level. In this article, we have shown that data obtained from the HaloJ program have a very good correlation with the data obtained using comet assay analysis program such as Comet Assay Software Project. To the best of our knowledge, this will be the first program to quantify DNA damage of halo images. This program will be of great use for researchers working on the DNA damage and repair, radiation biology, toxicology, cancer biology, and so on.

  14. New spiro-acridines: DNA interaction, antiproliferative activity and inhibition of human DNA topoisomerases.

    Science.gov (United States)

    Almeida, Sinara Mônica Vitalino de; Lafayette, Elizabeth Almeida; Silva, Willams Leal; Lima Serafim, Vanessa de; Menezes, Thais Meira; Neves, Jorge Luiz; Ruiz, Ana Lucia Tasca Gois; Carvalho, João Ernesto de; Moura, Ricardo Olímpio de; Beltrão, Eduardo Isidoro Carneiro; Carvalho Júnior, Luiz Bezerra de; Lima, Maria do Carmo Alves de

    2016-11-01

    Two new spiro-acridines were synthesized by introducing cyano-N-acylhydrazone between the acridine and phenyl rings followed by spontaneous cyclization. The final compounds (E)-1'-(benzylideneamino)-5'-oxo-1',5'-dihydro-10H-spiro[acridine-9,2'-pyrrole]-4'-carbonitrile (AMTAC-01) and (E)-1'-((4-methoxybenzylidene)amino)-5'-oxo-1',5'-dihydro-10H-spiro[acridine-9,2'-pyrrole]-4'-carbonitrile (AMTAC-02) were evaluated for their interactions with calf thymus DNA, antiproliferative and human topoisomerase I and IIα inhibitory activities. Both compounds presented ability to bind DNA. The binding constant determined by UV-vis spectroscopy was found to be 10(4)M(-1). Antiproliferative assay demonstrated that AMTAC-01 and AMTAC-02 were most active against prostate and melanoma tumor cell lines, respectively. The compound did not present Topo I inhibitory activity. However, both derivatives displayed topoisomerase IIα inhibitory activity comparable to amsacrine, and AMTAC-02 was more potent than AMTAC-01 with methoxy substituent group on phenyl ring. This study demonstrates that the new derivatives are promising molecules with topoisomerase IIα inhibitory and antiproliferative activities.

  15. The Serengeti food web : Empirical quantification and analysis of topological changes under increasing human impact

    NARCIS (Netherlands)

    de Visser, Sara N.; Freymann, Bernd P.; Olff, Han

    2011-01-01

    P>1. To address effects of land use and human overexploitation on wildlife populations, it is essential to better understand how human activities have changed species composition, diversity and functioning. Theoretical studies modelled how network properties change under human-induced, non-random sp

  16. Distinct kinetics of human DNA ligases I, IIIalpha, IIIbeta, and IV reveal direct DNA sensing ability and differential physiological functions in DNA repair

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Xi; Ballin, Jeff D.; Della-Maria, Julie; Tsai, Miaw-Sheue; White, Elizabeth J.; Tomkinson, Alan E.; Wilson, Gerald M.

    2009-05-11

    The three human LIG genes encode polypeptides that catalyze phosphodiester bond formation during DNA replication, recombination and repair. While numerous studies have identified protein partners of the human DNA ligases (hLigs), there has been little characterization of the catalytic properties of these enzymes. In this study, we developed and optimized a fluorescence-based DNA ligation assay to characterize the activities of purified hLigs. Although hLigI joins DNA nicks, it has no detectable activity on linear duplex DNA substrates with short, cohesive single-strand ends. By contrast, hLigIII{beta} and the hLigIII{alpha}/XRCC1 and hLigIV/XRCC4 complexes are active on both nicked and linear duplex DNA substrates. Surprisingly, hLigIV/XRCC4, which is a key component of the major non-homologous end joining (NHEJ) pathway, is significantly less active than hLigIII on a linear duplex DNA substrate. Notably, hLigIV/XRCC4 molecules only catalyze a single ligation event in the absence or presence of ATP. The failure to catalyze subsequent ligation events reflects a defect in the enzyme-adenylation step of the next ligation reaction and suggests that, unless there is an in vivo mechanism to reactivate DNA ligase IV/XRCC4 following phosphodiester bond formation, the cellular NHEJ capacity will be determined by the number of adenylated DNA ligaseIV/XRCC4 molecules.

  17. DNA damage in human cells. Progress report, August 1983-August 1984

    Energy Technology Data Exchange (ETDEWEB)

    Loeb, L.A.

    1986-08-01

    Studies reported center on the relationship between DNA damage and mutagenesis. The mutagenic potential of apurinic sites was documented in a variety of systems. Studies on the enhancement of depurination by metal ions was continued. Recombiant DNA techniques were used for measuring nucleotide substitution in human mitochondrial DNA.

  18. Quantification of etodolac in human plasma for pharmacokinetics and bioequivalence studies in 27 Korean subjects.

    Science.gov (United States)

    Song, Il-Dong; Kang, Je-Seop; Kim, Hyun-Jin; Kim, Se-Mi; Zhao, Dong-Xu; Kim, Shin-Hee; Chun, Min-Young; Lee, Kyuhyun

    2017-01-16

    We developed a simple and validated liquid chromatography tandem mass spectrometry(LC-MS/MS) for quantification of etodolac using pioglitazone as an internal standard (IS) to assess pharmacokinetics and to appraise bioequivalence of two formulations of etodolac (reference and tested) in 27 healthy Korean subjects. Isocratic mobile phase consisted of 10 mM ammonium formate and acetonitrile were used to separate the analytes on a Gemini C18 column. Also, analytes were analyzed by MS/MS in multiple reaction monitoring (MRM) mode using the transitions of (M+H)+ ions, m/z 288.2→172.3 and m/z 357.1→134.2 for quantification of etodolac and IS each. The standard calibration curves displayed significant linearity within the range of 0.2-30.0 μg/mL (r2=0.9956, 1/x2 weighting) with LLOQ of 0.1 μg/mL. The retention times of etodolac and the IS were 0.77 min and 0.57 min each, indicating the high-throughput potential of the proposed method. The pharmacokinetic parameters were calculated from the plasma samples and data form the reference and test drugs were represented as follows; Area under plasma concentration-time curve (AUCt) (78.03 vs. 84.00 μg×h/mL), AUC∞ (86.67 vs. 93.92 μg×h/mL), maximal plasma concentration (Cmax) (19.49 vs. 18.94 μg/mL), time for maximal concentrations (Tmax) (2.13 vs. 2.26 h), Plasma elimination half-life (T1/2) (8.12 vs. 8.47 h), elimination rate constant (λz) (0.0853 vs. 0.0818 h-1). Pharmacokinetic parameters with 90% confidence interval fall within the bioequivalence range of 80-125%. Thus, the new testified method was successfully applied for the pharmacokinetic and bioequivalence studies for two etodolac formulations.

  19. Study on Performance Shaping Factors (PSFs) Quantification Method in Human Reliability Analysis (HRA)

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ar Ryum; Jang, Inseok Jang; Seong, Poong Hyun [Korea Advanced Institute of Science and Technology, Daejon (Korea, Republic of); Park, Jinkyun [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of); Kim, Jong Hyun [KEPCO, Ulsan (Korea, Republic of)

    2015-05-15

    The purpose of HRA implementation is 1) to achieve the human factor engineering (HFE) design goal of providing operator interfaces that will minimize personnel errors and 2) to conduct an integrated activity to support probabilistic risk assessment (PRA). For these purposes, various HRA methods have been developed such as technique for human error rate prediction (THERP), simplified plant analysis risk human reliability assessment (SPAR-H), cognitive reliability and error analysis method (CREAM) and so on. In performing HRA, such conditions that influence human performances have been represented via several context factors called performance shaping factors (PSFs). PSFs are aspects of the human's individual characteristics, environment, organization, or task that specifically decrements or improves human performance, thus respectively increasing or decreasing the likelihood of human errors. Most HRA methods evaluate the weightings of PSFs by expert judgment and explicit guidance for evaluating the weighting is not provided. It has been widely known that the performance of the human operator is one of the critical factors to determine the safe operation of NPPs. HRA methods have been developed to identify the possibility and mechanism of human errors. In performing HRA methods, the effect of PSFs which may increase or decrease human error should be investigated. However, the effect of PSFs were estimated by expert judgment so far. Accordingly, in order to estimate the effect of PSFs objectively, the quantitative framework to estimate PSFs by using PSF profiles is introduced in this paper.

  20. Quantification of ultraviolet radiation-induced DNA damage in the urine of Swedish adults and children following exposure to sunlight.

    Science.gov (United States)

    Liljendahl, Tove Sandberg; Kotova, Natalia; Segerbäck, Dan

    2012-11-01

    DNA damage following exposure to ultraviolet radiation (UVR) is important in skin cancer development. The predominant photoproduct, cyclobutane thymine dimer (T=T), is repaired and excreted in the urine, where it provides a biomarker of exposure. To quantify urinary T=T levels after recreational sunlight exposure in adults and children. Average UVR doses were measured with personal dosimeters. Urinary T=T was analysed with (32)P-postlabelling. Background levels of T=T increased significantly following exposure to sunlight. Amounts of T=T in urine of children and adults were not significantly different after adjusting for area of skin exposed and physiological differences. UVR dose and amounts of T=T correlated for both adults and children. Recreational exposure to sunlight in Sweden induces levels of DNA damage, clearly detectable in urine.

  1. PLASMID DNA DAMAGE CAUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITIN

    Science.gov (United States)

    PLASMID DNA DAMAGE CAOUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITINABSTRACT Both dimethylarsinic acid (DMA(V)) and dimethylarsinous acid (DMA(III)) release iron from human liver ferritin (HLF) with or without the presence of ascorbic acid. ...

  2. Methods for the identification of mutations in the human phenylalanine hydroxylase gene using DNA probes

    Energy Technology Data Exchange (ETDEWEB)

    Woo, S.L.C.; Dilella, A.G.

    1990-10-23

    This patent describes a method of detecting a mutation in a phenylalanine hydroxylase gene of human genomic DNA. Also described is an automated method of detecting PKU affected, PKU helerozgotes and normals in fetal to adult human samples.

  3. Characterization of the human HOX 7 cDNA and identification of polymorphic markers.

    Science.gov (United States)

    Padanilam, B J; Stadler, H S; Mills, K A; McLeod, L B; Solursh, M; Lee, B; Ramirez, F; Buetow, K H; Murray, J C

    1992-09-01

    cDNA clones for a human HOX 7 gene obtained with homologous clones of Drosophila were used in human gene mapping studies. The human cDNA clone was isolated from a library constructed from human embryonic craniofacial material. The sequence of the cDNA demonstrates significant homology with mouse HOX 7. A search for RFLPs identified MboII and BstEII variants. A CA dinucleotide repeat with 5 alleles was also identified and allowed placement of HOX 7 into a defined linkage map. Evidence for linkage disequilibrium was found with markers tested. These results place the human HOX 7 gene in a defined position on 4p.

  4. GHK and DNA: Resetting the Human Genome to Health

    Directory of Open Access Journals (Sweden)

    Loren Pickart

    2014-01-01

    Full Text Available During human aging there is an increase in the activity of inflammatory, cancer promoting, and tissue destructive genes plus a decrease in the activity of regenerative and reparative genes. The human blood tripeptide GHK possesses many positive effects but declines with age. It improves wound healing and tissue regeneration (skin, hair follicles, stomach and intestinal linings, and boney tissue, increases collagen and glycosaminoglycans, stimulates synthesis of decorin, increases angiogenesis, and nerve outgrowth, possesses antioxidant and anti-inflammatory effects, and increases cellular stemness and the secretion of trophic factors by mesenchymal stem cells. Recently, GHK has been found to reset genes of diseased cells from patients with cancer or COPD to a more healthy state. Cancer cells reset their programmed cell death system while COPD patients’ cells shut down tissue destructive genes and stimulate repair and remodeling activities. In this paper, we discuss GHK’s effect on genes that suppress fibrinogen synthesis, the insulin/insulin-like system, and cancer growth plus activation of genes that increase the ubiquitin-proteasome system, DNA repair, antioxidant systems, and healing by the TGF beta superfamily. A variety of methods and dosages to effectively use GHK to reset genes to a healthier state are also discussed.

  5. Semi-automated quantification of methylmalonic acid in human serum by LC-MS/MS.

    Science.gov (United States)

    Nelson, Dick; Xu, Ning; Carlson, Joyce

    2012-10-01

    Methylmalonic acid (MMA), a sensitive biomarker of functional vitamin B12 deficiency, is commonly determined by gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods using manual extraction and derivatization of MMA to reduce polarity prior to separation. In the present study we introduce a semi-automated extraction on a strong anion exchanger, HPLC separation on a BEH-amide column to separate serum MMA from its abundant isoform, succinic acid, followed by MS/MS detection and quantification. The extraction of MMA plus internal standard provides full recovery and the method is linear between 0.03 μmol/L and 20.0 μmol/L (r(2) = 1.0) with intra-and inter-assay imprecision of 2.2%. Agreement with other laboratories has been demonstrated in external proficiency testing. Compared to both conventional GC-MS and LC-MS/MS methods, the correlation is r(2) > 0.99. The use of robotic pipetting, elimination of derivatization and improved separation by the BEH-amide column combined with HILIC chromatographic conditions significantly improve sample throughput compared to conventional methods. Using a single pipetting robot and LC-MS/MS instrument, this method is currently performing 180 analyses per day from 10 regional hospitals and several additional distant sites.

  6. Recurrence quantification analysis of human postural fluctuations in older fallers and non-fallers.

    Science.gov (United States)

    Ramdani, Sofiane; Tallon, Guillaume; Bernard, Pierre Louis; Blain, Hubert

    2013-08-01

    We investigate postural sway data dynamics in older adult fallers and non-fallers. Center of pressure (COP) signals were recorded during quiet standing in 28 older adults. The subjects were divided in two groups: with and without history of falls. COP time series were analyzed using recurrence quantification analysis (RQA) in both anteroposterior and mediolateral (ML) directions. Classical stabilometric variables (path length and range) were also computed. The results showed that RQA outputs quantifying predictability of COP fluctuations and Shannon entropy of recurrence plot diagonal line length distribution, were significantly higher in fallers, only for ML direction. In addition, the range of ML COP signals was also significantly higher in fallers. This result is in accordance with some findings of the literature and could be interpreted as an increased hip strategy in fallers. The RQA results seem coherent with the theory of loss of complexity with aging and disease. Our results suggest that RQA is a promising approach for the investigation of COP fluctuations in a frail population.

  7. More on contamination: the use of asymmetric molecular behavior to identify authentic ancient human DNA

    DEFF Research Database (Denmark)

    Malmström, Helena; Svensson, Emma M; Gilbert, M Thomas P;

    2007-01-01

    the reliability of one of the proposed criteria, that of appropriate molecular behavior. Using real-time polymerase chain reaction (PCR) and pyrosequencing, we have quantified the relative levels of authentic aDNA and contaminant human DNA sequences recovered from archaeological dog and cattle remains. In doing....... Furthermore, we find that there is a substantial increase in the relative proportions of authentic DNA to contaminant DNA as the PCR target fragment size is decreased. We therefore conclude that the degradation pattern in aDNA provides a quantifiable difference between authentic aDNA and modern contamination...

  8. A novel LC-MS/MS method for the simultaneous quantification of topiramate and its main metabolites in human plasma.

    Science.gov (United States)

    Milosheska, Daniela; Roškar, Robert

    2017-05-10

    The aim of the present report was to develop and validate simple, sensitive and reliable LC-MS/MS method for quantification of topiramate (TPM) and its main metabolites: 2,3-desisopropylidene TPM, 4,5-desisopropylidene TPM, 10-OH TPM and 9-OH TPM in human plasma samples. The most abundant metabolite 2,3-desisopropylidene TPM was isolated from patients urine, characterized and afterwards used as an authentic standard for method development and validation. Sample preparation method employs 100μL of plasma sample and liquid-liquid extraction with a mixture of ethyl acetate and diethyl ether as extraction solvent. Chromatographic separation was achieved on a 1290 Infinity UHPLC coupled to 6460 Triple Quad Mass Spectrometer operated in negative MRM mode using Kinetex C18 column (50×2.1mm, 2.6μm) by gradient elution using water and methanol as a mobile phase and stable isotope labeled TPM as internal standard. The method showed to be selective, accurate, precise and linear over the concentration ranges of 0.10-20μg/mL for TPM, 0.01-2.0μg/mL for 2,3-desisopropylidene TPM, and 0.001-0.200μg/mL for 4,5-desisopropylidene TPM, 10-OH TPM and 9-OH TPM. The described method is the first fully validated method capable of simultaneous determination of TPM and its main metabolites in plasma over the selected analytical range. The suitability of the method was successfully demonstrated by the quantification of all analytes in plasma samples of patients with epilepsy and can be considered as reliable analytical tool for future investigations of the TPM metabolism. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Quantification of clenbuterol at trace level in human urine by ultra-high pressure liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Nicoli, Raul; Petrou, Michael; Badoud, Flavia; Dvorak, Jiri; Saugy, Martial; Baume, Norbert

    2013-05-31

    Clenbuterol is a β2 agonist agent with anabolic properties given by the increase in the muscular mass in parallel to the decrease of the body fat. For this reason, the use of clenbuterol is forbidden by the World Anti-Doping Agency (WADA) in the practice of sport. This compound is of particular interest for anti-doping authorities and WADA-accredited laboratories due to the recent reporting of risk of unintentional doping following the eating of meat contaminated with traces of clenbuterol in some countries. In this work, the development and the validation of an ultra-high pressure liquid chromatography coupled to electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) method for the quantification of clenbuterol in human urine is described. The analyte was extracted from urine samples by liquid-liquid extraction (LLE) in basic conditions using tert butyl-methyl ether (TBME) and analyzed by UHPLC-MS/MS with a linear gradient of acetonitrile in 9min only. The simple and rapid method presented here was validated in compliance with authority guidelines and showed a limit of quantification at 5pg/mL and a linearity range from 5pg/mL to 300pg/mL. Good trueness (85.8-105%), repeatability (5.7-10.6% RSD) and intermediate precision (5.9-14.9% RSD) results were obtained. The method was then applied to real samples from eighteen volunteers collecting urines after single oral doses administration (1, 5 and 10μg) of clenbuterol-enriched yogurts.

  10. Kaempferol induces DNA damage and inhibits DNA repair associated protein expressions in human promyelocytic leukemia HL-60 cells.

    Science.gov (United States)

    Wu, Lung-Yuan; Lu, Hsu-Feng; Chou, Yu-Cheng; Shih, Yung-Luen; Bau, Da-Tian; Chen, Jaw-Chyun; Hsu, Shu-Chun; Chung, Jing-Gung

    2015-01-01

    Numerous evidences have shown that plant flavonoids (naturally occurring substances) have been reported to have chemopreventive activities and protect against experimental carcinogenesis. Kaempferol, one of the flavonoids, is widely distributed in fruits and vegetables, and may have cancer chemopreventive properties. However, the precise underlying mechanism regarding induced DNA damage and suppressed DNA repair system are poorly understood. In this study, we investigated whether kaempferol induced DNA damage and affected DNA repair associated protein expression in human leukemia HL-60 cells in vitro. Percentages of viable cells were measured via a flow cytometry assay. DNA damage was examined by Comet assay and DAPI staining. DNA fragmentation (ladder) was examined by DNA gel electrophoresis. The changes of protein levels associated with DNA repair were examined by Western blotting. Results showed that kaempferol dose-dependently decreased the viable cells. Comet assay indicated that kaempferol induced DNA damage (Comet tail) in a dose-dependent manner and DAPI staining also showed increased doses of kaempferol which led to increased DNA condensation, these effects are all of dose-dependent manners. Western blotting indicated that kaempferol-decreased protein expression associated with DNA repair system, such as phosphate-ataxia-telangiectasia mutated (p-ATM), phosphate-ataxia-telangiectasia and Rad3-related (p-ATR), 14-3-3 proteins sigma (14-3-3σ), DNA-dependent serine/threonine protein kinase (DNA-PK), O(6)-methylguanine-DNA methyltransferase (MGMT), p53 and MDC1 protein expressions, but increased the protein expression of p-p53 and p-H2AX. Protein translocation was examined by confocal laser microscopy, and we found that kaempferol increased the levels of p-H2AX and p-p53 in HL-60 cells. Taken together, in the present study, we found that kaempferol induced DNA damage and suppressed DNA repair and inhibited DNA repair associated protein expression in HL-60

  11. Conservation of DNA Methylation Programming Between Mouse and Human Gametes and Preimplantation Embryos.

    Science.gov (United States)

    White, Carlee R; MacDonald, William A; Mann, Mellissa R W

    2016-09-01

    In mice, assisted reproductive technologies (ARTs) applied during gametogenesis and preimplantation development can result in disruption of genomic imprinting. In humans, these technologies and/or subfertility have been linked to perturbations in genomic imprinting. To understand how ARTs and infertility affect DNA methylation, it is important to understand DNA methylation dynamics and the role of regulatory factors at these critical stages. Recent genome studies performed using mouse and human gametes and preimplantation embryos have shed light onto these processes. Here, we comprehensively review the current state of knowledge regarding global and imprinted DNA methylation programming in the mouse and human. Available data highlight striking similarities in mouse and human DNA methylation dynamics during gamete and preimplantation development. Just as fascinating, these studies have revealed sex-, gene-, and allele-specific differences in DNA methylation programming, warranting future investigation to untangle the complex regulation of DNA methylation dynamics during gamete and preimplantation development.

  12. Fine resolution mapping of double-strand break sites for human ribosomal DNA units

    OpenAIRE

    Pope, Bernard J; Khalid Mahmood; Chol-hee Jung; Park, Daniel J

    2016-01-01

    DNA breakage arises during a variety of biological processes, including transcription, replication and genome rearrangements. In the context of disease, extensive fragmentation of DNA has been described in cancer cells and during early stages of neurodegeneration (Stephens et al., 2011 Stephens et al. (2011) [5]; Blondet et al., 2001 Blondet et al. (2001) [1]). Stults et al. (2009) Stults et al. (2009) [6] reported that human rDNA gene clusters are hotspots for recombination and that rDNA res...

  13. Human POLD1 modulates cell cycle progression and DNA damage repair

    OpenAIRE

    Song, Jing; Hong, Ping; Liu, Chengeng; Zhang, Yueqi; Wang, Jinling; Wang, Peichang

    2015-01-01

    Background The activity of eukaryotic DNA polymerase delta (Pol ?) plays an essential role in genome stability through its effects on DNA replication and repair. The p125 catalytic subunit of Pol ? is encoded by POLD1 gene in human cells. To clarify biological functions of POLD1, we investigated the effects of POLD1 overexpression or downregulation on cell proliferation, cell cycle progression, DNA synthesis and oxidative DNA damage induced by H2O2. Methods HEK293 cells were transfected with ...

  14. Human Papilloma Viral DNA Replicates as a Stable Episome in Cultured Epidermal Keratinocytes

    Science.gov (United States)

    Laporta, Robert F.; Taichman, Lorne B.

    1982-06-01

    Human papilloma virus (HPV) is poorly understood because systems for its growth in tissue culture have not been developed. We report here that cultured human epidermal keratinocytes could be infected with HPV from plantar warts and that the viral DNA persisted and replicated as a stable episome. There were 50-200 copies of viral DNA per cell and there was no evidence to indicate integration of viral DNA into the cellular genome. There was also no evidence to suggest that viral DNA underwent productive replication. We conclude that cultured human epidermal keratinocytes may be a model for the study of certain aspects of HPV biology.

  15. Quantification of Ethanol's Anti-Punishment Effect in Humans Using the Generalized Matching Equation

    Science.gov (United States)

    Rasmussen, Erin B.; Newland, M. Christopher

    2009-01-01

    Increases in rates of punished behavior by the administration of drugs with anxiolytic effects (called antipunishment effects) are well established in animals but not humans. The present study examined antipunishment effects of ethanol in humans using a choice procedure. The behavior of 5 participants was placed under six concurrent…

  16. Capacitive DNA sensor for rapid and sensitive detection of whole genome human herpesvirus-1 dsDNA in serum.

    Science.gov (United States)

    Cheng, Cheng; Oueslati, Rania; Wu, Jayne; Chen, Jiangang; Eda, Shigetoshi

    2017-06-01

    This work presents a rapid, highly sensitive, low-cost, and specific capacitive DNA sensor for detection of whole genome human herpesvirus-1 DNA. This sensor is capable of direct DNA detection with a response time of 30 s, and it can be used to test standard buffer or serum samples. The sensing approach for DNA detection is based on alternating current (AC) electrokinetics. By applying an inhomogeneous AC electric field on sensor electrodes, positive dielectrophoresis is induced to accelerate DNA hybridization. The same applied AC signal also directly measures the hybridization of target with the probe on the sensor surface. Experiments are conducted to optimize the AC signal, as well as the buffers for probe immobilization and target DNA hybridization. The assay is highly sensitive and specific, with no response to human herpesvirus-2 DNA at 5 ng/mL and a LOD of 1.0 pg/mL (6.5 copies/μL or 10.7 aM) in standard buffer. When testing the double stranded (ds) DNA spiked in human serum samples, the sensor yields a LOD of 20.0 pg/mL (129.5 copies/μL or 0.21 femtomolar (fM)) in neat serum. In this work, the target is whole genome dsDNA, consequently the test can be performed without the use of enzyme or amplification, which considerably simplifies the sensor operation and is highly suitable for point of care disease diagnosis. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. No evidence of Neandertal mtDNA contribution to early modern humans.

    Directory of Open Access Journals (Sweden)

    David Serre

    2004-03-01

    Full Text Available The retrieval of mitochondrial DNA (mtDNA sequences from four Neandertal fossils from Germany, Russia, and Croatia has demonstrated that these individuals carried closely related mtDNAs that are not found among current humans. However, these results do not definitively resolve the question of a possible Neandertal contribution to the gene pool of modern humans since such a contribution might have been erased by genetic drift or by the continuous influx of modern human DNA into the Neandertal gene pool. A further concern is that if some Neandertals carried mtDNA sequences similar to contemporaneous humans, such sequences may be erroneously regarded as modern contaminations when retrieved from fossils. Here we address these issues by the analysis of 24 Neandertal and 40 early modern human remains. The biomolecular preservation of four Neandertals and of five early modern humans was good enough to suggest the preservation of DNA. All four Neandertals yielded mtDNA sequences similar to those previously determined from Neandertal individuals, whereas none of the five early modern humans contained such mtDNA sequences. In combination with current mtDNA data, this excludes any large genetic contribution by Neandertals to early modern humans, but does not rule out the possibility of a smaller contribution.

  18. Detection of human papillomavirus DNA by the hybrid capture assay

    Directory of Open Access Journals (Sweden)

    Carvalho Maria Odete O.

    2003-01-01

    Full Text Available Human Papillomavirus (HPV infection is the main cause of cervical cancers and cervical intraepithelial neoplasias (CIN worldwide. Consequently, it would be useful to evaluate HPV testing to screen for cervical cancer. Recently developed, the second-generation Hybrid Capture (HCA II test is a non-radioactive, relatively rapid, liquid hybridization assay designed to detect 18 HPV types, divided into high and low-risk groups. We evaluated 1055 women for HPV infection with the HCA II test. Five hundred and ten (48.3% of these women had HPV infection; 60 (11.8% had low cancer-risk HPV DNA; 269 (52.7% had high-risk HPV types and 181 (35.5% had both groups. Hence, 450 women (88.2% in this HPV-infected group had at least one high risk HPV type, and were therefore considered to be at high risk for cancer. Among the group with Papanicolaou (Pap test results, the overall prevalence of HPV DNA was 58.4%. Significant differences in HPV infection of the cervix were detected between Pap I (normal smears and Pap IV (carcinomas (p<0.0001. Values of HPV viral load obtained for Pap I and SILs were significantly different, with an upward trend (p<0.0001, suggesting a positive correlation between high viral load values and risk of SIL. Because of the high costs of the HCA II test, its use for routine cervical mass screening cannot be recommended in poor countries. Nevertheless, it is a useful tool when combined with cytology, diagnosing high-risk infections in apparently normal tissues. Use of this technique could help reduce the risk of cancer.

  19. Monitoring of transplanted liver health by quantification of organ-specific genomic marker in circulating DNA from receptor.

    Directory of Open Access Journals (Sweden)

    Hada C Macher

    Full Text Available BACKGROUND: Health assessment of the transplanted organ is very important due to the relationship of long-term survival of organ transplant recipients and health organ maintenance. Nowadays, the measurement of cell-free DNA from grafts in the circulation of transplant recipients has been considered a potential biomarker of organ rejection or transplant associated complications in an attempt to replace or reduce liver biopsy. However, methods developed to date are expensive and extremely time-consuming. Our approach was to measure the SRY gene, as a male organ biomarker, in a setting of sex-mismatched female recipients of male donor organs. METHODS: Cell-free DNA quantization of the SRY gene was performed by real-time quantitative PCR beforehand, at the moment of transplantation during reperfusion (day 0 and during the stay at the intensive care unit. Beta-globin cell-free DNA levels, a general cellular damage marker, were also quantified. RESULTS: Beta-globin mean values of patients, who accepted the graft without any complications during the first week after surgery, diminished from day 0 until patient stabilization. This decrease was not so evident in patients who suffered some kind of post-transplantation complications. All patients showed an increase in SRY levels at day 0, which decreased during hospitalization. Different complications that did not compromise donated organs showed increased beta-globin levels but no SRY gene levels. However, when a donated organ was damaged the patients exhibited high levels of both genes. CONCLUSION: Determination of a SRY gene in a female recipient's serum is a clear and specific biomarker of donated organs and may give us important information about graft health in a short period of time by a non-expensive technique. This approach may permit clinicians to maintain a close follow up of the transplanted patient.

  20. Monitoring of Transplanted Liver Health by Quantification of Organ-Specific Genomic Marker in Circulating DNA from Receptor

    Science.gov (United States)

    Macher, Hada C.; Suárez-Artacho, Gonzalo; Guerrero, Juan M.; Gómez-Bravo, Miguel A.; Álvarez-Gómez, Sara; Bernal-Bellido, Carmen; Dominguez-Pascual, Inmaculada; Rubio, Amalia

    2014-01-01

    Background Health assessment of the transplanted organ is very important due to the relationship of long-term survival of organ transplant recipients and health organ maintenance. Nowadays, the measurement of cell-free DNA from grafts in the circulation of transplant recipients has been considered a potential biomarker of organ rejection or transplant associated complications in an attempt to replace or reduce liver biopsy. However, methods developed to date are expensive and extremely time-consuming. Our approach was to measure the SRY gene, as a male organ biomarker, in a setting of sex-mismatched female recipients of male donor organs. Methods Cell-free DNA quantization of the SRY gene was performed by real-time quantitative PCR beforehand, at the moment of transplantation during reperfusion (day 0) and during the stay at the intensive care unit. Beta-globin cell-free DNA levels, a general cellular damage marker, were also quantified. Results Beta-globin mean values of patients, who accepted the graft without any complications during the first week after surgery, diminished from day 0 until patient stabilization. This decrease was not so evident in patients who suffered some kind of post-transplantation complications. All patients showed an increase in SRY levels at day 0, which decreased during hospitalization. Different complications that did not compromise donated organs showed increased beta-globin levels but no SRY gene levels. However, when a donated organ was damaged the patients exhibited high levels of both genes. Conclusion Determination of a SRY gene in a female recipient's serum is a clear and specific biomarker of donated organs and may give us important information about graft health in a short period of time by a non-expensive technique. This approach may permit clinicians to maintain a close follow up of the transplanted patient. PMID:25489845

  1. The DNA-binding box of human SPARTAN contributes to the targeting of Polη to DNA damage sites.

    Science.gov (United States)

    Toth, Agnes; Hegedus, Lili; Juhasz, Szilvia; Haracska, Lajos; Burkovics, Peter

    2017-01-01

    Inappropriate repair of UV-induced DNA damage results in human diseases such as Xeroderma pigmentosum (XP), which is associated with an extremely high risk of skin cancer. A variant form of XP is caused by the absence of Polη, which is normally able to bypass UV-induced DNA lesions in an error-free manner. However, Polη is highly error prone when replicating undamaged DNA and, thus, the regulation of the proper targeting of Polη is crucial for the prevention of mutagenesis and UV-induced cancer formation. Spartan is a novel regulator of the damage tolerance pathway, and its association with Ub-PCNA has a role in Polη targeting; however, our knowledge about its function is only rudimentary. Here, we describe a new biochemical property of purified human SPARTAN by showing that it is a DNA-binding protein. Using a DNA binding mutant, we provide in vivo evidence that DNA binding by SPARTAN regulates the targeting of Polη to damage sites after UV exposure, and this function contributes highly to its DNA-damage tolerance function. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. The architecture of the human Rad54-DNA complex provides evidence for protein translocation along DNA.

    NARCIS (Netherlands)

    D. Ristic (Dejan); C. Wyman (Claire); C. Paulusma (Coen); R. Kanaar (Roland)

    2001-01-01

    textabstractProper maintenance and duplication of the genome require accurate recombination between homologous DNA molecules. In eukaryotic cells, the Rad51 protein mediates pairing between homologous DNA molecules. This reaction is assisted by the Rad54 protein. To gai

  3. Cloning of human brevican cDNA and expression of its mRNA in human glioma

    Institute of Scientific and Technical Information of China (English)

    韩唏; 董艳; 由振东; 何成; 卢亦成

    2003-01-01

    Objective:To clone the cDNA of human brevican secreting isoform and to investigate its mRNA expression in human glioma.Methods:The full-length cDNA of human brevican secreted isoform was cloned from a human ahaplastic astrocytoma by RT-PCR,and the expression of human brevican mRNA in 22 cases of human glioma and 13 cases of non-glial brain tumors were investigated by in situ hybridization.Results:The cDNA which including the whole open reading frame of human brevican secreted isoform was obtained.In situ hybridization showed that brevican positive cells were present in all of the 22 cases of gliomas(100%),whereas none were found in the 13 cases of non-glial and metastasis brain tumors examined.Conclusion:The results suggest that brevican mRNA is highly and specifically expressed in human glioma.

  4. Quantification of rolling circle amplified DNA using magnetic nanobeads and a Blu-ray optical pick-up unit

    DEFF Research Database (Denmark)

    Donolato, Marco; Antunes, Paula Soares Martins; Gómez del a Torre, Teresa Zardán

    2015-01-01

    We present the first implementation of a Blu-ray optical pickup unit (OPU) for the high-performance low-cost readout of a homogeneous assay in a multichamber microfluidic disc with a chamber thickness of 600 μm. The assay relies on optical measurements of the dynamics of magnetic nanobeads...... the optimum magnetic bead concentration to 0.1 mg/mL and have measured the response vs. concentration of DNA coils formed from Escherichia Coli. We have found a limit of detection of 10 pM and a dynamic range of about two orders of magnitude, which is comparable to the performance obtained using costly...

  5. DNA replication, repair, and repair tests. [Rat; human leukocytes

    Energy Technology Data Exchange (ETDEWEB)

    Lambert, B.

    1980-09-01

    The rate of inhibition and recovery of DNA synthesis can be used in a rapid assay system to detect genotoxic potentials of chemicals. Also, the observation that an agent stimulates DNA repair in a test system indicates its ability to cause damage in DNA. Different experimental approaches to the study of repair synthesis are discussed.

  6. Space Radiation Effects on Human Cells: Modeling DNA Breakage, DNA Damage Foci Distribution, Chromosomal Aberrations and Tissue Effects

    Science.gov (United States)

    Ponomarev, A. L.; Huff, J. L.; Cucinotta, F. A.

    2011-01-01

    Future long-tem space travel will face challenges from radiation concerns as the space environment poses health risk to humans in space from radiations with high biological efficiency and adverse post-flight long-term effects. Solar particles events may dramatically affect the crew performance, while Galactic Cosmic Rays will induce a chronic exposure to high-linear-energy-transfer (LET) particles. These types of radiation, not present on the ground level, can increase the probability of a fatal cancer later in astronaut life. No feasible shielding is possible from radiation in space, especially for the heavy ion component, as suggested solutions will require a dramatic increase in the mass of the mission. Our research group focuses on fundamental research and strategic analysis leading to better shielding design and to better understanding of the biological mechanisms of radiation damage. We present our recent effort to model DNA damage and tissue damage using computational models based on the physics of heavy ion radiation, DNA structure and DNA damage and repair in human cells. Our particular area of expertise include the clustered DNA damage from high-LET radiation, the visualization of DSBs (DNA double strand breaks) via DNA damage foci, image analysis and the statistics of the foci for different experimental situations, chromosomal aberration formation through DSB misrepair, the kinetics of DSB repair leading to a model-derived spectrum of chromosomal aberrations, and, finally, the simulation of human tissue and the pattern of apoptotic cell damage. This compendium of theoretical and experimental data sheds light on the complex nature of radiation interacting with human DNA, cells and tissues, which can lead to mutagenesis and carcinogenesis later in human life after the space mission.

  7. Structural basis of human transcription factor Sry-related box 17 binding to DNA.

    Science.gov (United States)

    Gao, Nana; Jiang, Wei; Gao, Hai; Cheng, Zhong; Qian, Huolian; Si, Shuyi; Xie, Yong

    2013-04-01

    Sry-related box (Sox) transcription factors share a conserved high-mobility-group box domain (HMG-domain) that binds DNA in the minor groove and bends DNA for further assembly of transcriptional machineries. During organogenesis, each member of the Sox family triggers a specific cell lineage differentiation, indicating that their interactions with DNA are different from each other. Therefore, investigating structural rearrangement of each Sox transcription factor HMG-domain upon binding to DNA would help to elucidate the distinctive molecular mechanism by which they interact with DNA. Previous studies have determined the crystal structures of Sox2 HMG-domain/DNA, Sox4 HMGdomain/ DNA, Sox9 HMG-domain/DNA and Sox17 HMG-domain/DNA complexes. However, major gaps remain in the structural information on the Sox transcription factor HMG-domains. Here, we report the crystal structure of the human Sox17 HMG-domain alone at 2.4 A resolution. Comparing this structure and the structure of the mouse Sox17 HMGdomain/ DNA complex provides structural understanding of the mechanism of Sox17 binding to DNA. Specifically, after electrostatic interactions attract Sox17 to DNA, Asn73, Ser99, and Trp106 form hydrogen bonds with DNA, Arg70, Lys80, Arg83, His94, and Asn95 on Sox17 undergo conformational changes and form hydrogen bonds with DNA, contributing to the electrostatic interaction between Sox17 and DNA.

  8. Quantification of roxatidine in human plasma by liquid chromatography electrospray ionization tandem mass spectrometry: application to a bioequivalence study.

    Science.gov (United States)

    Ryu, Ju-Hee; Choi, Sang-Jun; Lee, Heon-Woo; Choi, Seung-Ki; Lee, Kyung-Tae

    2008-12-01

    A sensitive and specific method using a one-step liquid-liquid extraction (LLE) with ethyl acetate followed by high-performance liquid chromatography (HPLC) coupled with positive ion electrospray ionization tandem mass spectrometry (ESI-MS/MS) detection was developed and validated for the determination of roxatidine in human plasma using famotidine as an internal standard (IS). Data acquisition was carried out in multiple reaction monitoring (MRM) mode, by monitoring the transitions m/z 307.3-->107.1 for roxatidine and m/z 338.4-->189.1 for famotidine. Chromatographic separation was performed on a reverse phase Hydrosphere C(18) column at 0.2 mL min(-1) using a mixture of methanol-ammonium formate buffer as mobile phase (20:80, v/v; adjusted to pH 3.9 with formic acid). The achieved lower limit of quantification (LLOQ) was 1.0 ng mL(-1) and the standard calibration curve for roxatidine was linear (r(2)=0.998) over the studied range (1-1000 ng mL(-1)) with acceptable accuracy and precision. Roxatidine was found to be stable in human plasma samples under short-, long-term storage and processing conditions. The developed method was validated and successfully applied to the bioequivalence study of roxatidine administrated as a single oral dose (75 mg as roxatidine acetate hydrochloride) to healthy female Korean volunteers.

  9. Development and Validation of a Chromatographic Ultraviolet Method for the Simultaneous Quantification of Dolutegravir and Rilpivirine in Human Plasma.

    Science.gov (United States)

    Cozzi, Valeria; Charbe, Nitin; Baldelli, Sara; Castoldi, Simone; Atzori, Chiara; Cattaneo, Dario; Clementi, Emilio

    2016-06-01

    We have developed and validated a high-performance liquid chromatographic method for the simultaneous quantification, in human plasma, of dolutegravir, a new human immunodeficiency virus (HIV) integrase inhibitor, and rilpivirine, a novel HIV nonnucleoside reverse transcriptase inhibitor. An internal standard (quinoxaline) was added to plasma aliquots (500 μL), and a simple solid-phase extraction procedure was applied. Chromatographic separation of the drugs and internal standard was achieved with a gradient of acetonitrile and acetate buffer, and with an analytical run time of 25 minutes using an XBridge C18 column. The column eluate was monitored at 260 nm for dolutegravir and the internal standard and at 305 nm for rilpivirine. The method was linear in the range of 20-8000 and 20-2000 ng/mL for dolutegravir and rilpivirine, respectively (mean r ≥ 0.993 on 10 replicates for both analytes). Mean intraday and interday precision and inaccuracy were <15% for both compounds. The mean recovery was 73% and 80% for dolutegravir and rilpivirine, respectively. The high-performance liquid chromatography-ultraviolet method we developed showed a good analytical performance required for therapeutic drug monitoring of antiretrovirals, leading to potential improvements in HIV-infected patient care and laboratory management.

  10. Novel and sensitive UPLC-MS/MS method for quantification of sofosbuvir in human plasma: application to a bioequivalence study.

    Science.gov (United States)

    Rezk, Mamdouh R; Basalious, Emad B; Amin, Mohammed E

    2016-09-01

    A novel and sensitive LC-MS/MS method was developed and validated for determination of sofosbuvir (SF) using eplerenone as an internal standard. The Xevo TQD LC-MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. Extraction with tert-butyl methyl ether was used in sample preparation. The prepared samples were chromatographed on Acquity UPLC BEH C18 (50 × 2.1 mm, 1.7 μm) column by pumping 0.1% formic acid and acetonitrile in an isocratic mode at a flow rate of 0.35 mL/min. Method validation was performed as per the US Food and Drug Administration guidelines and the standard curves were found to be linear in the range of 0.25-3500 ng/mL for SF. The intra- and inter-day precision and accuracy results were within the acceptable limits. A very short run time of 1 min made it possible to analyze more than 500 human plasma samples per day. A very low quantification limit of SF allowed the applicability of the developed method for determination of SF in a bioequivalence study in human volunteers. Copyright © 2016 John Wiley & Sons, Ltd.

  11. Collaborating functions of BLM and DNA topoisomerase I in regulating human rDNA transcription

    Energy Technology Data Exchange (ETDEWEB)

    Grierson, Patrick M. [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States); Acharya, Samir, E-mail: samir.acharya@osumc.edu [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States); Groden, Joanna [Department of Microbiology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States)

    2013-03-15

    Bloom's syndrome (BS) is an inherited disorder caused by loss of function of the recQ-like BLM helicase. It is characterized clinically by severe growth retardation and cancer predisposition. BLM localizes to PML nuclear bodies and to the nucleolus; its deficiency results in increased intra- and inter-chromosomal recombination, including hyper-recombination of rDNA repeats. Our previous work has shown that BLM facilitates RNA polymerase I-mediated rRNA transcription in the nucleolus (Grierson et al., 2012 [18]). This study uses protein co-immunoprecipitation and in vitro transcription/translation (IVTT) to identify a direct interaction of DNA topoisomerase I with the C-terminus of BLM in the nucleolus. In vitro helicase assays demonstrate that DNA topoisomerase I stimulates BLM helicase activity on a nucleolar-relevant RNA:DNA hybrid, but has an insignificant effect on BLM helicase activity on a control DNA:DNA duplex substrate. Reciprocally, BLM enhances the DNA relaxation activity of DNA topoisomerase I on supercoiled DNA substrates. Our study suggests that BLM and DNA topoisomerase I function coordinately to modulate RNA:DNA hybrid formation as well as relaxation of DNA supercoils in the context of nucleolar transcription.

  12. Quantification of Leishmania (Viannia) Kinetoplast DNA in Ulcers of Cutaneous Leishmaniasis Reveals Inter-site and Inter-sampling Variability in Parasite Load

    Science.gov (United States)

    Suárez, Milagros; Valencia, Braulio M.; Jara, Marlene; Alba, Milena; Boggild, Andrea K.; Dujardin, Jean-Claude; Llanos-Cuentas, Alejandro; Arevalo, Jorge; Adaui, Vanessa

    2015-01-01

    Background Cutaneous leishmaniasis (CL) is a skin disease caused by the protozoan parasite Leishmania. Few studies have assessed the influence of the sample collection site within the ulcer and the sampling method on the sensitivity of parasitological and molecular diagnostic techniques for CL. Sensitivity of the technique can be dependent upon the load and distribution of Leishmania amastigotes in the lesion. Methodology/Principal Findings We applied a quantitative real-time PCR (qPCR) assay for Leishmania (Viannia) minicircle kinetoplast DNA (kDNA) detection and parasite load quantification in biopsy and scraping samples obtained from 3 sites within each ulcer (border, base, and center) as well as in cytology brush specimens taken from the ulcer base and center. A total of 248 lesion samples from 31 patients with laboratory confirmed CL of recent onset (≤3 months) were evaluated. The kDNA-qPCR detected Leishmania DNA in 97.6% (242/248) of the examined samples. Median parasite loads were significantly higher in the ulcer base and center than in the border in biopsies (P<0.0001) and scrapings (P = 0.0002). There was no significant difference in parasite load between the ulcer base and center (P = 0.80, 0.43, and 0.07 for biopsy, scraping, and cytology brush specimens, respectively). The parasite load varied significantly by sampling method: in the ulcer base and center, the descending order for the parasite load levels in samples was: cytology brushes, scrapings, and biopsies (P<0.0001); in the ulcer border, scrapings had higher parasite load than biopsies (P<0.0001). There was no difference in parasite load according to L. braziliensis and L. peruviana infections (P = 0.4). Conclusion/Significance Our results suggest an uneven distribution of Leishmania amastigotes in acute CL ulcers, with higher parasite loads in the ulcer base and center, which has implications for bedside collection of diagnostic specimens. The use of scrapings and cytology brushes is

  13. Human cultured cells are capable to incorporate isolated plant mitochondria loaded with exogenous DNA

    Directory of Open Access Journals (Sweden)

    Laktionov P. P.

    2012-07-01

    Full Text Available Aim. To investigate the possibility of human cultured cells to incorporate isolated mitochondria together with exogenous DNA introduced into organelles. Methods. Two approaches were used for this purpose, fluorescent labelling of mitochondria and/or DNA with subsequent analysis of the cells subjected to incubation by microscopy or by quantitative PCR. Results. We have shown that human cultured cells lines, HeLa and HUVEC, are capable to uptake isolated plant mitochondria and that this process depends on the incubation time and concentration of organelles present in medium. The incorporated mitochondria can serve as vehicles to deliver exogenous DNA into human cells, this DNA is then distributed in different cell compartments. Conclusions. These results are preliminary and need further investigations, including testing the possibility of human cells to incorporate the mitochondria of human or animal origin and creating genetic construction which could provide certain selectivity or stability of the transferred exogenous DNA upon cell uptake of the mitochondria as vectors.

  14. Discovery of human inversion polymorphisms by comparative analysis of human and chimpanzee DNA sequence assemblies.

    Directory of Open Access Journals (Sweden)

    2005-10-01

    Full Text Available With a draft genome-sequence assembly for the chimpanzee available, it is now possible to perform genome-wide analyses to identify, at a submicroscopic level, structural rearrangements that have occurred between chimpanzees and humans. The goal of this study was to investigate chromosomal regions that are inverted between the chimpanzee and human genomes. Using the net alignments for the builds of the human and chimpanzee genome assemblies, we identified a total of 1,576 putative regions of inverted orientation, covering more than 154 mega-bases of DNA. The DNA segments are distributed throughout the genome and range from 23 base pairs to 62 mega-bases in length. For the 66 inversions more than 25 kilobases (kb in length, 75% were flanked on one or both sides by (often unrelated segmental duplications. Using PCR and fluorescence in situ hybridization we experimentally validated 23 of 27 (85% semi-randomly chosen regions; the largest novel inversion confirmed was 4.3 mega-bases at human Chromosome 7p14. Gorilla was used as an out-group to assign ancestral status to the variants. All experimentally validated inversion regions were then assayed against a panel of human samples and three of the 23 (13% regions were found to be polymorphic in the human genome. These polymorphic inversions include 730 kb (at 7p22, 13 kb (at 7q11, and 1 kb (at 16q24 fragments with a 5%, 30%, and 48% minor allele frequency, respectively. Our results suggest that inversions are an important source of variation in primate genome evolution. The finding of at least three novel inversion polymorphisms in humans indicates this type of structural variation may be a more common feature of our genome than previously realized.

  15. Comparative Study on the Immunogenicity between Hsp70 DNA Vaccine and Hsp65 DNA Vaccine in Human Mycobacterium Tuberculosis

    Institute of Scientific and Technical Information of China (English)

    DAI; Wuxing; HUANG; Hailang; YUAN; Ye; HU; Jiajie; HUANGFU; Yongmu

    2001-01-01

    The BALB/c mice were immunized with Hsp70 DNA and Hsp65 DNA vaccines in human Mycobacterium tuberculosis. Eight weeks after immunization, the eyeballs were removed, blood and spleen taken, and intraperitoneal macrophages were harvested. The lymphocytic stimulating index(SI) was used to measure the cellular proliferating ability and NO release to measure the phagocytic activity of the macrophages. With ELISA kit, the levels of interleukin-2 (IL-2) and interferon-γ(IFN-γ) in serum and the splenic lymphocytic cultured supernatant were detected. The results showed that after the mice were immunized with 100 μg/mouse of Hsp70 DNA vaccine intramuscularly, the splenic lymphocytic proliferating ability in the mice was significantly increased as compared with that in the control group, vector group and Hsp65 DNA vaccine group (P<0. 01); The contents of NO in the intraperitoneal macrophages of the mice were significantly lower than in the control group and Hsp65 DNA vaccine group (P<0. 01); The levels of serum IL-2 in the mice were significantly higher than in the control group, but there was no statistical difference between Hsp65 DNA group and vector group (P>0. 05); The contents of serum IFN-γ in the mice were significantly higher than in the control group, but significantly lower than in the Hsp65 DNA vaccine group (P<0. 05). It was indicated that immunization with Hsp70 DNA vaccine could obviously enhance the immune response, but its intensity seemed inferior to Hsp65 DNA vaccine. The anti-infection mechanisms and clinical use in the future of the vaccines of Hsp70 DNA and Hsp65 DNA are worth further studying.

  16. Buspirone, fexofenadine, and omeprazole: Quantification of probe drugs and their metabolites in human plasma

    OpenAIRE

    Gor, Parul; Alnouti, Yazen; Reed, Gregory A.

    2011-01-01

    Probe drugs are critical tools for the measurement of drug metabolism and transport activities in human subjects. Often several probe drugs are administered simultaneously in a —cocktail . This cocktail approach requires efficient analytical methods for the simultaneous quantitation of multiple analytes. We have developed and validated a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of three probe drugs and their metabolites in human plasma. The anal...

  17. DNA fragmentation induced by all-trans retinoic acid and its steroidal analogue EA-4 in C2 C12 mouse and HL-60 human leukemic cells in vitro.

    Science.gov (United States)

    Alakhras, Raghda S; Stephanou, Georgia; Demopoulos, Nikos A; Grintzalis, Konstantinos; Georgiou, Christos D; Nikolaropoulos, Sotirios S

    2014-08-01

    We have recently shown that retinoic acid induces micronucleation mainly via chromosome breakage (Alakhras et al. Cancer Lett 2011; 306: 15-26). To further study retinoic acid clastogenicity and evaluate DNA damaging potential we investigated the ability of (a) all-trans retinoic acid and its steroidal analogue EA-4 to induce DNA fragmentation by using Comet assay under alkaline unwinding and neutral condition electrophoresis, and (b) the retinoids under study to induce small (0-1 kb) DNA fragments. Two cell lines, C2C12 mouse cells and HL-60 human leukemic cells were used in this study. We found that all-trans retinoic acid and its steroidal analogue EA-4 (a) provoke DNA migration due to DNA fragmentation as it is shown by the increased values of Comet parameters, and (b) induce significantly small-size fragmented genomic DNA as indicated by the quantification of necrotic/apoptotic small DNA segments in both cell systems. A different response between the two cell lines was observed in relation to retinoid ability to increase the percentage of DNA in the tail as well as break DNA in to small fragments. Our findings confirm the ability of retinoic acid to provoke micronucleation by disrupting DNA into fragments, among which small pieces of double-stranded DNA up to 1 kb are identified. Copyright © 2013 John Wiley & Sons, Ltd.

  18. DNA degradation in genetically modified rice with Cry1Ab by food processing methods: implications for the quantification of genetically modified organisms.

    Science.gov (United States)

    Xing, Fuguo; Zhang, Wei; Selvaraj, Jonathan Nimal; Liu, Yang

    2015-05-01

    Food processing methods contribute to DNA degradation, thereby affecting genetically modified organism detection and quantification. This study evaluated the effect of food processing methods on the relative transgenic content of genetically modified rice with Cry1Ab. In steamed rice and rice noodles, the levels of Cry1Ab were ⩾ 100% and <83%, respectively. Frying and baking in rice crackers contributed to a reduction in Pubi and Cry1Ab, while microwaving caused a decrease in Pubi and an increase in Cry1Ab. The processing methods of sweet rice wine had the most severe degradation effects on Pubi and Cry1Ab. In steamed rice and rice noodles, Cry1Ab was the most stable, followed by SPS and Pubi. However, in rice crackers and sweet rice wine, SPS was the most stable, followed by Cry1Ab and Pubi. Therefore, Cry1Ab is a better representative of transgenic components than is Pubi because the levels of Cry1Ab were less affected compared to Pubi. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. DNA Damage Signaling Is Required for Replication of Human Bocavirus 1 DNA in Dividing HEK293 Cells.

    Science.gov (United States)

    Deng, Xuefeng; Xu, Peng; Zou, Wei; Shen, Weiran; Peng, Jianxin; Liu, Kaiyu; Engelhardt, John F; Yan, Ziying; Qiu, Jianming

    2017-01-01

    Human bocavirus 1 (HBoV1), an emerging human-pathogenic respiratory virus, is a member of the genus Bocaparvovirus of the Parvoviridae family. In human airway epithelium air-liquid interface (HAE-ALI) cultures, HBoV1 infection initiates a DNA damage response (DDR), activating all three phosphatidylinositol 3-kinase-related kinases (PI3KKs): ATM, ATR, and DNA-PKcs. In this context, activation of PI3KKs is a requirement for amplification of the HBoV1 genome (X. Deng, Z. Yan, F. Cheng, J. F. Engelhardt, and J. Qiu, PLoS Pathog, 12:e1005399, 2016, https://doi.org/10.1371/journal.ppat.1005399), and HBoV1 replicates only in terminally differentiated, nondividing cells. This report builds on the previous discovery that the replication of HBoV1 DNA can also occur in dividing HEK293 cells, demonstrating that such replication is likewise dependent on a DDR. Transfection of HEK293 cells with the duplex DNA genome of HBoV1 induces hallmarks of DDR, including phosphorylation of H2AX and RPA32, as well as activation of all three PI3KKs. The large viral nonstructural protein NS1 is sufficient to induce the DDR and the activation of the three PI3KKs. Pharmacological inhibition or knockdown of any one of the PI3KKs significantly decreases both the replication of HBoV1 DNA and the downstream production of progeny virions. The DDR induced by the HBoV1 NS1 protein does not cause obvious damage to cellular DNA or arrest of the cell cycle. Notably, key DNA replication factors and major DNA repair DNA polymerases (polymerase η [Pol η] and polymerase κ [Pol κ]) are recruited to the viral DNA replication centers and facilitate HBoV1 DNA replication. Our study provides the first evidence of the DDR-dependent parvovirus DNA replication that occurs in dividing cells and is independent of cell cycle arrest.

  20. Acute hypoxia and hypoxic exercise induce DNA strand breaks and oxidative DNA damage in humans

    DEFF Research Database (Denmark)

    Møller, P; Loft, S; Lundby, C

    2001-01-01

    ; lymphocytes were isolated for analysis of DNA strand breaks and oxidatively altered nucleotides, detected by endonuclease III and formamidipyridine glycosylase (FPG) enzymes. Urine was collected for 24 h periods for analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), a marker of oxidative DNA damage....... Urinary excretion of 8-oxodG increased during the first day in altitude hypoxia, and there were more endonuclease III-sensitive sites on day 3 at high altitude. The subjects had more DNA strand breaks in altitude hypoxia than at sea level. The level of DNA strand breaks further increased immediately after...... exercise in altitude hypoxia. Exercise-induced generation of DNA strand breaks was not seen at sea level. In both environments, the level of FPG and endonuclease III-sensitive sites remained unchanged immediately after exercise. DNA strand breaks and oxidative DNA damage are probably produced by reactive...

  1. Impact of Personal Characteristics and Technical Factors on Quantification of Sodium 18F-Fluoride Uptake in Human Arteries

    DEFF Research Database (Denmark)

    Blomberg, Björn Alexander; Thomassen, Anders; de Jong, Pim A

    2015-01-01

    Sodium (18)F-fluoride ((18)F-NaF) PET/CT imaging is a promising imaging technique for assessment of atherosclerosis, but is hampered by a lack of validated quantification protocols. Both personal characteristics and technical factors can affect quantification of arterial (18)F-NaF uptake. This st...

  2. The effects of different maceration techniques on nuclear DNA amplification using human bone.

    Science.gov (United States)

    Lee, Esther J; Luedtke, Jennifer G; Allison, Jamie L; Arber, Carolyn E; Merriwether, D Andrew; Steadman, Dawnie Wolfe

    2010-07-01

    Forensic anthropologists routinely macerate human bone for the purposes of identity and trauma analysis, but the heat and chemical treatments used can destroy genetic evidence. As a follow-up to a previous study on nuclear DNA recovery that used pig ribs, this study utilizes human skeletal remains treated with various bone maceration techniques for nuclear DNA amplification using the standard Combined DNA Index System (CODIS) markers. DNA was extracted from 18 samples of human lower leg bones subjected to nine chemical and heat maceration techniques. Genotyping was carried out using the AmpFlSTR COfiler and AmpFlSTR Profiler Plus ID kits. Results showed that heat treatments via microwave or Biz/Na(2)CO(3) in sub-boiling water efficiently macerate bone and produce amplifiable nuclear DNA for genetic analysis. Long-term use of chemicals such as hydrogen peroxide is discouraged as it results in poor bone quality and has deleterious effects on DNA amplification.

  3. Vertically integrated analysis of human DNA. Final technical report

    Energy Technology Data Exchange (ETDEWEB)

    Olson, M.

    1997-10-01

    This project has been oriented toward improving the vertical integration of the sequential steps associated with the large-scale analysis of human DNA. The central focus has been on an approach to the preparation of {open_quotes}sequence-ready{close_quotes} maps, which is referred to as multiple-complete-digest (MCD) mapping, primarily directed at cosmid clones. MCD mapping relies on simple experimental steps, supported by advanced image-analysis and map-assembly software, to produce extremely accurate restriction-site and clone-overlap maps. We believe that MCD mapping is one of the few high-resolution mapping systems that has the potential for high-level automation. Successful automation of this process would be a landmark event in genome analysis. Once other higher organisms, paving the way for cost-effective sequencing of these genomes. Critically, MCD mapping has the potential to provide built-in quality control for sequencing accuracy and to make possible a highly integrated end product even if there are large numbers of discontinuities in the actual sequence.

  4. The DNA sequence and biology of human chromosome 19.

    Science.gov (United States)

    Grimwood, Jane; Gordon, Laurie A; Olsen, Anne; Terry, Astrid; Schmutz, Jeremy; Lamerdin, Jane; Hellsten, Uffe; Goodstein, David; Couronne, Olivier; Tran-Gyamfi, Mary; Aerts, Andrea; Altherr, Michael; Ashworth, Linda; Bajorek, Eva; Black, Stacey; Branscomb, Elbert; Caenepeel, Sean; Carrano, Anthony; Caoile, Chenier; Chan, Yee Man; Christensen, Mari; Cleland, Catherine A; Copeland, Alex; Dalin, Eileen; Dehal, Paramvir; Denys, Mirian; Detter, John C; Escobar, Julio; Flowers, Dave; Fotopulos, Dea; Garcia, Carmen; Georgescu, Anca M; Glavina, Tijana; Gomez, Maria; Gonzales, Eidelyn; Groza, Matthew; Hammon, Nancy; Hawkins, Trevor; Haydu, Lauren; Ho, Isaac; Huang, Wayne; Israni, Sanjay; Jett, Jamie; Kadner, Kristen; Kimball, Heather; Kobayashi, Arthur; Larionov, Vladimer; Leem, Sun-Hee; Lopez, Frederick; Lou, Yunian; Lowry, Steve; Malfatti, Stephanie; Martinez, Diego; McCready, Paula; Medina, Catherine; Morgan, Jenna; Nelson, Kathryn; Nolan, Matt; Ovcharenko, Ivan; Pitluck, Sam; Pollard, Martin; Popkie, Anthony P; Predki, Paul; Quan, Glenda; Ramirez, Lucia; Rash, Sam; Retterer, James; Rodriguez, Alex; Rogers, Stephanine; Salamov, Asaf; Salazar, Angelica; She, Xinwei; Smith, Doug; Slezak, Tom; Solovyev, Victor; Thayer, Nina; Tice, Hope; Tsai, Ming; Ustaszewska, Anna; Vo, Nu; Wagner, Mark; Wheeler, Jeremy; Wu, Kevin; Xie, Gary; Yang, Joan; Dubchak, Inna; Furey, Terrence S; DeJong, Pieter; Dickson, Mark; Gordon, David; Eichler, Evan E; Pennacchio, Len A; Richardson, Paul; Stubbs, Lisa; Rokhsar, Daniel S; Myers, Richard M; Rubin, Edward M; Lucas, Susan M

    2004-04-01

    Chromosome 19 has the highest gene density of all human chromosomes, more than double the genome-wide average. The large clustered gene families, corresponding high G + C content, CpG islands and density of repetitive DNA indicate a chromosome rich in biological and evolutionary significance. Here we describe 55.8 million base pairs of highly accurate finished sequence representing 99.9% of the euchromatin portion of the chromosome. Manual curation of gene loci reveals 1,461 protein-coding genes and 321 pseudogenes. Among these are genes directly implicated in mendelian disorders, including familial hypercholesterolaemia and insulin-resistant diabetes. Nearly one-quarter of these genes belong to tandemly arranged families, encompassing more than 25% of the chromosome. Comparative analyses show a fascinating picture of conservation and divergence, revealing large blocks of gene orthology with rodents, scattered regions with more recent gene family expansions and deletions, and segments of coding and non-coding conservation with the distant fish species Takifugu.

  5. The DNA sequence of the human X chromosome.

    Science.gov (United States)

    Ross, Mark T; Grafham, Darren V; Coffey, Alison J; Scherer, Steven; McLay, Kirsten; Muzny, Donna; Platzer, Matthias; Howell, Gareth R; Burrows, Christine; Bird, Christine P; Frankish, Adam; Lovell, Frances L; Howe, Kevin L; Ashurst, Jennifer L; Fulton, Robert S; Sudbrak, Ralf; Wen, Gaiping; Jones, Matthew C; Hurles, Matthew E; Andrews, T Daniel; Scott, Carol E; Searle, Stephen; Ramser, Juliane; Whittaker, Adam; Deadman, Rebecca; Carter, Nigel P; Hunt, Sarah E; Chen, Rui; Cree, Andrew; Gunaratne, Preethi; Havlak, Paul; Hodgson, Anne; Metzker, Michael L; Richards, Stephen; Scott, Graham; Steffen, David; Sodergren, Erica; Wheeler, David A; Worley, Kim C; Ainscough, Rachael; Ambrose, Kerrie D; Ansari-Lari, M Ali; Aradhya, Swaroop; Ashwell, Robert I S; Babbage, Anne K; Bagguley, Claire L; Ballabio, Andrea; Banerjee, Ruby; Barker, Gary E; Barlow, Karen F; Barrett, Ian P; Bates, Karen N; Beare, David M; Beasley, Helen; Beasley, Oliver; Beck, Alfred; Bethel, Graeme; Blechschmidt, Karin; Brady, Nicola; Bray-Allen, Sarah; Bridgeman, Anne M; Brown, Andrew J; Brown, Mary J; Bonnin, David; Bruford, Elspeth A; Buhay, Christian; Burch, Paula; Burford, Deborah; Burgess, Joanne; Burrill, Wayne; Burton, John; Bye, Jackie M; Carder, Carol; Carrel, Laura; Chako, Joseph; Chapman, Joanne C; Chavez, Dean; Chen, Ellson; Chen, Guan; Chen, Yuan; Chen, Zhijian; Chinault, Craig; Ciccodicola, Alfredo; Clark, Sue Y; Clarke, Graham; Clee, Chris M; Clegg, Sheila; Clerc-Blankenburg, Kerstin; Clifford, Karen; Cobley, Vicky; Cole, Charlotte G; Conquer, Jen S; Corby, Nicole; Connor, Richard E; David, Robert; Davies, Joy; Davis, Clay; Davis, John; Delgado, Oliver; Deshazo, Denise; Dhami, Pawandeep; Ding, Yan; Dinh, Huyen; Dodsworth, Steve; Draper, Heather; Dugan-Rocha, Shannon; Dunham, Andrew; Dunn, Matthew; Durbin, K James; Dutta, Ireena; Eades, Tamsin; Ellwood, Matthew; Emery-Cohen, Alexandra; Errington, Helen; Evans, Kathryn L; Faulkner, Louisa; Francis, Fiona; Frankland, John; Fraser, Audrey E; Galgoczy, Petra; Gilbert, James; Gill, Rachel; Glöckner, Gernot; Gregory, Simon G; Gribble, Susan; Griffiths, Coline; Grocock, Russell; Gu, Yanghong; Gwilliam, Rhian; Hamilton, Cerissa; Hart, Elizabeth A; Hawes, Alicia; Heath, Paul D; Heitmann, Katja; Hennig, Steffen; Hernandez, Judith; Hinzmann, Bernd; Ho, Sarah; Hoffs, Michael; Howden, Phillip J; Huckle, Elizabeth J; Hume, Jennifer; Hunt, Paul J; Hunt, Adrienne R; Isherwood, Judith; Jacob, Leni; Johnson, David; Jones, Sally; de Jong, Pieter J; Joseph, Shirin S; Keenan, Stephen; Kelly, Susan; Kershaw, Joanne K; Khan, Ziad; Kioschis, Petra; Klages, Sven; Knights, Andrew J; Kosiura, Anna; Kovar-Smith, Christie; Laird, Gavin K; Langford, Cordelia; Lawlor, Stephanie; Leversha, Margaret; Lewis, Lora; Liu, Wen; Lloyd, Christine; Lloyd, David M; Loulseged, Hermela; Loveland, Jane E; Lovell, Jamieson D; Lozado, Ryan; Lu, Jing; Lyne, Rachael; Ma, Jie; Maheshwari, Manjula; Matthews, Lucy H; McDowall, Jennifer; McLaren, Stuart; McMurray, Amanda; Meidl, Patrick; Meitinger, Thomas; Milne, Sarah; Miner, George; Mistry, Shailesh L; Morgan, Margaret; Morris, Sidney; Müller, Ines; Mullikin, James C; Nguyen, Ngoc; Nordsiek, Gabriele; Nyakatura, Gerald; O'Dell, Christopher N; Okwuonu, Geoffery; Palmer, Sophie; Pandian, Richard; Parker, David; Parrish, Julia; Pasternak, Shiran; Patel, Dina; Pearce, Alex V; Pearson, Danita M; Pelan, Sarah E; Perez, Lesette; Porter, Keith M; Ramsey, Yvonne; Reichwald, Kathrin; Rhodes, Susan; Ridler, Kerry A; Schlessinger, David; Schueler, Mary G; Sehra, Harminder K; Shaw-Smith, Charles; Shen, Hua; Sheridan, Elizabeth M; Shownkeen, Ratna; Skuce, Carl D; Smith, Michelle L; Sotheran, Elizabeth C; Steingruber, Helen E; Steward, Charles A; Storey, Roy; Swann, R Mark; Swarbreck, David; Tabor, Paul E; Taudien, Stefan; Taylor, Tineace; Teague, Brian; Thomas, Karen; Thorpe, Andrea; Timms, Kirsten; Tracey, Alan; Trevanion, Steve; Tromans, Anthony C; d'Urso, Michele; Verduzco, Daniel; Villasana, Donna; Waldron, Lenee; Wall, Melanie; Wang, Qiaoyan; Warren, James; Warry, Georgina L; Wei, Xuehong; West, Anthony; Whitehead, Siobhan L; Whiteley, Mathew N; Wilkinson, Jane E; Willey, David L; Williams, Gabrielle; Williams, Leanne; Williamson, Angela; Williamson, Helen; Wilming, Laurens; Woodmansey, Rebecca L; Wray, Paul W; Yen, Jennifer; Zhang, Jingkun; Zhou, Jianling; Zoghbi, Huda; Zorilla, Sara; Buck, David; Reinhardt, Richard; Poustka, Annemarie; Rosenthal, André; Lehrach, Hans; Meindl, Alfons; Minx, Patrick J; Hillier, Ladeana W; Willard, Huntington F; Wilson, Richard K; Waterston, Robert H; Rice, Catherine M; Vaudin, Mark; Coulson, Alan; Nelson, David L; Weinstock, George; Sulston, John E; Durbin, Richard; Hubbard, Tim; Gibbs, Richard A; Beck, Stephan; Rogers, Jane; Bentley, David R

    2005-03-17

    The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence.

  6. The DNA sequence and biology of human chromosome 19

    Energy Technology Data Exchange (ETDEWEB)

    Grimwood, J; Gordon, L A; Olsen, A; Terry, A; Schmutz, J; Lamerdin, J; Hellsten, U; Goodstein, D; Couronne, O; Tran-Gyamfi, M

    2004-04-06

    Chromosome 19 has the highest gene density of all human chromosomes, more than double the genome-wide average. The large clustered gene families, corresponding high GC content, CpG islands and density of repetitive DNA indicate a chromosome rich in biological and evolutionary significance. Here we describe 55.8 million base pairs of highly accurate finished sequence representing 99.9% of the euchromatin portion of the chromosome. Manual curation of gene loci reveals 1,461 protein-coding genes and 321 pseudogenes. Among these are genes directly implicated in Mendelian disorders, including familial hypercholesterolemia and insulin-resistant diabetes. Nearly one quarter of these genes belong to tandemly arranged families, encompassing more than 25% of the chromosome. Comparative analyses show a fascinating picture of conservation and divergence, revealing large blocks of gene orthology with rodents, scattered regions with more recent gene family expansions and deletions, and segments of coding and non-coding conservation with the distant fish species Takifugu.

  7. The DNA sequence and biology of human chromosome 19

    Energy Technology Data Exchange (ETDEWEB)

    Grimwood, Jane; Gordon, Laurie A.; Olsen, Anne; Terry, Astrid; Schmutz, Jeremy; Lamerdin, Jane; Hellsten, Uffe; Goodstein, David; Couronne, Olivier; Tran-Gyamfi, Mary; Aerts, Andrea; Altherr, Michael; Ashworth, Linda; Bajorek, Eva; Black, Stacey; Branscomb, Elbert; Caenepeel, Sean; Carrano, Anthony; Caoile, Chenier; Chan, Yee Man; Christensen, Mari; Cleland, Catherine A.; Copeland, Alex; Dalin, Eileen; Dehal, Paramvir; Denys, Mirian; Detter, John C.; Escobar, Julio; Flowers, Dave; Fotopulos, Dea; Garcia, Carmen; Georgescu, Anca M.; Glavina, Tijana; Gomez, Maria; Gonzales, Eldelyn; Groza, Matthew; Hammon, Nancy; Hawkins, Trevor; Haydu, Lauren; Ho, Issac; Huang, Wayne; Israni, Sanjay; Jett, Jamie; Kadner, Kristen; Kimball, Heather; Kobayashi, Arthur; Larionov, Vladimer; Leem, Sun-Hee; Lopez, Frederick; Lou, Yunian; Lowry, Steve; Malfatti, Stephanie; Martinez, Diego; McCready, Paula; Medina, Catherine; Morgan, Jenna; Nelson, Kathryn; Nolan, Matt; Ovcharenko, Ivan; Pitluck, Sam; Pollard, Martin; Popkie, Anthony P.; Predki, Paul; Quan, Glenda; Ramirez, Lucia; Rash, Sam; Retterer, James; Rodriguez, Alex; Rogers, Stephanine; Salamov, Asaf; Salazar, Angelica; She, Xinwei; Smith, Doug; Slezak, Tom; Solovyev, Victor; Thayer, Nina; Tice, Hope; Tsai, Ming; Ustaszewska, Anna; Vo, Nu; Wagner, Mark; Wheeler, Jeremy; Wu, Kevin; Xie, Gary; Yang, Joan; Dubchak, Inna; Furey, Terrence S.; DeJong, Pieter; Dickson, Mark; Gordon, David; Eichler, Evan E.; Pennacchio, Len A.; Richardson, Paul; Stubbs, Lisa; Rokhsar, Daniel S.; Myers, Richard M.; Rubin, Edward M.; Lucas, Susan M.

    2003-09-15

    Chromosome 19 has the highest gene density of all human chromosomes, more than double the genome-wide average. The large clustered gene families, corresponding high G1C content, CpG islands and density of repetitive DNA indicate a chromosome rich in biological and evolutionary significance. Here we describe 55.8 million base pairs of highly accurate finished sequence representing 99.9 percent of the euchromatin portion of the chromosome. Manual curation of gene loci reveals 1,461 protein-coding genes and 321 pseudogenes. Among these are genes directly implicated in mendelian disorders, including familial hypercholesterolaemia and insulin-resistant diabetes. Nearly one-quarter of these genes belong to tandemly arranged families, encompassing more than 25 percent of the chromosome. Comparative analyses show a fascinating picture of conservation and divergence, revealing large blocks of gene orthology with rodents, scattered regions with more recent gene family expansions and deletions, a nd segments of coding and non-coding conservation with the distant fish species Takifugu.

  8. Light chain editors of anti-DNA receptors in human B cells.

    Science.gov (United States)

    Kalinina, Olga; Wang, Yue; Sia, Kevin; Radic, Marko; Cazenave, Pierre-André; Weigert, Martin

    2014-02-10

    Receptor editing is a mechanism of self-tolerance used in newly generated B cells. The expressed heavy (H) or light (L) chain of an autoreactive receptor is replaced by upstream V genes which eliminate or modify autoreactivity. Editing of anti-DNA receptors has been characterized in anti-DNA transgenic mouse models including 3H9, 3H9/56R, and their revertant 3H9GL. Certain L chains, termed editors, rescue anti-DNA B cells by neutralizing or modifying DNA binding of the H chain. This editing mechanism acts on the natural H chain repertoire; endogenous H chains with anti-DNA features are expressed primarily in combination with editor L chains. We ask whether a similar set of L chains exists in the human repertoire, and if so, do they edit H chains with anti-DNA signatures? We compared the protein sequences of mouse editors to all human L chains and found several human L chains similar to mouse editors. These L chains diminish or veto anti-DNA binding when expressed with anti-DNA H chains. The human H chains expressed with these L chains also have relatively high arginine (Arg) content in the H chain complementarity determining region (H3), suggesting that receptor editing plays a role in establishing tolerance to DNA in humans.

  9. Quantification of MRI measured myocardial perfusion reserve in healthy humans: a comparison with positron emission tomography

    DEFF Research Database (Denmark)

    Fritz-Hansen, Thomas; Hove, Jens D; Kofoed, Klaus F

    2008-01-01

    PURPOSE: To validate a noninvasive quantitative MRI technique, the K(i) perfusion method, for myocardial perfusion in humans using (13)N-ammonia PET as a reference method. MATERIALS AND METHODS: Ten healthy males (64 +/- 8 years) were examined with combined PET and MRI perfusion imaging at rest...... and during stress induced by dipyridamole in order to determine the myocardial perfusion reserve. Myocardial and blood time concentration curves obtained by Gd-DTPA-enhanced MRI and (13)N-ammonia PET were fitted by a two-compartment perfusion model. RESULTS: Mean perfusion values (+/-SD) derived from the MRI...... as a quantitative marker for myocardial perfusion in healthy humans....

  10. Digital quantification of human eye color highlights genetic association of three new loci.

    NARCIS (Netherlands)

    F. Liu (Fan); A. Wollstein (Andreas); P.G. Hysi (Pirro); G.A. Ankra-Badu (Georgina); T.D. Spector (Timothy); D.J. Park (Daniel); G. Zhu; M. Larsson (Mats); D.L. Duffy (David); G.W. Montgomery (Grant); D.A. Mackey (David); S. Walsh (Susan); O. Lao Grueso (Oscar); A. Hofman (Albert); F. Rivadeneira Ramirez (Fernando); J.R. Vingerling (Hans); A.G. Uitterlinden (André); N.G. Martin (Nicholas); C.J. Hammond (Christopher); M.H. Kayser (Manfred)

    2010-01-01

    textabstractPrevious studies have successfully identified genetic variants in several genes associated with human iris (eye) color; however, they all used simplified categorical trait information. Here, we quantified continuous eye color variation into hue and saturation values using high-resolution

  11. Noninvasive quantification of ascorbate and glutathione concentration in the elderly human brain

    Science.gov (United States)

    In this study, Ascorbate (Asc) and glutathione (GSH) concentrations were non-invasively quantified using double edited 1H MRS at 4T in the occipital cortex of healthy young(age 18 – 22 years, N = 22) and elderly (age 70 – 89 years, N = 22) human subjects. Elderly subjects had a lower GSH concentrati...

  12. Quantification of Climate Changes and Human Activities That Impact Runoff in the Taihu Lake Basin, China

    Directory of Open Access Journals (Sweden)

    Dingzhi Peng

    2016-01-01

    Full Text Available Although a fragile climate region, the Taihu Lake Basin is among the most developed regions in China and is subjected to intense anthropogenic interference. In this basin, water resources encounter major challenges (e.g., floods, typhoons, and water pollution. In this study, the impacts of climate changes and human activities on hydrological processes were estimated to aid water resource management in developed regions in China. The Mann-Kendall test and cumulative anomaly curve were applied to detect the turning points in the runoff series. The year of 1982 divides the study period (1956~2008 into a baseline period (1956~1981 and a modified period (1982~2008. The double mass curve method and the hydrological sensitivity method based on the Budyko framework were applied to quantitatively attribute the runoff variation to climate changes and human activities. The results demonstrated that human activities are the dominant driving force of runoff variations in the basin, with a contribution of 83~89%; climate changes contributed to 11~17% of the variations. Moreover, the subregions of the basin indicated that humans severely disturbed the runoff variation, with contributions as high as 95~97%.

  13. Immunomagnetic separation and quantification of butyrylcholinesterase nerve agent adducts in human serum

    NARCIS (Netherlands)

    Sporty, J.L.S.; Lemire, S.W.; Jakubowski, E.M.; Renner, J.A.; Evans, R.A.; Williams, R.F.; Schmidt, J.G.; Schans, M.J. van der; Noort, D.; Johnson, R.C.

    2010-01-01

    A novel method for extracting butyrylcholinesterase (BuChE) from serum as a means of identifying and measuring nerve agent adducts to human BuChE is presented here. Antibutyrylcholinesterase monoclonal antibodies were conjugated to protein-G ferromagnetic particles and mixed with 500 μL serum sample

  14. ER-mitochondria contacts couple mtDNA synthesis with mitochondrial division in human cells.

    Science.gov (United States)

    Lewis, Samantha C; Uchiyama, Lauren F; Nunnari, Jodi

    2016-07-15

    Mitochondrial DNA (mtDNA) encodes RNAs and proteins critical for cell function. In human cells, hundreds to thousands of mtDNA copies are replicated asynchronously, packaged into protein-DNA nucleoids, and distributed within a dynamic mitochondrial network. The mechanisms that govern how nucleoids are chosen for replication and distribution are not understood. Mitochondrial distribution depends on division, which occurs at endoplasmic reticulum (ER)-mitochondria contact sites. These sites were spatially linked to a subset of nucleoids selectively marked by mtDNA polymerase and engaged in mtDNA synthesis--events that occurred upstream of mitochondrial constriction and division machine assembly. Our data suggest that ER tubules proximal to nucleoids are necessary but not sufficient for mtDNA synthesis. Thus, ER-mitochondria contacts coordinate licensing of mtDNA synthesis with division to distribute newly replicated nucleoids to daughter mitochondria.

  15. Nuclear Localization and DNA Binding Properties of a Protein Expressed by Human c-myc Oncogene

    Science.gov (United States)

    Persson, Hakan; Leder, Philip

    1984-08-01

    Antisera to the human cellular myc oncogene product were used to identify a human c-myc specific protein with a molecular weight of 65,000. Subcellular fractionation showed that the human c-myc protein is predominantly found in the cell nucleus. The p65 Kc-myc protein binds to double- and single-stranded DNA as measured by a DNA affinity chromatography assay.

  16. No increased sperm DNA fragmentation index in semen containing human papillomavirus or herpesvirus

    DEFF Research Database (Denmark)

    Kaspersen, Maja Døvling; Bungum, Mona; Fedder, Jens

    2013-01-01

    -based hybridization array that identifies all HHVs and 35 of the most common HPVs. Sperm DNA integrity was determined by the sperm chromatin structure assay. HPVs or HHVs, or both, were found in 57% of semen samples; however, sperm DNA fragmentation index was not increased in semen containing these viruses.......It remains unknown whether human papillomaviruses (HPVs) or human herpesviruses (HHVs) in semen affect sperm DNA integrity. We investigated whether the presence of these viruses in semen was associated with an elevated sperm DNA fragmentation index. Semen from 76 sperm donors was examined by a PCR...

  17. Extraction of high quality genomic DNA from microsamples of human blood.

    Science.gov (United States)

    Ma, H W; Cheng, J; Caddy, B

    1994-01-01

    A simple and efficient method for extracting human genomic DNA from microsamples of blood has been developed. This method used sodium perchlorate, chloroform, polymerised silica gel and a dumbbell-shape tube, instead of proteinase K and phenol. The entire process took less than two hours, and high molecular weight DNA, in high yield and purity, was obtained from a few microlitres of human blood. DNA prepared in this way can be easily digested with restriction endonucleases and has been employed for DNA profiling and the polymerase chain reaction.

  18. A DNA methylation signature associated with aberrant promoter DNA hypermethylation of DNMT3B in human colorectal cancer.

    Science.gov (United States)

    Huidobro, Covadonga; Urdinguio, Rocío G; Rodríguez, Ramón María; Mangas, Cristina; Calvanese, Vincenzo; Martínez-Camblor, Pablo; Ferrero, Cecilia; Parra-Blanco, Adolfo; Rodrigo, Luis; Obaya, Alvaro J; Suárez-Fernández, Laura; Astudillo, Aurora; Hernando, Henar; Ballestar, Esteban; Fernández, Agustín F; Fraga, Mario F

    2012-09-01

    Altered promoter DNA methylation, one of the most important molecular alterations in cancer, is proposed to correlate with deregulation of DNA methyltransferases, although the molecular mechanisms implicated are still poorly understood. Here we show that the de novo DNA methyltransferase DNMT3B is frequently repressed in human colorectal cancer cell lines (CCL) and primary tumours by aberrant DNA hypermethylation of its distal promoter. At the epigenome level, DNMT3B promoter hypermethylation was associated with the hypomethylation of gene promoters usually hypermethylated in the healthy colon. Forced DNMT3B overexpression in cancer cells restored the methylation levels of these promoters in the healthy colon. Our results show a new molecular mechanism of aberrant DNMT3B regulation in colon cancer and suggest that its expression is associated with the methylation of constitutively hypermethylated promoters in the healthy colon. Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. Induction of a mutant phenotype in human repair proficient cells after overexpression of a mutated human DNA repair gene.

    NARCIS (Netherlands)

    P.B.G.M. Belt; M.F. van Oostenrijk; H. Odijk (Hanny); J.H.J. Hoeijmakers (Jan); C.M.P. Backendorf (Claude)

    1991-01-01

    textabstractAntisense and mutated cDNA of the human excision repair gene ERCC-1 were overexpressed in repair efficient HeLa cells by means of an Epstein-Barr-virus derived CDNA expression vector. Whereas antisense RNA did not influence the survival of the transfected cells, a mutated cDNA generating

  20. Evaluating the Impact of Contaminant Dilution and Biodegradation in Uncertainty Quantification of Human Health Risk

    Science.gov (United States)

    Zarlenga, Antonio; de Barros, Felipe; Fiori, Aldo

    2016-04-01

    We present a probabilistic framework for assessing human health risk due to groundwater contamination. Our goal is to quantify how physical hydrogeological and biochemical parameters control the magnitude and uncertainty of human health risk. Our methodology captures the whole risk chain from the aquifer contamination to the tap water assumption by human population. The contaminant concentration, the key parameter for the risk estimation, is governed by the interplay between the large-scale advection, caused by heterogeneity and the degradation processes strictly related to the local scale dispersion processes. The core of the hazard identification and of the methodology is the reactive transport model: erratic displacement of contaminant in groundwater, due to the spatial variability of hydraulic conductivity (K), is characterized by a first-order Lagrangian stochastic model; different dynamics are considered as possible ways of biodegradation in aerobic and anaerobic conditions. With the goal of quantifying uncertainty, the Beta distribution is assumed for the concentration probability density function (pdf) model, while different levels of approximation are explored for the estimation of the one-point concentration moments. The information pertaining the flow and transport is connected with a proper dose response assessment which generally involves the estimation of physiological parameters of the exposed population. Human health response depends on the exposed individual metabolism (e.g. variability) and is subject to uncertainty. Therefore, the health parameters are intrinsically a stochastic. As a consequence, we provide an integrated in a global probabilistic human health risk framework which allows the propagation of the uncertainty from multiple sources. The final result, the health risk pdf, is expressed as function of a few relevant, physically-based parameters such as the size of the injection area, the Péclet number, the K structure metrics and

  1. Detection of DNA Damage by Space Radiation in Human Fibroblasts Flown on the International Space Station

    Science.gov (United States)

    Lu, Tao; Zhang, Ye; Wong, Michael; Feiveson, Alan; Gaza, Ramona; Stoffle, Nicholas; Wang, Huichen; Wilson, Bobby; Rohde, Larry; Stodieck, Louis; hide

    2017-01-01

    Space radiation consists of energetic charged particles of varying charges and energies. Exposure of astronauts to space radiation on future long duration missions to Mars, or missions back to the Moon, is expected to result in deleterious consequences such as cancer and comprised central nervous system (CNS) functions. Space radiation can also cause mutation in microorganisms, and potentially influence the evolution of life in space. Measurement of the space radiation environment has been conducted since the very beginning of the space program. Compared to the quantification of the space radiation environment using physical detectors, reports on the direct measurement of biological consequences of space radiation exposure have been limited, due primarily to the low dose and low dose rate nature of the environment. Most of the biological assays fail to detect the radiation effects at acute doses that are lower than 5 centiSieverts. In a recent study, we flew cultured confluent human fibroblasts in mostly G1 phase of the cell cycle to the International Space Station (ISS). The cells were fixed in space after arriving on the ISS for 3 and 14 days, respectively. The fixed cells were later returned to the ground and subsequently stained with the gamma-H2AX (Histone family, member X) antibody that are commonly used as a marker for DNA damage, particularly DNA double strand breaks, induced by both low-and high-linear energy transfer radiation. In our present study, the gamma-H2AX (Histone family, member X) foci were captured with a laser confocal microscope. To confirm that some large track-like foci were from space radiation exposure, we also exposed, on the ground, the same type of cells to both low-and high-linear energy transfer protons, and high-linear energy transfer Fe ions. In addition, we exposed the cells to low dose rate gamma rays, in order to rule out the possibility that the large track-like foci can be induced by chronic low-linear energy transfer

  2. Quantification of color alteration in human teeth with optical coherence tomography

    Science.gov (United States)

    Ni, Y. R.; Guo, Z. Y.; Shu, S. Y.; Zeng, C. C.; Zhong, H. Q.; Chen, B. L.; Liu, Z. M.; Bao, Y.

    2011-11-01

    It is necessary to develop a laboratory model to evaluate tooth discoloration, because there are several limitations to assessment methods at present stage. Therefore, in this letter, we report the results from a pilot study on using optical coherence tomography imaging method to quantify color alteration in the human teeth treatment with 35% hydrogen peroxide bleaching in vitro. Quantitative comparison of chromogens reduction in dental tissue showed that near infrared attenuation coefficient (μ) increased for enamel with the bleaching passage time and diminution for dentine. Therefore, the precise detection of the change in attenuation coefficient is can accurate quantitative chromogens alteration in tooth. OCT has a potential to become a useful tool for the assessment color alteration in human teeth.

  3. Identification and quantification of serum proteins secreted into the normal human jejunum

    DEFF Research Database (Denmark)

    Andersen, Vibeke; Hegnhøj, J H

    1990-01-01

    The in vivo transfer of serum proteins to the human intestinal lumen was characterized by crossed immunoelectrophoretic analyses of intestinal perfusates from four healthy volunteers. Serum proteins with molecular masses below 100 kDa and the immunoglobulins were found in human jejunal perfusates....... Larger serum proteins were either absent (alpha and beta lipoproteins) or present in small amounts (alpha 2-macroglobulin, haptoglobulin and ceruloplasmin). These results demonstrate the existence of a selective transfer of serum proteins to the intestinal lumen under physiological conditions....... The intestinal clearance rate was 0.1 ml serum per hour per 10 cm jejunum for albumin, prealbumin, alpha 1-antitrypsin, orosomucoid, transferrin and haemopexin. The rate of secretion of total protein to the jejunal lumen was 100 mg protein per hour per 10 cm jejunum. About 45% was due to immunoglobulins...

  4. Quantification of Climate Changes and Human Activities That Impact Runoff in the Taihu Lake Basin, China

    OpenAIRE

    Dingzhi Peng; Linghua Qiu; Jing Fang; Zhongyuan Zhang

    2016-01-01

    Although a fragile climate region, the Taihu Lake Basin is among the most developed regions in China and is subjected to intense anthropogenic interference. In this basin, water resources encounter major challenges (e.g., floods, typhoons, and water pollution). In this study, the impacts of climate changes and human activities on hydrological processes were estimated to aid water resource management in developed regions in China. The Mann-Kendall test and cumulative anomaly curve were applied...

  5. Gap-directed translesion DNA synthesis of an abasic site on circular DNA templates by a human replication complex.

    Directory of Open Access Journals (Sweden)

    Giuseppe Villani

    Full Text Available DNA polymerase ε (pol ε is believed to be the leading strand replicase in eukaryotes whereas pols λ and β are thought to be mainly involved in re-synthesis steps of DNA repair. DNA elongation by the human pol ε is halted by an abasic site (apurinic/apyrimidinic (AP site. We have previously reported that human pols λ, β and η can perform translesion synthesis (TLS of an AP site in the presence of pol ε. In the case of pol λ and β, this TLS requires the presence of a gap downstream from the product synthetized by the ε replicase. However, since these studies were conducted exclusively with a linear DNA template, we decided to test whether the structure of the template could influence the capacity of the pols ε, λ, β and η to perform TLS of an AP site. Therefore, we have investigated the replication of damaged "minicircle" DNA templates. In addition, replication of circular DNA requires, beyond DNA pols, the processivity clamp PCNA, the clamp loader replication factor C (RFC, and the accessory proteins replication protein A (RPA. Finally we have compared the capacity of unmodified versus monoubiquitinated PCNA in sustaining TLS by pols λ and η on a circular template. Our results indicate that in vitro gap-directed TLS synthesis by pols λ and β in the presence of pol ε, RPA and PCNA is unaffected by the structure of the DNA template. Moreover, monoubiquitination of PCNA does not affect TLS by pol λ while it appears to slightly stimulate TLS by pol η.

  6. cDNA cloning, sequence analysis, and chromosomal localization of the gene for human carnitine palmitoyltransferase

    Energy Technology Data Exchange (ETDEWEB)

    Finocchiaro, G.; Taroni, F.; Martin, A.L.; Colombo, I.; Tarelli, G.T.; DiDonato, S. (Istituto Nazionale Neurologico C. Besta, Milan (Italy)); Rocchi, M. (Istituto G. Gaslini, Genoa (Italy))

    1991-01-15

    The authors have cloned and sequenced a cDNA encoding human liver carnitine palmitoyltransferase an inner mitochondrial membrane enzyme that plays a major role in the fatty acid oxidation pathway. Mixed oligonucleotide primers whose sequences were deduced from one tryptic peptide obtained from purified CPTase were used in a polymerase chain reaction, allowing the amplification of a 0.12-kilobase fragment of human genomic DNA encoding such a peptide. A 60-base-pair (bp) oligonucleotide synthesized on the basis of the sequence from this fragment was used for the screening of a cDNA library from human liver and hybridized to a cDNA insert of 2255 bp. This cDNA contains an open reading frame of 1974 bp that encodes a protein of 658 amino acid residues including 25 residues of an NH{sub 2}-terminal leader peptide. The assignment of this open reading frame to human liver CPTase is confirmed by matches to seven different amino acid sequences of tryptic peptides derived from pure human CPTase and by the 82.2% homology with the amino acid sequence of rat CPTase. The NH{sub 2}-terminal region of CPTase contains a leucine-proline motif that is shared by carnitine acetyl- and octanoyltransferases and by choline acetyltransferase. The gene encoding CPTase was assigned to human chromosome 1, region 1q12-1pter, by hybridization of CPTase cDNA with a DNA panel of 19 human-hanster somatic cell hybrids.

  7. Quantification of the fate of dietary fiber in humans by a newly developed radiolabeled fiber marker

    Energy Technology Data Exchange (ETDEWEB)

    Carryer, P.W.; Brown, M.I.; Malagelada, J.R.; Carlson, G.L.; McCall, J.T.

    1982-06-01

    A radiolabeled cellulose (/sup 131/I-fiber) that retains the essential physical and chemical properties of this class of fiber was developed in our laboratory. Researchers quantified the fate of orally ingested /sup 131/I-fiber in healthy individuals by external gamma camera monitoring and fecal collections. The marker passes virtually intact through the human gastrointestinal tract with negligible release and absorption of the label in the gut. Comparison of the gastric emptying rate of /sup 131/I-fiber with that of a predominantly aqueous marker, /sup 99/mTc-diethylenetriamine pentaacetic acid (/sup 99/mTc-DTPA), showed that /sup 131/I-fiber strands were evacuated more slowly than intragastric fluids. An important finding was that some /sup 131/I-fiber emptying occurred during most time periods, even before liquids were completely evacuated. This suggests that the human stomach is able to empty simultaneously liquids and fiber strands (1-15 mm in length) that are resistant to grinding by antral mechanical forces and to digestion by acid-peptic secretion. Thus, some nondigestible solids may be emptied with the bulk of a meal, although at a slower rate. /sup 131/I-Fiber may be a useful marker for quantifying gastric emptying of nondigestible solids. Further, the stability of /sup 131/I-fiber in the gut, as opposed to most other physiologic solid labels, should enable future investigation of intestinal and colonic transit of fiber, which is an important component of the human diet.

  8. Propofol detection and quantification in human blood: the promise of feedback controlled, closed-loop anesthesia.

    Science.gov (United States)

    Kivlehan, Francine; Chaum, Edward; Lindner, Ernő

    2015-01-07

    The performance of a membrane-coated voltammetric sensor for propofol (2,6-diisopropylphenol) has been characterized in long term monitoring experiments using an automated flow analytical system (AFAS) and by analyzing human serum and whole blood samples by standard addition. It is shown that the signal of the membrane-coated electrochemical sensor for propofol is not influenced by the components of the pharmaceutical formulation of propofol (propofol injectable emulsion). The current values recorded with the electrochemical propofol sensor in buffer solutions and human serum samples spiked with propofol injectable emulsion showed excellent correlation with the peak heights recorded with an UV-Vis detector during the HPLC analysis of these samples (R(2) = 0.997 in PBS and R(2) = 0.975 in human serum). However, the determination of propofol using the electrochemical method is simpler, faster and has a better detection limit (0.08 ± 0.05 μM) than the HPLC method (0.4 ± 0.2 μM). As a first step towards feedback controlled closed-loop anesthesia, the membrane-coated electrochemical sensor has been implemented onto surface of an intravenous catheter. The response characteristics of the membrane-coated carbon fiber electrode on the catheter surface were very similar to those seen using a macroelectrode.

  9. Uncertainty quantification of adverse human health effects from continuously released contaminant sources in groundwater systems

    Science.gov (United States)

    Zarlenga, Antonio; de Barros, Felipe P. J.; Fiori, Aldo

    2016-10-01

    We propose a computationally efficient probabilistic modeling methodology to estimate the adverse effects on humans of exposure to contaminated groundwater. Our work is aligned with the standard suggested by the regulatory agencies and allows to propagate uncertainty from hydrogeological, toxicological and behavioral parameters to the final health risk endpoint. The problem under consideration consists of a contaminated aquifer supplying water to a population. Contamination stems from a continuous source that feeds a steady plume which constitutes the hazard source. This scenario is particularly suited for NAPL pollutants. The erratic displacement of the contaminant plume in groundwater, due to the spatial variability of hydraulic conductivity, is characterized within the Lagrangian stochastic framework which enables the complete probabilistic characterization of the contaminant concentration at an environmentally sensitive location. Following the probabilistic characterization of flow and transport, we quantify the adverse health effects on humans. The dose response assessment involves the estimation of the uncertain effects of the exposure to a given contaminant while accounting for the exposed individual's metabolism. The model integrates groundwater transport, exposure and human metabolism in a comprehensive probabilistic framework which allows the assessment of the risk probability through a novel simple analytical solution. Aside from its computational efficiency, the analytical features of the framework allows the assessment of uncertainty arising from the hydrogeological parameters.

  10. Ligand autoradiographical quantification of histamine H3 receptor in human dementia with Lewy bodies.

    Science.gov (United States)

    Lethbridge, Natasha L; Chazot, Paul L

    2016-11-01

    Dementia with Lewy bodies (DLB) is a serious age-dependent human neurodegenerative disease, with multiple debilitating symptoms, including dementia, psychosis and significant motor deficits, but with little or no effective treatments. This comparative ligand autoradiographical study has quantified histamine H3 receptors (H3R) in a series of major cortical and basal ganglia structures in human DLB and Alzheimer's (AD) post-mortem cases using the highly selective radioligand, [(3)H] GSK189254. In the main, the levels of H3 receptor were largely preserved in DLB cases when compared with aged-matched controls. However, we provide new evidence showing variable levels in the globus pallidus, and, moreover, raised levels of Pallidum H3 correlated with positive psychotic symptoms, in particular delusions and visual hallucinations, but not symptoms associated with depression. Furthermore, no correlation was detected for H3 receptor levels to MMSE or IUPRS symptom severity. This study suggests that H3R antagonists have scope for treating the psychotic symptomologies in DLB and other human brain disorders.

  11. Characterization and Quantification of Innate Lymphoid Cell Subsets in Human Lung.

    Directory of Open Access Journals (Sweden)

    Katrien C De Grove

    Full Text Available Innate lymphoid cells (ILC are a new family of innate immune cells that have emerged as important regulators of tissue homeostasis and inflammation. However, limited data are available concerning the relative abundance and characteristics of ILC in the human lung.The aim of this study was to characterize and enumerate the different ILC subsets in human lung by multi-color flow cytometry.Within the CD45+ Lin- CD127+ pulmonary ILC population, we identified group 1 (ILC1, group 2 (ILC2 and group 3 (ILC3 innate lymphoid cells using specific surface markers (i.e. IL12Rβ2, CRTH2 and CD117 respectively and key transcription factors (i.e. T-bet, GATA-3 and RORγT respectively. Based on the presence of NKp44, ILC3 were further subdivided in natural cytotoxicity receptor (NCR+ and NCR- ILC3. In addition, we demonstrated the production of signature cytokines IFN-γ, IL-5, IL-17A, IL-22 and GM-CSF in the pulmonary ILC population. Interestingly, we observed a tendency to a higher frequency of NCR- ILC3 in lungs of patients with chronic obstructive pulmonary disease (COPD compared with controls.We show that the three main ILC subsets are present in human lung. Importantly, the relative abundance of ILC subsets tended to change in COPD patients in comparison to control individuals.

  12. Isolation of 24 novel cDNA fragments from microdis—sected human chromosome band

    Institute of Scientific and Technical Information of China (English)

    ZHANGMIN; LONGYU; 等

    1998-01-01

    The strategy of isolating the band0specific expression fragments from a probe pool generated by human chromosome microdissection was reported.A chromosome 14q 24.3 band-specific single copy DNA pool was constructed based on this probe pool.Using total DNA of the pool as probe to hybridize the human marrow cDNA library,68 primary positive clones were selected from 5×105 cDNA clones.Among these primary clones,32 secondary clones were obtained after second-round screening and designed as cFD14-1-32.Finally,24 band-specific expression fragments were identified from these 32 positive clones by DNA hybridization.Those band-specific clones can hybridize to both 14q24.3 DNA and human genomic DNA but cann't hybridize to 17q11-12 DNA,Partial sequences of 13 fragments of them were sequenced and idenfified as novel cDNA sequences,and these sequences were proved to have some homology with known genes in NCBI database.Analysis of expression spectrum of cFD 14-1 suggested that the cDNA fragments thus obtained should be used to isolate the genes can not been cloned in 14q24.3 region.

  13. mtDNA variation predicts population size in humans and reveals a major Southern Asian chapter in human prehistory.

    Science.gov (United States)

    Atkinson, Quentin D; Gray, Russell D; Drummond, Alexei J

    2008-02-01

    The relative timing and size of regional human population growth following our expansion from Africa remain unknown. Human mitochondrial DNA (mtDNA) diversity carries a legacy of our population history. Given a set of sequences, we can use coalescent theory to estimate past population size through time and draw inferences about human population history. However, recent work has challenged the validity of using mtDNA diversity to infer species population sizes. Here we use Bayesian coalescent inference methods, together with a global data set of 357 human mtDNA coding-region sequences, to infer human population sizes through time across 8 major geographic regions. Our estimates of relative population sizes show remarkable concordance with the contemporary regional distribution of humans across Africa, Eurasia, and the Americas, indicating that mtDNA diversity is a good predictor of population size in humans. Plots of population size through time show slow growth in sub-Saharan Africa beginning 143-193 kya, followed by a rapid expansion into Eurasia after the emergence of the first non-African mtDNA lineages 50-70 kya. Outside Africa, the earliest and fastest growth is inferred in Southern Asia approximately 52 kya, followed by a succession of growth phases in Northern and Central Asia (approximately 49 kya), Australia (approximately 48 kya), Europe (approximately 42 kya), the Middle East and North Africa (approximately 40 kya), New Guinea (approximately 39 kya), the Americas (approximately 18 kya), and a second expansion in Europe (approximately 10-15 kya). Comparisons of relative regional population sizes through time suggest that between approximately 45 and 20 kya most of humanity lived in Southern Asia. These findings not only support the use of mtDNA data for estimating human population size but also provide a unique picture of human prehistory and demonstrate the importance of Southern Asia to our recent evolutionary past.

  14. Microbial DNA fingerprinting of human fingerprints: dynamic colonization of fingertip microflora challenges human host inferences for forensic purposes

    OpenAIRE

    Tims, S.; Wamel, van, JJ Jos; Endtz, H. P.; Belkum, van, A.; Kayser, M

    2009-01-01

    Human fingertip microflora is transferred to touched objects and may provide forensically relevant information on individual hosts, such as on geographic origins, if endogenous microbial skin species/strains would be retrievable from physical fingerprints and would carry geographically restricted DNA diversity. We tested the suitability of physical fingerprints for revealing human host information, with geographic inference as example, via microbial DNA fingerprinting. We showed that the tran...

  15. Inhibiting DNA-PKCS radiosensitizes human osteosarcoma cells.

    Science.gov (United States)

    Mamo, Tewodros; Mladek, Ann C; Shogren, Kris L; Gustafson, Carl; Gupta, Shiv K; Riester, Scott M; Maran, Avudaiappan; Galindo, Mario; van Wijnen, Andre J; Sarkaria, Jann N; Yaszemski, Michael J

    2017-04-29

    Osteosarcoma survival rate has not improved over the past three decades, and the debilitating side effects of the surgical treatment suggest the need for alternative local control approaches. Radiotherapy is largely ineffective in osteosarcoma, indicating a potential role for radiosensitizers. Blocking DNA repair, particularly by inhibiting the catalytic subunit of DNA-dependent protein kinase (DNA-PKCS), is an attractive option for the radiosensitization of osteosarcoma. In this study, the expression of DNA-PKCS in osteosarcoma tissue specimens and cell lines was examined. Moreover, the small molecule DNA-PKCS inhibitor, KU60648, was investigated as a radiosensitizing strategy for osteosarcoma cells in vitro. DNA-PKCS was consistently expressed in the osteosarcoma tissue specimens and cell lines studied. Additionally, KU60648 effectively sensitized two of those osteosarcoma cell lines (143B cells by 1.5-fold and U2OS cells by 2.5-fold). KU60648 co-treatment also altered cell cycle distribution and enhanced DNA damage. Cell accumulation at the G2/M transition point increased by 55% and 45%, while the percentage of cells with >20 γH2AX foci were enhanced by 59% and 107% for 143B and U2OS cells, respectively. These results indicate that the DNA-PKCS inhibitor, KU60648, is a promising radiosensitizing agent for osteosarcoma. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Analysis of epigenetic modifications of DNA in human cells

    DEFF Research Database (Denmark)

    Kristensen, Lasse Sommer; Treppendahl, Marianne Bach; Grønbæk, Kirsten

    2013-01-01

    Epigenetics, the study of somatically heritable changes in gene expression not related to changes in the DNA sequence, is a rapidly expanding research field that plays important roles in healthy as well as in diseased cells. DNA methylation and hydroxymethylation are epigenetic modifications found...

  17. Extraction of human genomic DNA from whole blood using a magnetic microsphere method.

    Science.gov (United States)

    Gong, Rui; Li, Shengying

    2014-01-01

    With the rapid development of molecular biology and the life sciences, magnetic extraction is a simple, automatic, and highly efficient method for separating biological molecules, performing immunoassays, and other applications. Human blood is an ideal source of human genomic DNA. Extracting genomic DNA by traditional methods is time-consuming, and phenol and chloroform are toxic reagents that endanger health. Therefore, it is necessary to find a more convenient and efficient method for obtaining human genomic DNA. In this study, we developed urea-formaldehyde resin magnetic microspheres and magnetic silica microspheres for extraction of human genomic DNA. First, a magnetic microsphere suspension was prepared and used to extract genomic DNA from fresh whole blood, frozen blood, dried blood, and trace blood. Second, DNA content and purity were measured by agarose electrophoresis and ultraviolet spectrophotometry. The human genomic DNA extracted from whole blood was then subjected to polymerase chain reaction analysis to further confirm its quality. The results of this study lay a good foundation for future research and development of a high-throughput and rapid extraction method for extracting genomic DNA from various types of blood samples.

  18. Polycyclic Aromatic Hydrocarbon (PAH Exposure and DNA Adduct Semi-Quantitation in Archived Human Tissues

    Directory of Open Access Journals (Sweden)

    M. Margaret Pratt

    2011-06-01

    Full Text Available Polycyclic aromatic hydrocarbons (PAHs are combustion products of organic materials, mixtures of which contain multiple known and probable human carcinogens. PAHs occur in indoor and outdoor air, as well as in char-broiled meats and fish. Human exposure to PAHs occurs by inhalation, ingestion and topical absorption, and subsequently formed metabolites are either rendered hydrophilic and excreted, or bioactivated and bound to cellular macromolecules. The formation of PAH-DNA adducts (DNA binding products, considered a necessary step in PAH-initiated carcinogenesis, has been widely studied in experimental models and has been documented in human tissues. This review describes immunohistochemistry (IHC studies, which reveal localization of PAH-DNA adducts in human tissues, and semi-quantify PAH-DNA adduct levels using the Automated Cellular Imaging System (ACIS. These studies have shown that PAH-DNA adducts concentrate in: basal and supra-basal epithelium of the esophagus, cervix and vulva; glandular epithelium of the prostate; and cytotrophoblast cells and syncitiotrophoblast knots of the placenta. The IHC photomicrographs reveal the ubiquitous nature of PAH-DNA adduct formation in human tissues as well as PAH-DNA adduct accumulation in specific, vulnerable, cell types. This semi-quantative method for PAH-DNA adduct measurement could potentially see widespread use in molecular epidemiology studies.

  19. Sunlight-induced DNA damage in human mononuclear cells

    DEFF Research Database (Denmark)

    Møller, Peter; Wallin, Hakan; Holst, Erik

    2002-01-01

    of sunlight was comparable to the interindividual variation, indicating that sunlight exposure and the individual's background were the two most important determinants for the basal level of DNA damage. Influence of other lifestyle factors such as exercise, intake of foods, infections, and age could......In this study of 301 blood samples from 21 subjects, we found markedly higher levels of DNA damage (nonpyrimidine dimer types) in the summer than in the winter detected by single-cell gel electrophoresis. The level of DNA damage was influenced by the average daily influx of sunlight ... to blood sampling. The 3 and 6 day periods before sampling influenced DNA damage the most. The importance of sunlight was further emphasized by a positive association of the DNA damage level to the amount of time the subjects had spent in the sun over a 3 day period prior to the sampling. The effect...

  20. Quantification of carbonic anhydrase gene expression in ventricle of hypertrophic and failing human heart

    Directory of Open Access Journals (Sweden)

    Alvarez Bernardo V

    2013-01-01

    Full Text Available Abstract Background Carbonic anhydrase enzymes (CA catalyze the reversible hydration of carbon dioxide to bicarbonate in mammalian cells. Trans-membrane transport of CA-produced bicarbonate contributes significantly to cellular pH regulation. A body of evidence implicates pH-regulatory processes in the hypertrophic growth pathway characteristic of hearts as they fail. In particular, Na+/H+ exchange (NHE activation is pro-hypertrophic and CA activity activates NHE. Recently Cardrase (6-ethoxyzolamide, a CA inhibitor, was found to prevent and revert agonist-stimulated cardiac hypertrophy (CH in cultured cardiomyocytes. Our goal thus was to determine whether hypertrophied human hearts have altered expression of CA isoforms. Methods We measured CA expression in hypertrophied human hearts to begin to examine the role of carbonic anhydrase in progression of human heart failure. Ventricular biopsies were obtained from patients undergoing cardiac surgery (CS, n = 14, or heart transplantation (HT, n = 13. CS patients presented mild/moderate concentric left ventricular hypertrophy and normal right ventricles, with preserved ventricular function; ejection fractions were ~60%. Conversely, HT patients with failing hearts presented CH or ventricular dilation accompanied by ventricular dysfunction and EF values of 20%. Non-hypertrophic, non-dilated ventricular samples served as controls. Results Expression of atrial and brain natriuretic peptide (ANP and BNP were markers of CH. Hypertrophic ventricles presented increased expression of CAII, CAIV, ANP, and BNP, mRNA levels, which increased in failing hearts, measured by quantitative real-time PCR. CAII, CAIV, and ANP protein expression also increased approximately two-fold in hypertrophic/dilated ventricles. Conclusions These results, combined with in vitro data that CA inhibition prevents and reverts CH, suggest that increased carbonic anhydrase expression is a prognostic molecular marker of cardiac

  1. Human impacts quantification on the coastal landforms of Gran Canaria Island (Canary Islands)

    Science.gov (United States)

    Ferrer-Valero, Nicolás; Hernández-Calvento, Luis; Hernández-Cordero, Antonio I.

    2017-06-01

    The coastal areas of the Canary Islands are particularly sensitive to changes, both from a natural perspective and for their potential socio-economic implications. In this paper, the state of conservation of an insular coast is approached from a geomorphological point of view, considering recent changes induced by urban and tourism development. The analysis is applied to the coast of Gran Canaria, a small Atlantic island of volcanic origin, subject to a high degree of human pressure on its coastal areas, especially in recent decades. Currently, much of the economic activity of Gran Canaria is linked to mass tourism, associated with climatic and geomorphological features of the coast. This work is addressed through detailed mapping of coastal landforms across the island (256 km perimeter), corresponding to the period before the urban and tourism development (late 19th century for the island's capital, mid-20th century for the rest of the island) and today. The comparison between the coastal geomorphology before and after the urban and tourism development was established through four categories of human impacts, related to their conservation state: unaltered, altered, semi-destroyed and extinct. The results indicate that 43% of coastal landforms have been affected by human impacts, while 57% remain unaltered. The most affected are sedimentary landforms, namely coastal dunes, palaeo-dunes, beaches and wetlands. Geodiversity loss was also evaluated by applying two diversity indices. The coastal geodiversity loss by total or partial destruction of landforms is estimated at - 15.2%, according to Shannon index (H‧), while it increases to - 32.1% according to an index proposed in this paper. We conclude that the transformations of the coast of Gran Canaria induced by urban and tourism development have heavily affected the most singular coastal landforms (dunes, palaeo-dunes and wetlands), reducing significantly its geodiversity.

  2. NOTE: A preliminary study for non-invasive quantification of manganese in human hand bones

    Science.gov (United States)

    Aslam; Pejović-Milić, A.; Chettle, D. R.; McNeill, F. E.; Pysklywec, M. W.; Oudyk, J.

    2008-10-01

    Manganese (Mn) is a nutrient essential for regulating neurological and skeletal functions in the human body, but it is also toxic when humans are excessively exposed to Mn. Blood (or serum/plasma) and other body fluids reflect only the most recent exposure and rapidly return to within normal ranges, even when there has been a temporary excursion in response to exposure. In this context, we have been developing a non-invasive measurement of Mn stored in bone, using in vivo neutron activation analysis. Following feasibility studies, a first pilot study, using neutron activation analysis to measure Mn in the bones of the hand of ten healthy male human subjects, was conducted with the approval of the concerned research ethics boards. The participants of this study had no known history of exposure to Mn. Two volunteers were excluded from this study due to technical problems with their measurements. The inverse variance weighted mean value of Mn/Ca for the participants of this study is 0.12 ± 0.68 µg Mn/g Ca which is comparable within uncertainties with the estimated range of 0.16 0.78 µg Mn/g Ca and mean value of 0.63 ± 0.30 µg Mn/g Ca derived from cadaver data. It is recommended to investigate the use of the diagnostic technique for in vivo measurements of workers exposed occupationally to excessive amounts of Mn who could develop many-fold increased levels of Mn in bones as demonstrated through various animal studies. The technique needs further development to improve the precision of in vivo measurements in the non-exposed population.

  3. A radiomicroassay for cytotoxic antibody to human spermatozoa. Quantification by tritiated actinomycin d.

    Science.gov (United States)

    Sung, J S; Shizuya, H; Black, D D; Mumford, D M

    1977-03-01

    A radiomicroassay for titration of spermocytotoxic antibody is described. The assay used [3H]AACTINOMYCIN D ([3H]Act D) to label damaged spermatozoa in a fashion analogous to penetration by vital dye. Optimal conditions for and some kinetics of the assay are presented. The assay is sensitive, reliable, simple to perform and uses only small amounts of serum and spermatozoa. Applied to sperm antibody positive human postvasectomy sera, the assay compared favourably in sensitivity eith vital dye microscopic observations and with parallel titration by the Isojima's immobilization tests.

  4. Internal strain estimation for quantification of human heel pad elastic modulus: A phantom study

    DEFF Research Database (Denmark)

    Holst, Karen; Liebgott, Hervé; Wilhjelm, Jens E.

    2013-01-01

    Shock absorption is the most important function of the human heel pad. However, changes in heel pad elasticity, as seen in e.g. long-distance runners, diabetes patients, and victims of Falanga torture are affecting this function, often in a painful manner. Assessment of heel pad elasticity......, and the three hard phantoms were 89kPa, 153kPa, and 168kPa, respectively. The combination of ultrasound images and force measurements provided an effective way of assessing the elastic properties of the heel pad due to the internal strain estimation....