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Sample records for human dna progress

  1. Human ribonuclease H1 resolves R-loops and thereby enables progression of the DNA replication fork.

    Science.gov (United States)

    Parajuli, Shankar; Teasley, Daniel C; Murali, Bhavna; Jackson, Jessica; Vindigni, Alessandro; Stewart, Sheila A

    2017-09-15

    Faithful DNA replication is essential for genome stability. To ensure accurate replication, numerous complex and redundant replication and repair mechanisms function in tandem with the core replication proteins to ensure DNA replication continues even when replication challenges are present that could impede progression of the replication fork. A unique topological challenge to the replication machinery is posed by RNA-DNA hybrids, commonly referred to as R-loops. Although R-loops play important roles in gene expression and recombination at immunoglobulin sites, their persistence is thought to interfere with DNA replication by slowing or impeding replication fork progression. Therefore, it is of interest to identify DNA-associated enzymes that help resolve replication-impeding R-loops. Here, using DNA fiber analysis, we demonstrate that human ribonuclease H1 (RNH1) plays an important role in replication fork movement in the mammalian nucleus by resolving R-loops. We found that RNH1 depletion results in accumulation of RNA-DNA hybrids, slowing of replication forks, and increased DNA damage. Our data uncovered a role for RNH1 in global DNA replication in the mammalian nucleus. Because accumulation of RNA-DNA hybrids is linked to various human cancers and neurodegenerative disorders, our study raises the possibility that replication fork progression might be impeded, adding to increased genomic instability and contributing to disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. High expression of the DNA methyltransferase gene characterizes human neoplastic cells and progression stages of colon cancer

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    El-Deiry, W.S.; Nelkin, B.D.; Celano, P.; Ray-Whay Chiu Yen; Falco, J.P.; Hamilton, S.R.; Baylin, S.B. (Johns Hopkins Medical Inst., Baltimore, MD (United States))

    1991-04-15

    DNA methylation abnormalities occur consistently in human neoplasia including widespread hypomethylation and more recently recognized local increases in DNA methylation that hold potential for gene inactivation events. To study this imbalance further, the authors have localized to chromosome 19 a portion of the human DNA methyltransferase gene that codes for the enzyme catalyzing DNA methylation. Expression of this gene is low in normal human cells, significantly increased (30- to 50-fold by PCR analysis) in virally transformed cells, and strikingly elevated in human cancer cells (several hundredfold). In comparison to colon mucosa from patients without neoplasia, median levels of DNA methyltransferase transcripts are 15-fold increased in histologically normal mucosa from patients with cancers or the benign polyps that can precede cancers, 60-fold increased in the premalignant polyps, and >200-fold increased in the cancers. Thus, increases in DNA methyltransferase gene expression precede development of colonic neoplasia and continue during progression of colonic neoplasms. These increases may play a role in the genetic instability of cancer and mark early events in cell transformation.

  3. S phase progression in human cells is dictated by the genetic continuity of DNA foci.

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    Apolinar Maya-Mendoza

    2010-04-01

    Full Text Available DNA synthesis must be performed with extreme precision to maintain genomic integrity. In mammalian cells, different genomic regions are replicated at defined times, perhaps to preserve epigenetic information and cell differentiation status. However, the molecular principles that define this S phase program are unknown. By analyzing replication foci within discrete chromosome territories during interphase, we show that foci which are active during consecutive intervals of S phase are maintained as spatially adjacent neighbors throughout the cell cycle. Using extended DNA fibers, we demonstrate that this spatial continuity of replication foci correlates with the genetic continuity of adjacent replicon clusters along chromosomes. Finally, we used bioinformatic tools to compare the structure of DNA foci with DNA domains that are seen to replicate during discrete time intervals of S phase using genome-wide strategies. Data presented show that a major mechanism of S phase progression involves the sequential synthesis of regions of the genome because of their genetic continuity along the chromosomal fiber.

  4. DNA technological progress toward advanced diagnostic tools to support human hookworm control.

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    Gasser, R B; Cantacessi, C; Loukas, A

    2008-01-01

    Blood-feeding hookworms are parasitic nematodes of major human health importance. Currently, it is estimated that 740 million people are infected worldwide, and more than 80 million of them are severely affected clinically by hookworm disease. In spite of the health problems caused and the advances toward the development of vaccines against some hookworms, limited attention has been paid to the need for improved, practical methods of diagnosis. Accurate diagnosis and genetic characterization of hookworms is central to their effective control. While traditional diagnostic methods have considerable limitations, there has been some progress toward the development of molecular-diagnostic tools. The present article provides a brief background on hookworm disease of humans, reviews the main methods that have been used for diagnosis and describes progress in establishing polymerase chain reaction (PCR)-based methods for the specific diagnosis of hookworm infection and the genetic characterisation of the causative agents. This progress provides a foundation for the rapid development of practical, highly sensitive and specific diagnostic and analytical tools to be used in improved hookworm prevention and control programmes.

  5. Physical state & copy number of high risk human papillomavirus type 16 DNA in progression of cervical cancer

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    Shirish Shukla

    2014-01-01

    Full Text Available Background & objectives: High-risk human papilloma virus (HR-HPV infection and its integration in host genome is a key event in malignant transformation of cervical cells. HPV16 being a dominant HR-HPV type, we undertook this study to analyze if viral load and physical state of the virus correlated with each other in the absence of other confounding variables and examined their potential as predictors of progressive cervical lesions. Methods: Both, viral load and integration status of HPV16 were determined by real time URR PCR and estimation of E2:E6 ratio in a total of 130 PGMY-RLB -confirmed, monotypic HPV16-infected cervical DNA samples from biopsies of cytology-confirmed low grade (LSIL, 30 and high grade (HSIL, 30, and invasive carcinoma, (squamous cell carcinoma SCC, 70 cases. Results: Investigation of DNA samples revealed a gradual increase in HPV16 viral load over several magnitudes and increased frequency of integration from LSIL to HSIL and HSIL to invasive cancer in relation to the severity of lesions in monotypic HPV16-infected cervical tissues. In a substantial number of precancer (11/60 and cancer cases (29/70, HPV16 was detected in concomitant mixed form. The concomitant form of HPV16 genome carried significantly higher viral load. Interpretation & conclusions: Overall, viral load and integration increased with disease severity and could be useful biomarkers in disease progression, at least, in HPV16-infected cervical pre-cancer and cancer lesions.

  6. Variations of Human DNA Polymerase Genes as Biomarkers of Prostate Cancer Progression

    Science.gov (United States)

    2013-07-01

    Athens, Greece, 07/10 Makridakis N. Error-prone polymerase mutations and prostate cancer progression, COBRE /Cancer Genetics group seminar, Tulane...New Orleans, LA, 03/11 12 Makridakis N. Error-prone polymerases, genomic instability and prostate cancer progression, COBRE / External...Orleans, LA, 09/11 Makridakis N. Error-prone polymerases, genomic instability and prostate cancer progression, COBRE / External Advisory Board

  7. Variations of Human DNA Polymerase Genes as Biomarkers of Prostate Cancer Progression

    Science.gov (United States)

    2011-07-01

    IRG); Grant number: 58-007-45. Current affiliation for Lúcio Fábio Caldas Ferraz: Centro de Biologia Molecular e Engenharia Genética, University of...8-9 References…………………………………………………………………………….9 Appendices………………………………………………………………………10-29 4 INTRODUCTION The early molecular ...Picopure® DNA Extraction Kit ( Molecular Devices; Sunnyvale, CA), and PCR amplified using the same primers that identified the POLB mutations shown on Table

  8. Human cDNA mapping using fluorescence in situ hybridization. Final progress report, April 1, 1994--July 31, 1997

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    Korenberg, J.R.

    1997-12-31

    The ultimate goal of this research is to generate and apply novel technologies to speed completion and integration of the human genome map and sequence with biomedical problems. To do this, techniques were developed and genome-wide resources generated. This includes a genome-wide Mapped and Integrated BAC/PAC Resource that has been used for gene finding, map completion and anchoring, breakpoint definition and sequencing. In the last period of the grant, the Human Mapped BAC/PAC Resource was also applied to determine regions of human variation and to develop a novel paradigm of primate evolution through to humans. Further, in order to more rapidly evaluate animal models of human disease, a BAC Map of the mouse was generated in collaboration with the MTI Genome Center, Dr. Bruce Birren.

  9. 'Progress' renders detrimental an ancient mitochondrial DNA genetic variant.

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    Pacheu-Grau, David; Gómez-Durán, Aurora; López-Gallardo, Ester; Pinós, Tomàs; Andreu, Antoni L; López-Pérez, Manuel J; Montoya, Julio; Ruiz-Pesini, Eduardo

    2011-11-01

    A human mitochondrial DNA (mtDNA) transition, m.1555A>G, in the 12S rRNA gene causes non-syndromic hearing loss. However, this pathological mutation is the wild-type allele in orangutan mtDNA. Here we rule out different genetic factors as the reason for its fixation in orangutans and show that aminoglycosides negatively affect the oxidative phosphorylation function by decreasing the synthesis of mtDNA-encoded proteins and the amount and activity of respiratory complex IV. These drugs also diminish the growth rate of orangutan cells. The m.1555G nucleotide is also the wild-type allele in other mammal species and they might be at risk of suffering a mitochondrial disorder if treated with aminoglycosides. Therefore, pharmacogenomic approaches should be used to confirm this possibility. These observations are important for human health. Due to the fact that old age and high frequency are criteria widely used in mitochondrial medicine to rule out a genetic change as being a pathological mutation, our results prevent against simplistic genetic approaches that do not consider the potential effect of environmental conditions. Hence, these results suggest that some ancient and highly frequent human population polymorphisms, such as those defining mtDNA haplogroups, in mitochondrial rRNA genes can be deleterious in association with new environmental conditions. Therefore, as the discovery of ribosomal antibiotics has allowed to fight infectious diseases and this breakthrough can be considered an important scientific advance or 'progress', our results suggest that 'progress' can also have a negative counterpart and render detrimental many of these mtDNA genotypes.

  10. "Human potential" and progressive pedagogy

    DEFF Research Database (Denmark)

    Øland, Trine

    2012-01-01

    This article examines the cultural constructs of progressive pedagogy in Danish school pedagogy and its emerging focus on the child’s human potential from the 1920s to the 1950s. It draws on Foucault’s notion of ‘dispositifs’ and the ‘elements of history’, encircling a complex transformation...... of continuity and discontinuity of progressive pedagogy. The Danish context is identified as being part of an international and scientific enlightenment movement circulating in, e.g., the New Education Fellowship (NEF). The cultural constructs embedded in progressivism are clarified in the article...

  11. Earnings progression, human capital and incentives

    DEFF Research Database (Denmark)

    Frederiksen, Anders

    progression by investigating the effects of on-the-job human capital acquisition, explicit short-run incentives and career concern incentives on earnings progression. The model leads to predictions about the incentive structure and the progression in both cross-sectional and individual earnings which...

  12. Correlation of chromosome patterns in human leukemic cells with exposure to chemicals and/or radiation. Progress report, July 1992--August 1993

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    Rowley, J.D.

    1993-09-01

    Progress in identification of chromosomal transformations associated with leukemogenesis is described. In particular progress in DNA cloning of chromosomal break points in human cancer patients is described.

  13. Correlation of DNA Ploidy with Progression of Cervical Cancer

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    Singh, M.; Mehrotra, S.; Kalra, N.; Singh, U.; Shukla, Y.

    2008-01-01

    The majority of squamous cell carcinomas of cervix are preceded by visible changes in the cervix, most often detected by cervical smear. As cervical cancer is preceded by long precancerous stages, identification of the high-risk population through detection of DNA ploidy may be of importance in effective management of this disease. Here we attempted to correlate aneuploid DNA patterns and their influence on biological behavior of flow-cytometry analysis of DNA ploidy which was carried out in cytologically diagnosed cases of mild (79), moderate (36), and severe (12) dysplasia, as well as “atypical squamous cells of unknown significance (ASCUS)” (57) along with controls (69), in order to understand its importance in malignant progression of disease. Cytologically diagnosed dysplasias, which were employed for DNA ploidy studies, 39 mild, 28 moderate, and 11 severe dysplasia cases were found to be aneuploid. Out of the 69 control subjects, 6 cases showed aneuploidy pattern and the rest 63 subjects were diploid. An aneuploidy pattern was observed in 8 out of 57 cases of cytologically evaluated ASCUS. The results of the followup studies showed that aberrant DNA content reliably predicts the occurrence of squamous cell carcinoma in cervical smear. Flow cytometric analysis of DNA ploidy may provide a strategic diagnostic tool for early detection of carcinoma cervix. Therefore, it is a concept of an HPV screening with reflex cytology in combination with DNA flow cytometry to detect progressive lesions with the greatest possible sensitivity and specificity. PMID:20445775

  14. Understanding human DNA sequence variation.

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    Kidd, K K; Pakstis, A J; Speed, W C; Kidd, J R

    2004-01-01

    Over the past century researchers have identified normal genetic variation and studied that variation in diverse human populations to determine the amounts and distributions of that variation. That information is being used to develop an understanding of the demographic histories of the different populations and the species as a whole, among other studies. With the advent of DNA-based markers in the last quarter century, these studies have accelerated. One of the challenges for the next century is to understand that variation. One component of that understanding will be population genetics. We present here examples of many of the ways these new data can be analyzed from a population perspective using results from our laboratory on multiple individual DNA-based polymorphisms, many clustered in haplotypes, studied in multiple populations representing all major geographic regions of the world. These data support an "out of Africa" hypothesis for human dispersal around the world and begin to refine the understanding of population structures and genetic relationships. We are also developing baseline information against which we can compare findings at different loci to aid in the identification of loci subject, now and in the past, to selection (directional or balancing). We do not yet have a comprehensive understanding of the extensive variation in the human genome, but some of that understanding is coming from population genetics.

  15. Challenges and progress in making DNA-based AIS early ...

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    The ability of DNA barcoding to find additional species in hard-to-sample locations or hard-to-identify samples is well established. Nevertheless, adoption of DNA barcoding into regular monitoring programs has been slow, in part due to issues of standardization and interpretation that need resolving. In this presentation, we describe our progress towards incorporating DNA-based identification into broad-spectrum aquatic invasive species early-detection monitoring in the Laurentian Great Lakes. Our work uses community biodiversity information as the basis for evaluating survey performance for various taxonomic groups. Issues we are tackling in bringing DNA-based data to bear on AIS monitoring design include: 1) Standardizing methodology and work flow from field collection and sample handling through bioinformatics post-processing; 2) Determining detection sensitivity and accounting for inter-species differences in DNA amplification and primer affinity; 3) Differentiating sequencing and barcoding errors from legitimate new finds when range and natural history information is limited; and 4) Accounting for the different nature of morphology- vs. DNA-based biodiversity information in subsequent analysis (e.g., via species accumulation curves, multi-metric indices). not applicable

  16. The potential use of DNA methylation biomarkers to identify risk and progression of type 2 diabetes

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    Linn eGillberg

    2015-03-01

    Full Text Available Type 2 diabetes mellitus (T2D is a slowly progressive disease that can be postponed or even avoided through lifestyle changes. Recent data demonstrate highly significant correlations between DNA methylation and the most important risk factors of T2D, including age and BMI, in blood and human tissues relevant to insulin resistance and T2D. Also, T2D patients and individuals with increased risk of the disease display differential DNA methylation profiles and plasticity compared to controls. Accordingly, the novel clues to DNA methylation fingerprints in blood and tissues with deteriorated metabolic capacity indicate that blood-borne epigenetic biomarkers of T2D progression might become a reality. This Mini Review will address the most recent associations between DNA methylation and diabetes-related traits in human tissues and blood. The overall focus is on the potential of future epigenome-wide studies, carried out across tissues and populations with correlations to pre-diabetes and T2D risk factors, to build up a library of epigenetic markers of risk and early progression of T2D. These markers may, tentatively in combination with other predictors of T2D development, increase the possibility of individual-based lifestyle prevention of T2D and associated metabolic diseases.

  17. Expression of Hyaluronan in human tumor progression

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    Boregowda Rajeev K

    2006-01-01

    Full Text Available Abstract Background The development and progression of human tumors is accompanied by various cellular, biochemical and genetic alterations. These events include tumor cells interaction with extracellular matrix molecules including hyaluronan (HA. Hyaluronan is a large polysaccharide associated with pericellular matrix of proliferating, migrating cells. Its implication in malignant transformation, tumor progression and with the degree of differentiation in various invasive tumors has well accepted. It has been well known the role HA receptors in tumor growth and metastasis in various cancer tissues. Previously we have observed the unified over expression of Hyaluronic Acid Binding Protein (HABP, H11B2C2 antigen by the tumor cells in various types progressing tumor tissues with different grades. However, the poor understanding of relation between HA and HA-binding protein expression on tumor cells during tumor progression as well as the asymmetric observations of the role of HA expression in tumor progression prompted us to examine the degree of HA expression on tumor cells vs. stroma in various types of human tumors with different grades. Methods In the present study clinically diagnosed tumor tissue samples of different grades were used to screen the histopathological expression of hyaluronan by using b-PG (biotinylated proteoglycan as a probe and we compared the relative HA expression on tumor cells vs. stroma in well differentiated and poorly differentiated tumors. Specificity of the reaction was confirmed either by pre-digesting the tissue sections with hyaluronidase enzyme or by staining the sections with pre-absorbed complex of the probe and HA-oligomers. Results We show here the down regulation of HA expression in tumor cells is associated with progression of tumor from well differentiated through poorly differentiated stage, despite the constant HA expression in the tumor associated stroma. Conclusion The present finding enlighten the

  18. A DNA methylation fingerprint of 1628 human samples

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    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

  19. Human Connectivity: The Key to Progress

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    Janani Harish

    2016-10-01

    Full Text Available Progress results from human interaction. Advances in knowledge, transportation, communication, technology and industry have aided in social development only to the extent that they have brought greater numbers of people closer. It is only human ingenuity that has achieved. Historically, every event that marks a step forward in social evolution coincides with a discovery, invention or organizational innovation that brings people closer in contact. When two people meet, the knowledge, resourcefulness and capacity that they bring together grow not cumulatively but exponentially. Thus every additional connection made to the human network adds to its depth and richness. When this is done at a larger scale, between entire cultures and civilizations, the result is an explosion of creativity. Urbanization and advances in technology have accelerated this interchange between people and cultures. Unlike all other resources, human capital is inexhaustible, self-replenishing and has the potential to meet and overcome every challenge the world faces today. The logistics of bringing human minds together effectively holds the key to consciously accelerating human progress.

  20. Comprehensive DNA methylation study identifies novel progression-related and prognostic markers for cutaneous melanoma.

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    Wouters, Jasper; Vizoso, Miguel; Martinez-Cardus, Anna; Carmona, F Javier; Govaere, Olivier; Laguna, Teresa; Joseph, Jesuchristopher; Dynoodt, Peter; Aura, Claudia; Foth, Mona; Cloots, Roy; van den Hurk, Karin; Balint, Balazs; Murphy, Ian G; McDermott, Enda W; Sheahan, Kieran; Jirström, Karin; Nodin, Bjorn; Mallya-Udupi, Girish; van den Oord, Joost J; Gallagher, William M; Esteller, Manel

    2017-06-05

    Cutaneous melanoma is the deadliest skin cancer, with an increasing incidence and mortality rate. Currently, staging of patients with primary melanoma is performed using histological biomarkers such as tumor thickness and ulceration. As disruption of the epigenomic landscape is recognized as a widespread feature inherent in tumor development and progression, we aimed to identify novel biomarkers providing additional clinical information over current factors using unbiased genome-wide DNA methylation analyses. We performed a comprehensive DNA methylation analysis during all progression stages of melanoma using Infinium HumanMethylation450 BeadChips on a discovery cohort of benign nevi (n = 14) and malignant melanoma from both primary (n = 33) and metastatic (n = 28) sites, integrating the DNA methylome with gene expression data. We validated the discovered biomarkers in three independent validation cohorts by pyrosequencing and immunohistochemistry. We identified and validated biomarkers for, and pathways involved in, melanoma development (e.g., HOXA9 DNA methylation) and tumor progression (e.g., TBC1D16 DNA methylation). In addition, we determined a prognostic signature with potential clinical applicability and validated PON3 DNA methylation and OVOL1 protein expression as biomarkers with prognostic information independent of tumor thickness and ulceration. Our data underscores the importance of epigenomic regulation in triggering metastatic dissemination through the inactivation of central cancer-related pathways. Inactivation of cell-adhesion and differentiation unleashes dissemination, and subsequent activation of inflammatory and immune system programs impairs anti-tumoral defense pathways. Moreover, we identify several markers of tumor development and progression previously unrelated to melanoma, and determined a prognostic signature with potential clinical utility.

  1. 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

  2. Physical mapping of human chromosome 16. Annual progress report

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    Sutherland, G.R.

    1993-08-01

    We aim to isolate cDNAs mapping to human chromosome 16 and localise such cDNAs on the high resolution physical map. In collaboration with LANL, PCR primers will be synthesised from cDNA sequences mapped to chromosome 16 and used as ESTs in the generation of mega-YAC contigs for this chromosome. Probing of high density cosmid grids will enable integration of the ESTs into cosmid contigs and location of the cosmid contigs on the YAC contig. A hn-cDNA library has been constructed from the hybrid CY18 which contains chromosome 16 as the only human chromosome. A modified screening protocol has been successfully developed and 15 hn-cDNA clones have been sequenced and localised on the hybrid map. Sequence analysis of four of these revealed that they were known cDNAs, which are now mapped to chromosome 16. Development of techniques to allow the isolation of longer cDNAs from the identified exons is in progress. This will depend on PCR amplification of cDNAs from a total human CDNA library.

  3. Human DNA Ligase III Recognizes DNA Ends by Dynamic Switching Between Two DNA Bound States†

    OpenAIRE

    Cotner-Gohara, Elizabeth; Kim, In-Kwon; Hammel, Michal; Tainer, John A.; Tomkinson, Alan E.; Ellenberger, Tom

    2010-01-01

    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 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)...

  4. Tenacity of exogenous human papillomavirus DNA in sperm washing.

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    Brossfield, J E; Chan, P J; Patton, W C; King, A

    1999-07-01

    Sperm cells have been shown to take up exogenous DNA readily. The hypothesis was that sperm washing would remove exogenous viral DNA infecting sperm cells. The objective was to compare three types of sperm washing procedures for their capacity to remove exogenous human papillomavirus (HPV) DNA from infected sperm. Prewashed sperm were equally divided and sperm in one portion were exposed to L1 HPV DNA fragments for 30 min at 37 degrees C. Untreated washed sperm served as the control. After transfection, the sperm were washed by either centrifuge, two-layer Isolate colloid wash, or test-yolk buffer procedures. Sperm parameters were measured on a Hamilton Thorn HTM-C analyzer. Sperm DNA were extracted and polymerase chain reaction (PCR) was carried out targeting the L1 consensus gene of HPV and the designated sentinel gene, 17q21 spanning the D17S855 gene. Amplified products were analyzed in 2% agarose gel electrophoresis. PCR analyses detected the consensus L1 HPV gene in sperm after they were processed through either of the three procedures. Controls were negative for the L1 gene. Extracted DNA were verified by PCR amplification of 17q21 spanning the D17S855 gene. Transfected sperm had higher percentages of total motility and progression compared with the control. Centrifuged, washed, transfected sperm exhibited a greater curvilinear velocity and hyperactivation. The data showed that washing would not remove exogenous HPV DNA from sperm cells. The viral DNA was tenaciously bound to the sperm, suggesting an internalization into the sperm. The viral DNA also increased the motility of the sperm by affecting the velocity and progression of the sperm, which suggested either an increase in metabolism, an enhancement of the calcium-regulated motility mechanism, or an artifact of PCR reagents. More studies are needed to elucidate the mechanism of DNA stimulated sperm motility.

  5. DNA-informed breeding of rosaceous crops: promises, progress and prospects

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    Peace, Cameron P

    2017-01-01

    Crops of the Rosaceae family provide valuable contributions to rural economies and human health and enjoyment. Sustained solutions to production challenges and market demands can be met with genetically improved new cultivars. Traditional rosaceous crop breeding is expensive and time-consuming and would benefit from improvements in efficiency and accuracy. Use of DNA information is becoming conventional in rosaceous crop breeding, contributing to many decisions and operations, but only after past decades of solved challenges and generation of sufficient resources. Successes in deployment of DNA-based knowledge and tools have arisen when the ‘chasm’ between genomics discoveries and practical application is bridged systematically. Key steps are establishing breeder desire for use of DNA information, adapting tools to local breeding utility, identifying efficient application schemes, accessing effective services in DNA-based diagnostics and gaining experience in integrating DNA information into breeding operations and decisions. DNA-informed germplasm characterization for revealing identity and relatedness has benefitted many programs and provides a compelling entry point to reaping benefits of genomics research. DNA-informed germplasm evaluation for predicting trait performance has enabled effective reallocation of breeding resources when applied in pioneering programs. DNA-based diagnostics is now expanding from specific loci to genome-wide considerations. Realizing the full potential of this expansion will require improved accuracy of predictions, multi-trait DNA profiling capabilities, streamlined breeding information management systems, strategies that overcome plant-based features that limit breeding progress and widespread training of current and future breeding personnel and allied scientists. PMID:28326185

  6. The role of geoscientists in human progress

    Directory of Open Access Journals (Sweden)

    Stefania Lucchesi

    2012-07-01

    Full Text Available Like any science, geology has a key role in the development and progress of human culture and society. In this context, scientists, professionals and practitioners of Earth sciences must inevitably confront themselves with the purposes, methods and results of their studies, concerning relationships between man and his environment, which thus deals with ethical questions. An essential base for any geo-environmental action should be respect for the natural ecosystem. This can be achieved by encouraging an ‘affectionate attitude’ towards Nature or ecology. Some ancient cultures had a great awareness of the close relationships between humankind and the Earth. The recent web-based ‘Museo Torino’ (Turin Museum multimedia product shows the ‘history of a city’ (Turin, Italy in a dimension of unity and as a continuum of space–time–life between the history of the Earth and of humans. Geoscientists are not limited to merely having a pragmatic vision of the Earth, but should pursue harmonious collaboration between man and Nature. Within complex Earth systems, scientists and professionals rediscover their geoethical roles by responsibly evaluating and managing georesources, for progress aimed at improving conditions of life and human dignity. Geoscientists can also promote respect for ‘human rights’ through appropriate educational and training actions, for the balanced exploitation of our georesources. An example from Mendoza (Argentina is presented here, to encourage opportunities for meetings on environmental issues among people of different backgrounds and cultures. Last, but not least, there is the ethical role of geoscientists in ‘the service of the truth’. A misleading view of Earth systems by geoscientists can lead to fatalism or myths that often affect people from the psychological and sociological points of view.

  7. MMSET is dynamically regulated during cell-cycle progression and promotes normal DNA replication.

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    Evans, Debra L; Zhang, Haoxing; Ham, Hyoungjun; Pei, Huadong; Lee, SeungBaek; Kim, JungJin; Billadeau, Daniel D; Lou, Zhenkun

    2016-01-01

    The timely and precise duplication of cellular DNA is essential for maintaining genome integrity and is thus tightly-regulated. During mitosis and G1, the Origin Recognition Complex (ORC) binds to future replication origins, coordinating with multiple factors to load the minichromosome maintenance (MCM) complex onto future replication origins as part of the pre-replication complex (pre-RC). The pre-RC machinery, in turn, remains inactive until the subsequent S phase when it is required for replication fork formation, thereby initiating DNA replication. Multiple myeloma SET domain-containing protein (MMSET, a.k.a. WHSC1, NSD2) is a histone methyltransferase that is frequently overexpressed in aggressive cancers and is essential for normal human development. Several studies have suggested a role for MMSET in cell-cycle regulation; however, whether MMSET is itself regulated during cell-cycle progression has not been examined. In this study, we report that MMSET is degraded during S phase in a cullin-ring ligase 4-Cdt2 (CRL4(Cdt2)) and proteasome-dependent manner. Notably, we also report defects in DNA replication and a decreased association of pre-RC factors with chromatin in MMSET-depleted cells. Taken together, our results suggest a dynamic regulation of MMSET levels throughout the cell cycle, and further characterize the role of MMSET in DNA replication and cell-cycle progression.

  8. DNA methylation markers for oral pre-cancer progression: A critical review.

    Science.gov (United States)

    Shridhar, Krithiga; Walia, Gagandeep Kaur; Aggarwal, Aastha; Gulati, Smriti; Geetha, A V; Prabhakaran, Dorairaj; Dhillon, Preet K; Rajaraman, Preetha

    2016-02-01

    Although oral cancers are generally preceded by a well-established pre-cancerous stage, there is a lack of well-defined clinical and morphological criteria to detect and signal progression from pre-cancer to malignant tumours. We conducted a critical review to summarize the evidence regarding aberrant DNA methylation patterns as a potential diagnostic biomarker predicting progression. We identified all relevant human studies published in English prior to 30th April 2015 that examined DNA methylation (%) in oral pre-cancer by searching PubMed, Web-of-Science and Embase databases using combined key-searches. Twenty-one studies (18-cross-sectional; 3-longitudinal) were eligible for inclusion in the review, with sample sizes ranging from 4 to 156 affected cases. Eligible studies examined promoter region hyper-methylation of tumour suppressor genes in pathways including cell-cycle-control (n=15), DNA-repair (n=7), cell-cycle-signalling (n=4) and apoptosis (n=3). Hyper-methylated loci reported in three or more studies included p16, p14, MGMT and DAPK. Two longitudinal studies reported greater p16 hyper-methylation in pre-cancerous lesions transformed to malignancy compared to lesions that regressed (57-63.6% versus 8-32.1%; pmethylation patterns reported three novel hyper-methylated loci (TRHDE; ZNF454; KCNAB3). The majority of reviewed studies were small, cross-sectional studies with poorly defined control groups and lacking validation. Whilst limitations in sample size and study design preclude definitive conclusions, current evidence suggests a potential utility of DNA methylation patterns as a diagnostic biomarker for oral pre-cancer progression. Robust studies such as large epigenome-wide methylation explorations of oral pre-cancer with longitudinal tracking are needed to validate the currently reported signals and identify new risk-loci and the biological pathways of disease progression. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  9. 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

  10. 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

  11. DNA Methylation Dynamics of Human Hematopoietic Stem Cell Differentiation.

    Science.gov (United States)

    Farlik, Matthias; Halbritter, Florian; Müller, Fabian; Choudry, Fizzah A; Ebert, Peter; Klughammer, Johanna; Farrow, Samantha; Santoro, Antonella; Ciaurro, Valerio; Mathur, Anthony; Uppal, Rakesh; Stunnenberg, Hendrik G; Ouwehand, Willem H; Laurenti, Elisa; Lengauer, Thomas; Frontini, Mattia; Bock, Christoph

    2016-12-01

    Hematopoietic stem cells give rise to all blood cells in a differentiation process that involves widespread epigenome remodeling. Here we present genome-wide reference maps of the associated DNA methylation dynamics. We used a meta-epigenomic approach that combines DNA methylation profiles across many small pools of cells and performed single-cell methylome sequencing to assess cell-to-cell heterogeneity. The resulting dataset identified characteristic differences between HSCs derived from fetal liver, cord blood, bone marrow, and peripheral blood. We also observed lineage-specific DNA methylation between myeloid and lymphoid progenitors, characterized immature multi-lymphoid progenitors, and detected progressive DNA methylation differences in maturing megakaryocytes. We linked these patterns to gene expression, histone modifications, and chromatin accessibility, and we used machine learning to derive a model of human hematopoietic differentiation directly from DNA methylation data. Our results contribute to a better understanding of human hematopoietic stem cell differentiation and provide a framework for studying blood-linked diseases. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  12. Bacterial DNA delays human eosinophil apoptosis

    OpenAIRE

    2009-01-01

    Bacterial DNA delays human eosinophil apoptosis correspondance: Corresponding author. Tel.: +358 3 3551 6687; fax: +358 3 3551 8082. (Ilmarinen, Pinja) (Ilmarinen, Pinja) The Immunopharmacology Research Group--> , Medical School--> , University of Tampere and Research Unit--> , Tampere University Hospital--> , Tampere--> - FINLAND (Ilmarinen, Pinja) The Immunopharmacology ...

  13. Human papillomavirus DNA in aerodigestive squamous carcinomas ...

    African Journals Online (AJOL)

    A series of 10 oesophageal and 10 laryngeal squamous carcinomas was examined by means of immuno cytochemistry and in situ DNA hybridisation to demonstrate human papillomavirus (HPV) infection. Changes in the epithelium adjacent to the carcinoma were found in 5 of 10 oesophageal and 7 of 10 laryngeal ...

  14. Extraction of DNA from Human Skeletal Material.

    Science.gov (United States)

    Pajnič, Irena Zupanič

    2016-01-01

    In recent years the recovery and analysis of DNA from skeletal remains has been applied to several contexts ranging from disaster victim identification to the identification of the victims of conflict. Here are described procedures for processing the bone and tooth samples including mechanical and chemical cleaning, cutting and powdering in the presence of liquid nitrogen, complete demineralization of bone and tooth powder, DNA extraction, DNA purification using magnetic beads, and the precautions and strategies implemented to avoid and detect contamination. It has proven highly successful in the analysis of bones and teeth from Second World War victims' skeletal remains that have been excavated from mass graves in Slovenia and is also suitable for genetic identification of relatively fresh human remains.

  15. 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. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  16. Peak Oil and the Everyday Complexity of Human Progress Narratives

    Directory of Open Access Journals (Sweden)

    John C. Pruit

    2012-11-01

    Full Text Available The “big” story of human progress has polarizing tendencies featuring the binary options of progress or decline. I consider human progress narratives in the context of everyday life. Analysis of the “little” stories from two narrative environments focusing on peak oil offers a more complex picture of the meaning and contours of the narrative. I consider the impact of differential blog site commitments to peak oil perspectives and identify five narrative types culled from two narrative dimensions. I argue that the lived experience complicates human progress narratives, which is no longer an either/or proposition.

  17. 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.

  18. Identification of Streptococcus parasanguinis DNA contamination in human buccal DNA samples.

    Science.gov (United States)

    Mahfuz, Istiak; Cheng, Wei; White, Stefan J

    2013-11-22

    The use of buccal swabs in clinical and scientific studies is a very popular method of collecting DNA, due to its non-invasive nature of collection. However, contamination of the DNA sample may interfere with analysis. Here we report the finding of Streptococcus parasanguinis bacterial DNA contamination in human buccal DNA samples, which led to preferential amplification of bacterial sequence with PCR primers designed against human sequence. Contamination of buccal-derived DNA with bacterial DNA can be significant, and may influence downstream genetic analysis. One needs to be aware of possible bacterial contamination when interpreting abnormal findings following PCR amplification of buccal swab DNA samples.

  19. 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.

  20. DNA copy-number control through inhibition of replication fork progression

    NARCIS (Netherlands)

    J.T. Nordman (Jared T.); E. Kozhevnikova (Elena); C.P. Verrijzer (Peter); A.V. Pindyurin (Alexey); E.N. Andreyeva (Evgeniya); V.V. Shloma (Victor); I.F. Zhimulev (Igor); T. Orr-Weaver (T.)

    2014-01-01

    textabstractProper control of DNA replication is essential to ensure faithful transmission of genetic material and prevent chromosomal aberrations that can drive cancer progression and developmental disorders. DNA replication is regulated primarily at the level of initiation and is under strict

  1. DNA banking and DNA databanking: Legal, ethical, and public policy issues. Progress report, [April 1, 1993--March 31, 1994

    Energy Technology Data Exchange (ETDEWEB)

    Reilly, P.R.; McEwen, J.E.; Small, D.

    1994-02-18

    The purpose of the grant was to provide support to enable us to: (1) perform legal and empirical research and critically analyze DNA banking and DNA databanking as those activities are conducted by state forensic laboratories, the military, academic researchers, and commercial enterprises; and (2) develop a broadcast quality educational videotape for viewing by the general public about DNA technology and the privacy and related issues that it raises. The grant thus has both a research and analysis component and a public education component. This report outlines the work completed since the inception of the project and describes the activities still in progress.

  2. Overlapping DNA methylation dynamics in mouse intestinal cell differentiation and early stages of malignant progression.

    Directory of Open Access Journals (Sweden)

    Marta Forn

    Full Text Available Mouse models of intestinal crypt cell differentiation and tumorigenesis have been used to characterize the molecular mechanisms underlying both processes. DNA methylation is a key epigenetic mark and plays an important role in cell identity and differentiation programs and cancer. To get insights into the dynamics of cell differentiation and malignant transformation we have compared the DNA methylation profiles along the mouse small intestine crypt and early stages of tumorigenesis. Genome-scale analysis of DNA methylation together with microarray gene expression have been applied to compare intestinal crypt stem cells (EphB2high, differentiated cells (EphB2negative, ApcMin/+ adenomas and the corresponding non-tumor adjacent tissue, together with small and large intestine samples and the colon cancer cell line CT26. Compared with late stages, small intestine crypt differentiation and early stages of tumorigenesis display few and relatively small changes in DNA methylation. Hypermethylated loci are largely shared by the two processes and affect the proximities of promoter and enhancer regions, with enrichment in genes associated with the intestinal stem cell signature and the PRC2 complex. The hypermethylation is progressive, with minute levels in differentiated cells, as compared with intestinal stem cells, and reaching full methylation in advanced stages. Hypomethylation shows different signatures in differentiation and cancer and is already present in the non-tumor tissue adjacent to the adenomas in ApcMin/+ mice, but at lower levels than advanced cancers. This study provides a reference framework to decipher the mechanisms driving mouse intestinal tumorigenesis and also the human counterpart.

  3. DNA Replication Origins and Fork Progression at Mammalian Telomeres

    Science.gov (United States)

    Higa, Mitsunori; Fujita, Masatoshi; Yoshida, Kazumasa

    2017-01-01

    Telomeres are essential chromosomal regions that prevent critical shortening of linear chromosomes and genomic instability in eukaryotic cells. The bulk of telomeric DNA is replicated by semi-conservative DNA replication in the same way as the rest of the genome. However, recent findings revealed that replication of telomeric repeats is a potential cause of chromosomal instability, because DNA replication through telomeres is challenged by the repetitive telomeric sequences and specific structures that hamper the replication fork. In this review, we summarize current understanding of the mechanisms by which telomeres are faithfully and safely replicated in mammalian cells. Various telomere-associated proteins ensure efficient telomere replication at different steps, such as licensing of replication origins, passage of replication forks, proper fork restart after replication stress, and dissolution of post-replicative structures. In particular, shelterin proteins have central roles in the control of telomere replication. Through physical interactions, accessory proteins are recruited to maintain telomere integrity during DNA replication. Dormant replication origins and/or homology-directed repair may rescue inappropriate fork stalling or collapse that can cause defects in telomere structure and functions. PMID:28350373

  4. [The human variome project and its progress].

    Science.gov (United States)

    Gao, Shan; Zhang, Ning; Zhang, Lei; Duan, Guang-You; Zhang, Tao

    2010-11-01

    The main goal of post genomics is to explain how the genome, the map of which has been constructed in the Human Genome Project, affacts activities of life. This leads to generate multiple "omics": structural genomics, functional genomics, proteomics, metabonomics, et al. In Jun. 2006, Melbourne, Australia, Human Genome Variation Society (HGVS) initiated the Human Variome Project (HVP) to collect all the sequence variation and polymorphism data worldwidely. HVP is to search and determine those mutations related with human diseases by association study between genetype and phenotype on the scale of genome level and other methods. Those results will be translated into clinical application. Considering the potential effects of this project on human health, this paper introduced its origin and main content in detail and discussed its meaning and prospect.

  5. Identification of Streptococcus parasanguinis DNA contamination in human buccal DNA samples

    OpenAIRE

    Mahfuz, Istiak; Cheng, Wei; White, Stefan J

    2013-01-01

    Background The use of buccal swabs in clinical and scientific studies is a very popular method of collecting DNA, due to its non-invasive nature of collection. However, contamination of the DNA sample may interfere with analysis. Findings Here we report the finding of Streptococcus parasanguinis bacterial DNA contamination in human buccal DNA samples, which led to preferential amplification of bacterial sequence with PCR primers designed against human sequence. Conclusion Contamination of buc...

  6. Human genome program report. Part 1, overview and progress

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1997-11-01

    This report contains Part 1 of a two-part report to reflect research and progress in the U.S. Department of Energy Human Genome Program from 1994 through 1996, with specified updates made just before publication. Part 1 consists of the program overview and report on progress.

  7. [Progress of the study on DNA electrochemical biosensor].

    Science.gov (United States)

    Zhao, Yanzhen; Zhang, Haiyan; Wu, Xiaoli; Liu, Zhongming; Wang, Jie

    2013-02-01

    With its rapid development, the electrochemical biosensor has recently been widely used in gene diagnosis, environmental monitoring, and medical sciences. More and more attention has been focused on how to improve the sensitivity and selectivity of biosensor. In this review, the principle and composition of DNA electrochemical biosensor is simply introduced, the preparation of biological membrane, the application of indicator are specially emphasized, and the future prospect for the development in this field is given.

  8. Reducing microbial and human contamination in DNA extractions from ancient bones and teeth.

    Science.gov (United States)

    Korlević, Petra; Gerber, Tobias; Gansauge, Marie-Theres; Hajdinjak, Mateja; Nagel, Sarah; Aximu-Petri, Ayinuer; Meyer, Matthias

    2015-08-01

    Although great progress has been made in improving methods for generating DNA sequences from ancient biological samples, many, if not most, samples are still not amenable for analyses due to overwhelming contamination with microbial or modern human DNA. Here we explore different DNA decontamination procedures for ancient bones and teeth for use prior to DNA library preparation and high-throughput sequencing. Two procedures showed promising results: (i) the release of surface-bound DNA by phosphate buffer and (ii) the removal of DNA contamination by sodium hypochlorite treatment. Exposure to phosphate removes on average 64% of the microbial DNA from bone powder but only 37% of the endogenous DNA (from the organism under study), increasing the percentage of informative sequences by a factor of two on average. An average 4.6-fold increase, in one case reaching 24-fold, is achieved by sodium hypochlorite treatment, albeit at the expense of destroying 63% of the endogenous DNA preserved in the bone. While both pretreatment methods described here greatly reduce the cost of genome sequencing from ancient material due to efficient depletion of microbial DNA, we find that the removal of human DNA contamination remains a challenging problem.

  9. Conference scene: progress with promising human antibodies.

    Science.gov (United States)

    Larrick, James W

    2012-03-01

    Antibodies and antibody-based therapeutics have become big business, with annual sales over US$50 billion, accounting for >6% of worldwide pharmaceutical revenues. Ten molecules have blockbuster status (>US$1 billion), with six generating more than US$6 billion in sales. In excess of 300 products based on this rapidly maturing technology are in clinical trials. The generation and manufacture of human antibodies is now routine, although the cost of goods remains an issue. Optimizing combinations of antibodies with other therapeutics (e.g., chemotherapy) is a major short-term goal, while target validation and product differentiation remain significant hurdles if growth is to continue. Some of the notable highlights of the recent 16th International Conference on Human Antibodies and Hybridomas meeting in Cannes, France are described below. The conference was sponsored by the international journal Human Antibodies, in association with the Integrative Medical Sciences Association (IMSA). The Program Chairman was Professor Mark Glassy, IMSA, San Diego, CA, USA.

  10. Repair of oxidative DNA damage and cancer: recent progress in DNA base excision repair.

    Science.gov (United States)

    Scott, Timothy L; Rangaswamy, Suganya; Wicker, Christina A; Izumi, Tadahide

    2014-02-01

    Reactive oxygen species (ROS) are generated by exogenous and environmental genotoxins, but also arise from mitochondria as byproducts of respiration in the body. ROS generate DNA damage of which pathological consequence, including cancer is well established. Research efforts are intense to understand the mechanism of DNA base excision repair, the primary mechanism to protect cells from genotoxicity caused by ROS. In addition to the notion that oxidative DNA damage causes transformation of cells, recent studies have revealed how the mitochondrial deficiencies and ROS generation alter cell growth during the cancer transformation. The emphasis of this review is to highlight the importance of the cellular response to oxidative DNA damage during carcinogenesis. Oxidative DNA damage, including 7,8-dihydro-8-oxoguanine, play an important role during the cellular transformation. It is also becoming apparent that the unusual activity and subcellular distribution of apurinic/apyrimidinic endonuclease 1, an essential DNA repair factor/redox sensor, affect cancer malignancy by increasing cellular resistance to oxidative stress and by positively influencing cell proliferation. Technological advancement in cancer cell biology and genetics has enabled us to monitor the detailed DNA repair activities in the microenvironment. Precise understanding of the intracellular activities of DNA repair proteins for oxidative DNA damage should provide help in understanding how mitochondria, ROS, DNA damage, and repair influence cancer transformation.

  11. Mitochondrial DNA Mutations in Epithelial Ovarian Tumor Progression

    Science.gov (United States)

    2007-12-01

    only in cystadenomas , 3/3; borderline, 4/4; stage IA, 2/2; stage IB, 0/3 and matched normal tissues, 4/4 of the ovarian serous subtype. Our findings...100% (7/7). Furthermore, two mutations were observed in serous tumors only at np 1657 in stage IV (10/10), and at np 8221delA in benign cystadenomas (3... cystadenomas , 3/3; borderline tumors, 4/4; stage I tumors, 2/5 and matched normal tissues 4/4). Conclusion: Our findings indicate that certain mtDNA

  12. DNA methylation analysis in human cancer

    OpenAIRE

    O'Sullivan, Eileen; Goggins, Michael

    2013-01-01

    The functional impact of aberrant DNA methylation and the widespread alterations in DNA methylation in cancer development has led to the development of a variety of methods to characterize the DNA methylation patterns. This chapter will critique and describe the major approaches to analyzing DNA methylation.

  13. Repair of Oxidative DNA Damage and Cancer: Recent Progress in DNA Base Excision Repair

    OpenAIRE

    Scott, Timothy L.; Rangaswamy, Suganya; Wicker, Christina A.; Izumi, Tadahide

    2014-01-01

    Significance: Reactive oxygen species (ROS) are generated by exogenous and environmental genotoxins, but also arise from mitochondria as byproducts of respiration in the body. ROS generate DNA damage of which pathological consequence, including cancer is well established. Research efforts are intense to understand the mechanism of DNA base excision repair, the primary mechanism to protect cells from genotoxicity caused by ROS. Recent Advances: In addition to the notion that oxidative DNA dama...

  14. Recent progress in atomistic simulation of electrical current DNA sequencing.

    Science.gov (United States)

    Kim, Han Seul; Kim, Yong-Hoon

    2015-07-15

    We review recent advances in the DNA sequencing method based on measurements of transverse electrical currents. Device configurations proposed in the literature are classified according to whether the molecular fingerprints appear as the major (Mode I) or perturbing (Mode II) current signals. Scanning tunneling microscope and tunneling electrode gap configurations belong to the former category, while the nanochannels with or without an embedded nanopore belong to the latter. The molecular sensing mechanisms of Modes I and II roughly correspond to the electron tunneling and electrochemical gating, respectively. Special emphasis will be given on the computer simulation studies, which have been playing a critical role in the initiation and development of the field. We also highlight low-dimensional nanomaterials such as carbon nanotubes, graphene, and graphene nanoribbons that allow the novel Mode II approach. Finally, several issues in previous computational studies are discussed, which points to future research directions toward more reliable simulation of electrical current DNA sequencing devices. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Plans and progress for building a Great Lakes fauna DNA ...

    Science.gov (United States)

    DNA reference libraries provide researchers with an important tool for assessing regional biodiversity by allowing unknown genetic sequences to be assigned identities, while also providing a means for taxonomists to validate identifications. Expanding the representation of Great Lakes species in such reference libraries is an explicit component of research at EPA’s Mid-Continent Ecology Division. Our DNA reference library building efforts began in 2012 with the goal of providing barcodes for at least 5 specimens of each native and nonindigenous fish and aquatic invertebrate species currently present in the Great Lakes. The approach is to pull taxonomically validated specimen for sequencing from EPA led sampling efforts of adult/juvenile fish, larval fish, benthic macroinvertebrates, and zooplankton; while also soliciting aid from state and federal agencies for tissue from “shopping list” organisms. The barcodes we generate are made available through the publicly accessible BOLD (Barcode of Life) database, and help inform a planned Great Lakes biodiversity inventory. To date, our submissions to BOLD are limited to fishes; of the 88 fish species listed as being present within Lake Superior, roughly half were successfully barcoded, while only 22 species met the desired quota of 5 barcoded specimens per species. As we continue to generate genomic information from our collections and the taxonomic representations become more complete, we will continue to

  16. The persistence of human DNA in soil following surface decomposition.

    Science.gov (United States)

    Emmons, Alexandra L; DeBruyn, Jennifer M; Mundorff, Amy Z; Cobaugh, Kelly L; Cabana, Graciela S

    2017-09-01

    Though recent decades have seen a marked increase in research concerning the impact of human decomposition on the grave soil environment, the fate of human DNA in grave soil has been relatively understudied. With the purpose of supplementing the growing body of literature in forensic soil taphonomy, this study assessed the relative persistence of human DNA in soil over the course of decomposition. Endpoint PCR was used to assess the presence or absence of human nuclear and mitochondrial DNA, while qPCR was used to evaluate the quantity of human DNA recovered from the soil beneath four cadavers at the University of Tennessee's Anthropology Research Facility (ARF). Human nuclear DNA from the soil was largely unrecoverable, while human mitochondrial DNA was detectable in the soil throughout all decomposition stages. Mitochondrial DNA copy abundances were not significantly different between decomposition stages and were not significantly correlated to soil edaphic parameters tested. There was, however, a significant positive correlation between mitochondrial DNA copy abundances and the human associated bacteria, Bacteroides, as estimated by 16S rRNA gene abundances. These results show that human mitochondrial DNA can persist in grave soil and be consistently detected throughout decomposition. Copyright © 2017 The Chartered Society of Forensic Sciences. Published by Elsevier B.V. All rights reserved.

  17. Progress in the international protection of human rights.

    Science.gov (United States)

    Suter, Keith

    2002-01-01

    Great progress has been made in the international protection of human rights since 10 December 1948 (when the United Nations General Assembly adopted the Universal Declaration of Human Rights). Amidst the doom and gloom of the media's reporting of current affairs, it is easy to overlook this progress. This article provides a definition of 'human rights' and examines early human rights campaigns. It then considers the areas of progress: human rights are now part of the international political vocabulary, there is a recognition that respect for human rights can assist a country's economic and social development, there has been a growth of human rights treaties and techniques and non-governmental organizations (NGOs) see protecting human rights as a major activity. State sovereignty has been eroded as national governments are being held accountable to the international community for their human rights policies. A new challenge is to ensure respect for human rights by non-state entities, such as transnational corporations. The growing culture of international protection of human rights is here to stay. This is not a reason for complacency, but it is a sign of hope.

  18. Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication.

    Science.gov (United States)

    Zou, Wei; Wang, Zekun; Xiong, Min; Chen, Aaron Yun; Xu, Peng; Ganaie, Safder S; Badawi, Yomna; Kleiboeker, Steve; Nishimune, Hiroshi; Ye, Shui Qing; Qiu, Jianming

    2017-12-13

    Human parvovirus B19 (B19V) infection of human erythroid progenitor cells (EPCs) induces a DNA damage response and cell cycle arrest at late S phase, which facilitates viral DNA replication. However, it is not clear exactly which cellular factors are employed by this single-stranded DNA virus. Here, we used microarrays to systematically analyze the dynamic transcriptome of EPCs infected with B19V. We found that DNA metabolism, DNA replication, DNA repair, DNA damage response, cell cycle, and cell cycle arrest pathways were significantly regulated after B19V infection. Confocal microscopy analyses revealed that most cellular DNA replication proteins were recruited to the centers of viral DNA replication, but not the DNA repair DNA polymerases. Our results suggest that DNA replication polymerase δ and polymerase α are responsible for B19V DNA replication by knocking down its expression in EPCs. We further showed that although RPA32 is essential for B19V DNA replication and the phosphorylated forms of RPA32 colocalized with the replicating viral genomes, RPA32 phosphorylation was not necessary for B19V DNA replication. Thus, this study provides evidence that B19V uses the cellular DNA replication machinery for viral DNA replication.IMPORTANCE Human parvovirus B19 (B19V) infection can cause transient aplastic crisis, persistent viremia, and pure red-cell aplasia. In fetuses, B19V infection can result in non-immune hydrops fetalis and fetal death. These clinical manifestations of B19V infection are a direct outcome of the death of human erythroid progenitors that host B19V replication. B19V infection induces a DNA damage response that is important for cell cycle arrest at late S phase. Here, we analyzed dynamic changes in cellular gene expression, and found that DNA metabolic processes are tightly regulated during B19V infection. Although genes involved in cellular DNA replication were downregulated overall, the cellular DNA replication machinery was tightly

  19. 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

  20. Triplet repeat DNA structures and human genetic disease: dynamic ...

    Indian Academy of Sciences (India)

    Triplet repeat DNA structures and human genetic disease: dynamic mutations from dynamic DNA. Richard R Sinden Vladimir N ... Different models have been proposed for the expansion of triplet repeats, most of which presume the formation of alternative DNA structures in repeat tracts. One of the most likely structures, ...

  1. Tenacity of Exogenous Human Papillomavirus DNA in Sperm Washing

    OpenAIRE

    Brossfield, Jeralyn E.; Philip J. Chan; Patton, William C.; King, Alan

    1999-01-01

    Purpose:Sperm cells have been shown to take up exogenous DNA readily. The hypothesis was that sperm washing would remove exogenous viral DNA infecting sperm cells. The objective was to compare three types of sperm washing procedures for their capacity to remove exogenous human papillomavirus (HPV) DNA from infected sperm.

  2. Search for DNA damage by human alkyladenine DNA glycosylase involves early intercalation by an aromatic residue.

    Science.gov (United States)

    Hendershot, Jenna M; O'Brien, Patrick J

    2017-09-29

    DNA repair enzymes recognize and remove damaged bases that are embedded in the duplex. To gain access, most enzymes use nucleotide flipping, whereby the target nucleotide is rotated 180° into the active site. In human alkyladenine DNA glycosylase (AAG), the enzyme that initiates base excision repair of alkylated bases, the flipped-out nucleotide is stabilized by intercalation of the side chain of tyrosine 162 that replaces the lesion nucleobase. Previous kinetic studies provided evidence for the formation of a transient complex that precedes the stable flipped-out complex, but it is not clear how this complex differs from nonspecific complexes. We used site-directed mutagenesis and transient-kinetic approaches to investigate the timing of Tyr 162 intercalation for AAG. The tryptophan substitution (Y162W) appeared to be conservative, because the mutant protein retained a highly favorable equilibrium constant for flipping the 1, N 6 -ethenoadenine (ϵA) lesion, and the rate of N -glycosidic bond cleavage was identical to that of the wild-type enzyme. We assigned the tryptophan fluorescence signal from Y162W by removing two native tryptophan residues (W270A/W284A). Stopped-flow experiments then demonstrated that the change in tryptophan fluorescence of the Y162W mutant is extremely rapid upon binding to either damaged or undamaged DNA, much faster than the lesion-recognition and nucleotide flipping steps that were independently determined by monitoring the ϵA fluorescence. These observations suggest that intercalation by this aromatic residue is one of the earliest steps in the search for DNA damage and that this interaction is important for the progression of AAG from nonspecific searching to specific-recognition complexes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. 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.

  4. [Progress of research on human tolerance to impact acceleration].

    Science.gov (United States)

    Liu, Bingkun; Ma, Honglei; Jiang, Shizhong

    2010-04-01

    Impact acceleration is one of the factors to which human body is exposed in aerospace exploring. When the impact level is greater than human tolerance, it usually results in human injuries which may be fatal. Therefore, in order to reduce or avert the risk of serious injury from crash impact, human tolerance to impact acceleration is a crucial consideration in aircraft since the beginning of aviation. The study on human tolerance to impact acceleration has become a cynosure in the realm of modern biomechanics. So this paper reviews the progress of the researches.

  5. Earnings progression, human capital and incentives: Theory and evidence

    OpenAIRE

    Frederiksen, Anders

    2010-01-01

    The career prospects of newly recruited employees differ substantially within an organization. The stars experience a considerable growth in earnings; others can hardly maintain their entry salaries. This article sheds light on the mechanisms generating the observed heterogeneity in earnings progression by investigating the effects of on-the-job human capital acquisition, explicit short-run incentives and career concern incentives on earnings progression. The model leads to predictions about ...

  6. 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

  7. Progress on molecular mechanism of T-DNA transport and integration.

    Science.gov (United States)

    Zhan, Ya-Guang; Zeng, Fan-Suo; Xin, Ying

    2005-06-01

    Agrobacterium-mediated transformation is probably the most widely used method to introduce genes into plants. Great progress has been made in recent years in studies on the mechanism of Agrobacterium-mediated transformation. Agrobacterium genetically transforms plants by transferring a portion of the resident Ti-plasmid, the T-DNA, to the plant. VirD2 and VirE2 accompany the T-DNA into the plant cell. Both proteins may aid in T-DNA transfer, nuclear targeting and integration into the plant genome. In recent years, some Arabidopsis rat (resistant to transformation) mutants are deficient in T-DNA integration according to some studies. These results showed that plant genes participate in the T-DNA transport and integration process. This paper discusses our current knowledge about the functions of virulence protein, namely VirD2 and VirE2, and plant genes in several aspect of Agrobacterium transformation. The paper discusses two different classes of integration patterns in detail: one is T-DNA right border being linked to genomic sequences by the VirD2 protein, the other is T-DNA integration via SDSA (synthesis-dependent strand-annealing). According to the latest studies we elaborated the T-DNA integration model based on genomic DSB (double-strand breaks) and proposed a new opinion about the mechanism of T-DNA integration.

  8. Human DNA repair disorders in dermatology: A historical perspective, current concepts and new insight.

    Science.gov (United States)

    Moriwaki, Shinichi

    2016-02-01

    Products of DNA damage, such as cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts (6-4 PPs), are continually formed in genomes after exposure to UV radiation. When these DNA damages remain unrepaired in essential DNA sites for prolonged periods, DNA replication and transcription are hampered or mutation is induced, which may cause cell death, cellular senescence, and carcinogenesis of the skin. To protect against such UV-induced DNA damage, living organisms nicely retain "DNA repair systems", which can efficiently repair "harmful" DNA damage through precise mechanisms by the integrated functions of many proteins. In humans, the failure of DNA repair systems causes a variety of disorders. Dermatological conditions such as hereditary photodermatoses, xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are caused by congenital functional defects in the nucleotide excision repair (NER) system or the translesion synthesis (TLS) system. In this review, we describe the historical progress, recent findings, and future prospects of studies of human diseases associated with DNA-repair defects. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  9. Methylation of insulin DNA in response to proinflammatory cytokines during the progression of autoimmune diabetes in NOD mice.

    Science.gov (United States)

    Rui, Jinxiu; Deng, Songyan; Lebastchi, Jasmin; Clark, Pamela L; Usmani-Brown, Sahar; Herold, Kevan C

    2016-05-01

    Type 1 diabetes is caused by the immunological destruction of pancreatic beta cells. Preclinical and clinical data indicate that there are changes in beta cell function at different stages of the disease, but the fate of beta cells has not been closely studied. We studied how immune factors affect the function and epigenetics of beta cells during disease progression and identified possible triggers of these changes. We studied FACS sorted beta cells and infiltrating lymphocytes from NOD mouse and human islets. Gene expression was measured by quantitative real-time RT-PCR (qRT-PCR) and methylation of the insulin genes was investigated by high-throughput and Sanger sequencing. To understand the role of DNA methyltransferases, Dnmt3a was knocked down with small interfering RNA (siRNA). The effects of cytokines on methylation and expression of the insulin gene were studied in humans and mice. During disease progression in NOD mice, there was an inverse relationship between the proportion of infiltrating lymphocytes and the beta cell mass. In beta cells, methylation marks in the Ins1 and Ins2 genes changed over time. Insulin gene expression appears to be most closely regulated by the methylation of Ins1 exon 2 and Ins2 exon 1. Cytokine transcription increased with age in NOD mice, and these cytokines could induce methylation marks in the insulin DNA by inducing methyltransferases. Similar changes were induced by cytokines in human beta cells in vitro. Epigenetic modification of DNA by methylation in response to immunological stressors may be a mechanism that affects insulin gene expression during the progression of type 1 diabetes.

  10. 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.

  11. Recent progress with the DNA repair mutants of Chinese hamster ovary cells

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, L.H.; Salazar, E.P.; Brookman, K.W.; Collins, C.C.; Stewart, S.A.; Busch, D.B.; Weber, C.A.

    1986-04-02

    Repair deficient mutants of Chinese hamster ovary (CHO) cells are being used to identify human genes that correct the repair defects and to study mechanisms of DNA repair and mutagenesis. Five independent tertiary DNA transformants were obtained from the EM9 mutant. In these clones a human DNA sequence was identified that correlated with the resistance of the cells to CldUrd. After Eco RI digestion, Southern transfer, and hybridization of transformant DNAs with the BLUR-8 Alu family sequence, a common fragment of 25 to 30 kb was present. 37 refs., 4 figs., 3 tabs.

  12. [A single deletion of mitochondrial DNA in a Brazilian patient with chronic progressive external ophthalmoplegia].

    Science.gov (United States)

    Carod-Artal, F J; Solano-Palacios, A; Playán-Ariso, A; Viana-Brandi, I; López-Gallardo, E; Andreu, A; López-Pérez, M; Montoya, J

    The syndrome of chronic progressive external ophthalmoplegia (CPEO) has been associated to the presence of large deletion, single or multiple, in the mitochondrial DNA of skeletal muscle. We report a sporadic case of chronic progressive external ophthalmoplegia that began at age 19 years and was associated with ragged red fibers in skeletal muscle. Genetic analysis of mitochondrial DNA revealed the presence of a single deletion of 4237 bp that encompasses the nucleotide positions 9486 to 13722, a location that has not been described before, and flanked by a direct repeat sequence. The deletion is flanked by a direct repeat. The amount of deleted mitochondrial DNA (55%) in this patient's muscle suggests that this deletion is the molecular cause of the phenotypic presentation of this patient.

  13. [Mitochondrial DNA mutations in patients with chronic progressive external ophthalmoplegia and Kearns-Sayre syndrome].

    Science.gov (United States)

    Wang, Zhao-xia; Yuan, Yun; Gao, Feng; Qi, Yu; Shen, Ding-guo; Chen, Qing-tang

    2003-08-01

    Kearns-Sayre syndrome (KSS) and chronic progressive external ophthalmoplegia (CPEO) belong to neurological diseases caused by a defect in the energy-producing system of mitochondria, and are known to be associated with a deletion in the mitochondrial genome. This study was aimed to understand with greater clearness the characteristics of mitochondrial DNA (mtDNA) mutations in 11 Chinese patients with CPEO (7 cases) or KSS (4 cases). Densitometry of the bands on Southern blot, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and sequencing were performed to search large scale deletions and A3243G point mutation in patients' muscle mtDNA. Large deletions in mtDNA were detected in 2 CPEO and 3 KSS patients, the size of deletion ranged from 3.0 kb to 8.0 kb. Moreover, mtDNA A3243G point mutation was identified in 1 KSS patient. The proportion of mutant mtDNA was 37.6%-87.0%. Direct sequencing of the PCR products revealed 5 novel large deletions not reported by others. The findings in this study being consistent with the reports by others, large scale deletions of mtDNA are frequently found in Chinese patients with KSS and CPEO. mtDNA A3243G mutation may also exist in some patients with KSS and CPEO.

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

    Science.gov (United States)

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

    2012-07-24

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

  15. 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.

  16. Glutathione dysregulation and the etiology and progression of human diseases.

    NARCIS (Netherlands)

    Ballatori, N.; Krance, S.M.; Notenboom, S.; Shi, S.; Tieu, K.; Hammond, C.L.

    2009-01-01

    Glutathione (GSH) plays an important role in a multitude of cellular processes, including cell differentiation, proliferation, and apoptosis, and as a result, disturbances in GSH homeostasis are implicated in the etiology and/or progression of a number of human diseases, including cancer, diseases

  17. Mechanism of Error-Free DNA Replication Past Lucidin-Derived DNA Damage by Human DNA Polymerase κ.

    Science.gov (United States)

    Yockey, Oliver P; Jha, Vikash; Ghodke, Pratibha P; Xu, Tianzuo; Xu, Wenyan; Ling, Hong; Pradeepkumar, P I; Zhao, Linlin

    2017-11-20

    DNA damage impinges on genetic information flow and has significant implications in human disease and aging. Lucidin-3-O-primeveroside (LuP) is an anthraquinone derivative present in madder root, which has been used as a coloring agent and food additive. LuP can be metabolically converted to genotoxic compound lucidin, which subsequently forms lucidin-specific N2-2'-deoxyguanosine (N2-dG) and N6-2'-deoxyadenosine (N6-dA) DNA adducts. Lucidin is mutagenic and carcinogenic in rodents but has low carcinogenic risks in humans. To understand the molecular mechanism of low carcinogenicity of lucidin in humans, we performed DNA replication assays using site-specifically modified oligodeoxynucleotides containing a structural analogue (LdG) of lucidin-N2-dG DNA adduct and determined the crystal structures of DNA polymerase (pol) κ in complex with LdG-bearing DNA and an incoming nucleotide. We examined four human pols (pol η, pol ι, pol κ, and Rev1) in their efficiency and accuracy during DNA replication with LdG; these pols are key players in translesion DNA synthesis. Our results demonstrate that pol κ efficiently and accurately replicates past the LdG adduct, whereas DNA replication by pol η, pol ι is compromised to different extents. Rev1 retains its ability to incorporate dCTP opposite the lesion albeit with decreased efficiency. Two ternary crystal structures of pol κ illustrate that the LdG adduct is accommodated by pol κ at the enzyme active site during insertion and postlesion-extension steps. The unique open active site of pol κ allows the adducted DNA to adopt a standard B-form for accurate DNA replication. Collectively, these biochemical and structural data provide mechanistic insights into the low carcinogenic risk of lucidin in humans.

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

    Science.gov (United States)

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

    2018-02-01

    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.

  19. Chronic progressive external ophthalmoplegia with T9957C mitochondrial DNA mutation in a Taiwanese patient.

    Science.gov (United States)

    Liu, Ching-Hsiung; Liou, Chia-Wei; Liu, Chi-Hung; Kuo, Hung-Chou; Chu, Chun-Che; Huang, Chin-Chang

    2011-03-01

    Mitochondrial T9957C mutations have been reported in patients with nonarteritic ischemic optic neuropathy and seizures and in patients with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes. However, thus far, this mutation has not been reported in patients with chronic progressive external ophthalmoplegia (CPEO). Here we report a female patient with CPEO and agenesis of the corpus callosum. Although no ragged-red fibers were found upon muscle biopsy, sequencing of the entire mitochondrial DNA genome was done. The molecular genetic study revealed a nonsynonymous mitochondrial T9957C mutation. a new genotype of CPEO was identified with varied clinical presentations. Although the effect of the nuclear genome remains unknown, we believe that the nonsynonymous mitochondrial DNA (mtDNA) T9957C mutation may have a role in the clinical manifestations of this patient. This study extends the phenotype of T9957C mtDNA mutation.

  20. HIV-1 DNA predicts disease progression and post-treatment virological control

    Science.gov (United States)

    Williams, James P; Hurst, Jacob; Stöhr, Wolfgang; Robinson, Nicola; Brown, Helen; Fisher, Martin; Kinloch, Sabine; Cooper, David; Schechter, Mauro; Tambussi, Giuseppe; Fidler, Sarah; Carrington, Mary; Babiker, Abdel; Weber, Jonathan

    2014-01-01

    In HIV-1 infection, a population of latently infected cells facilitates viral persistence despite antiretroviral therapy (ART). With the aim of identifying individuals in whom ART might induce a period of viraemic control on stopping therapy, we hypothesised that quantification of the pool of latently infected cells in primary HIV-1 infection (PHI) would predict clinical progression and viral replication following ART. We measured HIV-1 DNA in a highly characterised randomised population of individuals with PHI. We explored associations between HIV-1 DNA and immunological and virological markers of clinical progression, including viral rebound in those interrupting therapy. In multivariable analyses, HIV-1 DNA was more predictive of disease progression than plasma viral load and, at treatment interruption, predicted time to plasma virus rebound. HIV-1 DNA may help identify individuals who could safely interrupt ART in future HIV-1 eradication trials. Clinical trial registration: ISRCTN76742797 and EudraCT2004-000446-20 DOI: http://dx.doi.org/10.7554/eLife.03821.001 PMID:25217531

  1. Dietary boron: progress in establishing essential roles in human physiology.

    Science.gov (United States)

    Hunt, Curtiss D

    2012-06-01

    This review summarizes the progress made in establishing essential roles for boron in human physiology and assesses that progress in view of criteria for essentiality of elements. The evidence to date suggests that humans and at least some higher animals may use boron to support normal biological functions. These include roles in calcium metabolism, bone growth and maintenance, insulin metabolism, and completion of the life cycle. The biochemical mechanisms responsible for these effects are poorly understood but the nature of boron biochemistry suggests further characterization of the cell signaling molecules capable of complexing with boron. Such characterization may provide insights into the biochemical function(s) of boron in humans. Copyright © 2012 Elsevier GmbH. All rights reserved.

  2. Extraction of human nuclear DNA from feces samples using the QIAamp DNA Stool Mini Kit.

    Science.gov (United States)

    Vandenberg, Nicholas; van Oorschot, Roland A H

    2002-09-01

    The use of a QIAamp DNA Stool Mini Kit (QIAGEN) for extracting human nuclear DNA from feces samples is reported. This method employs a stool lysis buffer and a unique matrix (InhibitEX tablet) to remove PCR inhibitory substances specific to feces samples. DNA extracted from various amounts of stool and from stool samples exposed to different environmental impacts was successfully amplified and typed using the Profiler Plus Amplification Kit and ABI PRISM 310 Genetic Analyser.

  3. 3D replicon distributions arise from stochastic initiation and domino-like DNA replication progression.

    Science.gov (United States)

    Löb, D; Lengert, N; Chagin, V O; Reinhart, M; Casas-Delucchi, C S; Cardoso, M C; Drossel, B

    2016-04-07

    DNA replication dynamics in cells from higher eukaryotes follows very complex but highly efficient mechanisms. However, the principles behind initiation of potential replication origins and emergence of typical patterns of nuclear replication sites remain unclear. Here, we propose a comprehensive model of DNA replication in human cells that is based on stochastic, proximity-induced replication initiation. Critical model features are: spontaneous stochastic firing of individual origins in euchromatin and facultative heterochromatin, inhibition of firing at distances below the size of chromatin loops and a domino-like effect by which replication forks induce firing of nearby origins. The model reproduces the empirical temporal and chromatin-related properties of DNA replication in human cells. We advance the one-dimensional DNA replication model to a spatial model by taking into account chromatin folding in the nucleus, and we are able to reproduce the spatial and temporal characteristics of the replication foci distribution throughout S-phase.

  4. Characterization of circulating DNA in healthy human plasma.

    Science.gov (United States)

    Suzuki, Nobuyasu; Kamataki, Akihisa; Yamaki, Junko; Homma, Yoshimi

    2008-01-01

    The importance of circulating DNA has been recognized since the detection of mutated oncogene products in cancer patients; however, there is little information about circulating DNA in normal human plasma. We characterized circulating DNA in normal human plasma to obtain basic information. Circulating DNA was purified from plasma samples obtained from 10 healthy donors and examined. Purified DNA was cloned and their sequence determined and analyzed. The terminal structure was examined by a labeling method. The DNA levels in normal plasma samples were quite low (3.6-5.0 ng/ml). All 556 clones analyzed were independent, and obtained from various chromosomes and various regions of the gene. The mean values of their length and GC content were 176 bp and 53.7%, respectively. Their 5' and 3' ends were rich in C and G, respectively, and they presented as 5' protruding forms of double-stranded DNA in plasma. Circulating DNA in normal human plasma is derived from apoptotic cells but not from necrotic cells. Structural characteristics of the circulating DNA might be associated with their stability in plasma.

  5. 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. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  6. Characterisation of the histone methyltransferase SET8 in cell cycle progression and the DNA damage response

    DEFF Research Database (Denmark)

    Jørgensen, Stine

    2008-01-01

    component of the replication fork, further supporting the involvement of SET8 in replication, suggesting that SET8 may be required to support replication fork progression. Finally, we showed that SET8 was rapidly degraded by DNA damage such as UV and ionizing radiation (IR), which was accompanied...... recombination and repair. I therefore initiated a mass spectrometry based study to identify changes in histone modifications after DNA damage. By using SILAC labelling of cells to quantatively measure the changes in histone modifications, we observed a marked reduction in the level of monomethylated Histone H4...

  7. 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.

  8. DNA content in human uterine cervical dysplasias.

    Science.gov (United States)

    Palan, P R; Romney, S L

    1979-01-01

    The DNA content of nuclei extracted from separate biopsies of normal cervical epithelium and from dysplastic sites in the same patient was determined biochemically. Mean percentage increase in the DNA content of dysplasias (mild: 143.3 +/- 14.9; moderate: 225.8 +/- 51.1; and severe: 359.8 +/- 45.8) was found to be statistically significant (F-Test: p less than 0.001) over the normal values in control cervical tissues.

  9. Artificial Intelligence, DNA Mimicry, and Human Health.

    Science.gov (United States)

    Stefano, George B; Kream, Richard M

    2017-08-14

    The molecular evolution of genomic DNA across diverse plant and animal phyla involved dynamic registrations of sequence modifications to maintain existential homeostasis to increasingly complex patterns of environmental stressors. As an essential corollary, driver effects of positive evolutionary pressure are hypothesized to effect concerted modifications of genomic DNA sequences to meet expanded platforms of regulatory controls for successful implementation of advanced physiological requirements. It is also clearly apparent that preservation of updated registries of advantageous modifications of genomic DNA sequences requires coordinate expansion of convergent cellular proofreading/error correction mechanisms that are encoded by reciprocally modified genomic DNA. Computational expansion of operationally defined DNA memory extends to coordinate modification of coding and previously under-emphasized noncoding regions that now appear to represent essential reservoirs of untapped genetic information amenable to evolutionary driven recruitment into the realm of biologically active domains. Additionally, expansion of DNA memory potential via chemical modification and activation of noncoding sequences is targeted to vertical augmentation and integration of an expanded cadre of transcriptional and epigenetic regulatory factors affecting linear coding of protein amino acid sequences within open reading frames.

  10. Interaction of clinically important human DNA topoisomerase I poison, topotecan, with double-stranded DNA.

    Science.gov (United States)

    Streltsov, Sergei; Oleinikov, Vladimir; Ermishov, Mikhail; Mochalov, Konstantin; Sukhanova, Alyona; Nechipurenko, Yuri; Grokhovsky, Sergei; Zhuze, Alexei; Pluot, Michel; Nabiev, Igor

    2003-01-01

    Topotecan (TPT), a water-soluble derivative of camptothecin, is a potent antitumor poison of human DNA topoisomerase I (top1) that stabilizes the cleavage complex between the enzyme and DNA. The role of the recently discovered TPT affinity to DNA remains to be defined. The aim of this work is to clarify the molecular mechanisms of the TPT-DNA interaction and to propose the models of TPT-DNA complexes in solution in the absence of top1. It is shown that TPT molecules form dimers with a dimerization constant of (4.0 +/- 0.7) x 10(3) M(-1) and the presence of DNA provokes more than a 400-fold increase of the effective dimerization constant. Flow linear dichroism spectroscopy accompanied by circular dichroism, fluorescence, and surface-enhanced Raman scattering experiments provide evidence that TPT dimers are able to bind DNA by bridging different DNA molecules or distant DNA structural domains. This effect may provoke modification of the intrinsic geometry of the cruciform DNA structures, leading to the appearance of new crossover points that serve as the sites of the top1 loading position. The data presume the hypothesis of TPT-mediated modulation of top1-DNA recognition before ternary complex formation. Copyright 2003 Wiley Periodicals, Inc.

  11. Mouse DNA contamination in human tissue tested for XMRV.

    Science.gov (United States)

    Robinson, Mark J; Erlwein, Otto W; Kaye, Steve; Weber, Jonathan; Cingoz, Oya; Patel, Anup; Walker, Marjorie M; Kim, Wun-Jae; Uiprasertkul, Mongkol; Coffin, John M; McClure, Myra O

    2010-12-20

    We used a PCR-based approach to study the prevalence of genetic sequences related to a gammaretrovirus, xenotropic murine leukemia virus-related virus, XMRV, in human prostate cancer. This virus has been identified in the US in prostate cancer patients and in those with chronic fatigue syndrome. However, with the exception of two patients in Germany, XMRV has not been identified in prostate cancer tissue in Europe. Most putative associations of new or old human retroviruses with diseases have turned out to be due to contamination. We have looked for XMRV sequences in DNA extracted from formalin-fixed paraffin- embedded prostate tissues. To control for contamination, PCR assays to detect either mouse mitochondrial DNA (mtDNA) or intracisternal A particle (IAP) long terminal repeat DNA were run on all samples, owing to their very high copy number in mouse cells. In general agreement with the US prevalence, XMRV-like sequences were found in 4.8% of prostate cancers. However, these were also positive, as were 21.5% of XMRV-negative cases, for IAP sequences, and many, but not all were positive for mtDNA sequences. These results show that contamination with mouse DNA is widespread and detectable by the highly sensitive IAP assay, but not always with less sensitive assays, such as murine mtDNA PCR. This study highlights the ubiquitous presence of mouse DNA in laboratory specimens and offers a means of rigorous validation for future studies of murine retroviruses in human disease.

  12. 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.

  13. Human exonuclease 1 and BLM helicase interact to resect DNA and initiate DNA repair

    Science.gov (United States)

    Nimonkar, Amitabh V.; Özsoy, A. Zeynep; Genschel, Jochen; Modrich, Paul; Kowalczykowski, Stephen C.

    2008-01-01

    The error-free repair of double-stranded DNA breaks by homologous recombination requires processing of broken ends. These processed ends are substrates for assembly of DNA strand exchange proteins that mediate DNA strand invasion. Here, we establish that human BLM helicase, a member of the RecQ family, stimulates the nucleolytic activity of human exonuclease 1 (hExo1), a 5′→3′ double-stranded DNA exonuclease. The stimulation is specific because other RecQ homologs fail to stimulate hExo1. Stimulation of DNA resection by hExo1 is independent of BLM helicase activity and is, instead, mediated by an interaction between the 2 proteins. Finally, we show that DNA ends resected by hExo1 and BLM are used by human Rad51, but not its yeast or bacterial counterparts, to promote homologous DNA pairing. This in vitro system recapitulates initial steps of homologous recombination and provides biochemical evidence for a role of BLM and Exo1 in the initiation of recombinational DNA repair. PMID:18971343

  14. Recovery of DNA from human teeth by cryogenic grinding.

    Science.gov (United States)

    Sweet, D; Hildebrand, D

    1998-11-01

    DNA has been previously recovered from human teeth for RFLP and PCR-based forensic analysis. In some cases, the maximum amount of undisturbed tooth structure is required for ulterior forensic analysis. But, in most cases, following comprehensive documentation, it is possible to section the tooth longitudinally or horizontally, or crush it to access the DNA-rich core. This technical report describes an alternative method to recover DNA from whole extracted human molar teeth. A 6700 freezer mill was used to pulverize 20 teeth under frozen preparation in liquid nitrogen and sterile conditions. The mean yield of DNA was 30.9 micrograms (18.4 micrograms DNA per gm tooth powder). The resulting fine powder was subjected to organic extraction and subsequently quantified using slot blot hybridization. Aliquots were successfully amplified at three short tandem repeat polymorphic loci. The technique is simple and relatively rapid. Isolation of the samples during pulverization minimizes the risk of contamination.

  15. 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...... in human cells, which are deeply implicated in normal cellular processes as well as in several major human diseases. Here, a range of different methods for the analyses of DNA methylation and hydroxymethylation at locus-specific and genome-wide scales is described....

  16. [The cytogenetics of human oocytes: 40 years of progress].

    Science.gov (United States)

    Pellestor, F; Andréo, B; Anahory, T; Déchaud, H; Hédon, B; Hamamah, S

    2005-05-01

    Chromosomal abnormalities account for the majority of pre- and post- implantation embryo wastage in humans. Most of these abnormalities result from maternal meiotic errors, which preferentially occur during the first meiotic division. Consequently, the cytogenetic analysis of human oocytes has then been considered as a highly valuable source of data for the investigation of both the occurrence and the origin of chromosomal abnormalities in human. During the last 4 decades, the cytogenetic analysis of human oocytes has never stopped progressing, according to the advents of new technologies. Both karyotyping and molecular cytogenetic studies have been reported to date, providing a large body of data on the incidence and the distribution of chromosomal abnormalities in human female gametes. However, these studies display a great variability in results, which may be essentially attributable to the limitations of these techniques when applied to human oocytes. The most relevant analysis have led to the estimate that 15-20% of human oocytes present chromosome abnormalities, and they have emphasized the implication of both whole chromosome non-disjunction and chromatid separation in the occurrence of aneuploidy in human oocytes. The effect of advanced maternal age on the incidence of aneuploidy in human oocytes has also been clearly evidenced by recent reports based on large sample of oocytes or polar bodies.

  17. Non-DBS DNA Repair Genes Regulate Radiation-induced Cytogenetic Damage Repair and Cell Cycle Progression

    Science.gov (United States)

    Zhang, Ye; Rohde, Larry H.; Emami, Kamal; Casey, Rachael; Wu, Honglu

    2008-01-01

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have shown that genes up-regulated by IR may play important roles in DNA damage repair, the relationship between the regulation of gene expression by IR, particularly genes not known for their roles in DSB repair, and its impact on cytogenetic responses has not been systematically studied. In the present study, the expression of 25 genes selected on the basis of their transcriptional changes in response to IR was individually knocked down by transfection with small interfering RNA in human fibroblast cells. The purpose of this study is to identify new roles of these selected genes on regulating DSB repair and cell cycle progression , as measured in the micronuclei formation and chromosome aberration. In response to IR, the formation of MN was significantly increased by suppressed expression of 5 genes: Ku70 in the DSB repair pathway, XPA in the NER pathway, RPA1 in the MMR pathway, and RAD17 and RBBP8 in cell cycle control. Knocked-down expression of 4 genes (MRE11A, RAD51 in the DSB pathway, SESN1, and SUMO1) significantly inhibited cell cycle progression, possibly because of severe impairment of DNA damage repair. Furthermore, loss of XPA, P21, or MLH1 expression resulted in both significantly enhanced cell cycle progression and increased yields of chromosome aberrations, indicating that these gene products modulate both cell cycle control and DNA damage repair. Most of the 11 genes that affected cytogenetic responses are not known to have clear roles influencing DBS repair. Nine of these 11 genes were up-regulated in cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulate the biological consequences after IR.

  18. A rapid DNA extraction method suitable for human papillomavirus detection.

    Science.gov (United States)

    Brestovac, Brian; Wong, Michelle E; Costantino, Paul S; Groth, David

    2014-04-01

    Infection with oncogenic human papillomavirus (HPV) genotypes is necessary for the development of cervical cancer. Testing for HPV DNA from liquid based cervical samples can be used as an adjunct to traditional cytological screening. In addition there are ongoing viral load, genotyping, and prevalence studies. Therefore, a sensitive DNA extraction method is needed to maximize the efficiency of HPV DNA detection. The XytXtract Tissue kit is a DNA extraction kit that is rapid and so could be useful for HPV testing, particularly in screening protocols. This study was undertaken to determine the suitability of this method for HPV detection. DNA extraction from HeLa and Caski cell lines containing HPV 18 and 16 respectively together with DNA from five liquid based cervical samples were used in a HPV PCR assay. DNA was also extracted using the QIAamp DNA mini kit (Qiagen, Hilden, Germany) as a comparison. DNA extracts were serially diluted and assayed. HPV DNA was successfully detected in cell lines and cervical samples using the XytXtract Tissue kit. In addition, the XytXtract method was found to be more sensitive than the QIAmp method as determined by a dilution series of the extracted DNA. While the XytXtract method is a closed, the QIAamp method uses a spin column with possible loss of DNA through DNA binding competition of the matrix, which could impact on the final extraction efficiency. The XytXtract is a cheap, rapid and efficient method for extracting HPV DNA from both cell lines and liquid based cervical samples. © 2014 Wiley Periodicals, Inc.

  19. Human DNA polymerase θ grasps the primer terminus to mediate DNA repair.

    Science.gov (United States)

    Zahn, Karl E; Averill, April M; Aller, Pierre; Wood, Richard D; Doublié, Sylvie

    2015-04-01

    DNA polymerase θ protects against genomic instability via an alternative end-joining repair pathway for DNA double-strand breaks. Polymerase θ is overexpressed in breast, lung and oral cancers, and reduction of its activity in mammalian cells increases sensitivity to double-strand break-inducing agents, including ionizing radiation. Reported here are crystal structures of the C-terminal polymerase domain from human polymerase θ, illustrating two potential modes of dimerization. One structure depicts insertion of ddATP opposite an abasic-site analog during translesion DNA synthesis. The second structure describes a cognate ddGTP complex. Polymerase θ uses a specialized thumb subdomain to establish unique upstream contacts to the primer DNA strand, including an interaction with the 3'-terminal phosphate from one of five distinctive insertion loops. These observations demonstrate how polymerase θ grasps the primer to bypass DNA lesions or extend poorly annealed DNA termini to mediate end-joining.

  20. [Diagnostic value of mitochondrial DNA analysis in chronic progressive external ophthalmoplegia (CPEO)].

    Science.gov (United States)

    Kornblum, C; Kunz, W S; Klockgether, T; Roggenkämper, P; Schröder, R

    2004-12-01

    Chronic progressive external ophthalmoplegia (CPEO) is a mitochondrial cytopathy presenting with ptosis and external ophthalmoparesis. Mitochondrial disorders are characterized by a broad clinical spectrum and genetic background with marked genotype/phenotype variability. The routine diagnostic work-up usually includes clinical and laboratory examinations as well as histological and histochemical analysis of skeletal muscle biopsy. In our case only an additional molecular biological examination allowed the diagnosis of CPEO. We report a 35-year-old woman with a 7-years history of slowly progressive diplopia due to impaired ocular motility and bilateral ptosis. We performed ophthalmological and neurological examinations, laboratory testing, lower limb skeletal muscle biopsy including histological and histochemical investigations, biochemical analysis of respiratory chain enzymes in skeletal muscle homogenate and molecular genetic testing of skeletal muscle DNA. Although clinical, laboratory, histological and biochemical analyses did not give any hints suggesting a mitochondrial cytopathy, molecular genetic testing of total DNA from skeletal muscle tissue by Southern blot analysis finally revealed a 3.8 kb mitochondrial DNA deletion with a degree of heteroplasmy of 45 %. In patients with unexplained binocular diplopia , oculomotor deficits and/or acquired ptosis, an underlying mitochondrial cytopathy should be considered. Even in the case of inconspicuous skeletal muscle histology and biochemistry, molecular genetic testing of skeletal muscle DNA is advisable in order to establish the diagnosis.

  1. The Human Cytomegalovirus UL44 C Clamp Wraps around DNA

    Science.gov (United States)

    Komazin-Meredith, Gloria; Petrella, Robert J.; Santos, Webster L.; Filman, David J.; Hogle, James M.; Verdine, Gregory L.; Karplus, Martin; Coen, Donald M.

    2010-01-01

    Summary Processivity factors tether the catalytic subunits of DNA polymerases to DNA so that continuous synthesis of long DNA strands is possible. The human cytomegalovirus DNA polymerase subunit UL44 forms a C clamp-shaped dimer intermediate in structure between monomeric herpes simplex virus UL42, which binds DNA directly via a basic surface, and the trimeric sliding clamp PCNA, which encircles DNA. To investigate how UL44 interacts with DNA, calculations were performed in which a 12 bp DNA oligonucleotide was docked to UL44. The calculations suggested that UL44 encircles DNA, which interacts with basic residues both within the cavity of the C clamp and in flexible loops of UL44 that complete the “circle.” The results of mutational and crosslinking studies were consistent with this model. Thus, UL44 is a “hybrid” of UL42 and PCNA: its structure is intermediate between the two and its mode of interaction with DNA has elements of both. PMID:18682223

  2. A DNA Vaccine Protects Human Immune Cells against Zika Virus Infection in Humanized Mice

    Directory of Open Access Journals (Sweden)

    Guohua Yi

    2017-11-01

    Full Text Available A DNA vaccine encoding prM and E protein has been shown to induce protection against Zika virus (ZIKV infection in mice and monkeys. However, its effectiveness in humans remains undefined. Moreover, identification of which immune cell types are specifically infected in humans is unclear. We show that human myeloid cells and B cells are primary targets of ZIKV in humanized mice. We also show that a DNA vaccine encoding full length prM and E protein protects humanized mice from ZIKV infection. Following administration of the DNA vaccine, humanized DRAG mice developed antibodies targeting ZIKV as measured by ELISA and neutralization assays. Moreover, following ZIKV challenge, vaccinated animals presented virtually no detectable virus in human cells and in serum, whereas unvaccinated animals displayed robust infection, as measured by qRT-PCR. Our results utilizing humanized mice show potential efficacy for a targeted DNA vaccine against ZIKV in humans.

  3. 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

  4. Damage-specific DNA-binding proteins from human cells

    Energy Technology Data Exchange (ETDEWEB)

    Kanjilal, S.

    1992-01-01

    The primary objective of the study was to detect and characterize factors from human cells that bind DNA damaged by ultraviolet radiation. An application of the gel-shift assay was devised in which a DNA probe was UV-irradiated and compared with non-irradiated probe DNA for the ability to bind to such factors in cell extracts. UV-dose dependent binding proteins were identified. Formation of the DNA-protein complexes was independent of the specific sequence, form or source of the DNA. There was a marked preference for lesions on double stranded DNA over those on single stranded DNA. DNA irradiated with gamma rays did not compete with UV-irradiated DNA for the binding activities. Cell lines from patients with genetic diseases associated with disorders of the DNA repair system were screened for the presence of damaged-DNA-binding activities. Simultaneous occurrence of the clinical symptoms of some of these diseases had been previously documented and possible links between the syndromes proposed. However, supporting biochemical or molecular evidence for such associations were lacking. The data from the present investigations indicate that some cases of Xeroderma Pigmentosum group A, Cockayne's Syndrome, Bloom's Syndrome and Ataxia Telangiectasia, all of which exhibit sensitivity to UV or gamma radiation, share an aberrant damaged-DNA-binding factor. These findings support the hypothesis that some of the repair disorder diseases are closely related and may have arisen from a common defect. Partial purification of the binding activities from HeLa cells was achieved. Size-exclusion chromatography resolved the activities into various peaks, one of which was less damage-specific than the others as determined by competition studies using native or UV-irradiated DNA. Some of the activities were further separated by ion-exchange chromatography. On using affinity chromatography methods, the major damage-binding factor could be eluted in the presence of 2 M KCl and 1

  5. 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.

  6. Human Rap1 modulates TRF2 attraction to telomeric DNA.

    Science.gov (United States)

    Janoušková, Eliška; Nečasová, Ivona; Pavloušková, Jana; Zimmermann, Michal; Hluchý, Milan; Marini, Victoria; Nováková, Monika; Hofr, Ctirad

    2015-03-11

    More than two decades of genetic research have identified and assigned main biological functions of shelterin proteins that safeguard telomeres. However, a molecular mechanism of how each protein subunit contributes to the protecting function of the whole shelterin complex remains elusive. Human Repressor activator protein 1 (Rap1) forms a multifunctional complex with Telomeric Repeat binding Factor 2 (TRF2). Rap1-TRF2 complex is a critical part of shelterin as it suppresses homology-directed repair in Ku 70/80 heterodimer absence. To understand how Rap1 affects key functions of TRF2, we investigated full-length Rap1 binding to TRF2 and Rap1-TRF2 complex interactions with double-stranded DNA by quantitative biochemical approaches. We observed that Rap1 reduces the overall DNA duplex binding affinity of TRF2 but increases the selectivity of TRF2 to telomeric DNA. Additionally, we observed that Rap1 induces a partial release of TRF2 from DNA duplex. The improved TRF2 selectivity to telomeric DNA is caused by less pronounced electrostatic attractions between TRF2 and DNA in Rap1 presence. Thus, Rap1 prompts more accurate and selective TRF2 recognition of telomeric DNA and TRF2 localization on single/double-strand DNA junctions. These quantitative functional studies contribute to the understanding of the selective recognition of telomeric DNA by the whole shelterin complex. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  7. Vitrification in human and domestic animal embryology: work in progress.

    Science.gov (United States)

    Vajta, Gábor

    2013-01-01

    According to the analysis of papers published in major international journals, rapidly increasing application of vitrification is one of the greatest achievements in domestic animal and especially human embryology during the first decade of our century. This review highlights factors supporting or hampering this progress, summarises results achieved with vitrification and outlines future tasks to fully exploit the benefits of this amazing approach that has changed or will change many aspects of laboratory (and also clinical) embryology. Supporting factors include the simplicity, cost efficiency and convincing success of vitrification compared with other approaches in all species and developmental stages in mammalian embryology, while causes that slow down the progress are mostly of human origin: inadequate tools and solutions, superficial teaching, improper application and unjustified concerns resulting in legal restrictions. Elimination of these hindrances seems to be a slower process and more demanding task than meeting the biological challenge. A key element of future progress will be to pass the pioneer age, establish a consensus regarding biosafety requirements, outline the indispensable features of a standard approach and design fully-automated vitrification machines executing all phases of the procedure, including equilibration, cooling, warming and dilution steps.

  8. Increased DNA damage in progression of COPD: a response by poly(ADP-ribose polymerase-1.

    Directory of Open Access Journals (Sweden)

    Ingrid Oit-Wiscombe

    Full Text Available Chronic oxidative stress (OS, a major mechanism of chronic obstructive pulmonary disease (COPD, may cause significant damage to DNA. Poly(ADP-ribose polymerase (PARP-1 is rapidly activated by OS-induced DNA lesions. However, the degree of DNA damage along with the evolution of COPD is unclear. In peripheral blood mononuclear cells of non-smoking individuals, non-obstructive smokers, patients with COPD of all stages and those with COPD exacerbation, we evaluated DNA damage, PARP activity and PARP-1 mRNA expression using Comet Assay IV, biotinylated-NAD incorporation assay and qRT-PCR, respectively and subjected results to ordinal logistic regression modelling. Adjusted for demographics, smoking-related parameters and lung function, novel comet parameters, tail length/cell length ratio and tail migration/cell length ratio, showed the greatest increase along the study groups corresponding to the evolution of COPD [odds ratio (OR 7.88, 95% CI 4.26-14.57; p<0.001 and OR 3.91, 95% CI 2.69-5.66; p<0.001, respectively]. Analogously, PARP activity increased significantly over the groups (OR = 1.01; 95%; p<0.001. An antioxidant tetrapeptide UPF17 significantly reduced the PARP-1 mRNA expression in COPD, compared to that in non-obstructive individuals (p = 0.040. Tail length/cell length and tail migration/cell length ratios provide novel progression-sensitive tools for assessment of DNA damage. However, it remains to be elucidated whether inhibition of an elevated PARP-1 activity has a safe enough potential to break the vicious cycle of the development and progression of COPD.

  9. Genetics and biochemistry of DNA repair in Neurospora crassa. Progress report

    Energy Technology Data Exchange (ETDEWEB)

    Mishra, N.C.

    1979-04-01

    Mutants of Neurospora, presumably defective for deoxyribonuclease and DNA polymerase are being biochemically characterized and compared with the wild type enzyme to establish which enzymes control a particular function in DNA repair and other aspects of DNA biosynthesis. After biochemical characterization, these mutants will be also examined for their ability to repair damage caused by exposure to radiation, radiomimetic drugs and carcinogens. Also, a number of Neurospora mutants which are known to be sensitive to uv radiation or to MMS (methyl methane sulfonate) or to histidine or defective in recombination are being characterized for DNA polymerases and nucleases to elucidate the biochemical basis of these defects. These studies will provide a detailed knowledge of the different aspects of DNA repair and the role of the key enzymes therein. The results of present studies involving a eukaryote can be meaningfully generalized for similar studies in humans. Efficient DNA repair in man is necessary for the maintenance of normal growth and for exposure to the sunlight and to various radiomimetic chemicals present in our environment. Due to the introduction of various chemicals in our environment, there is increased risk of damage to our own genetic material (including that of our crop and cattle) and the outcome of the proposed study may provide a meaningful insight into these problems.

  10. 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.

  11. [Research progress on free radicals in human body].

    Science.gov (United States)

    Wang, Q B; Xu, F P; Wei, C X; Peng, J; Dong, X D

    2016-08-10

    Free radicals are the intermediates of metabolism, widely exist in the human bodies. Under normal circumstances, the free radicals play an important role in the metabolic process on human body, cell signal pathway, gene regulation, induction of cell proliferation and apoptosis, so as to maintain the normal growth and development of human body and to inhibit the growth of bacteria, virus and cancer. However, when organic lesion occurs affected by external factors or when equilibrium of the free radicals is tipped in the human body, the free radicals will respond integratedly with lipids, protein or nucleic acid which may jeopardize the health of human bodies. This paper summarizes the research progress of the free radicals conducted in recent years, in relations to the perspective of the types, origins, test methods of the free radicals and their relationship with human's health. In addition, the possible mechanisms of environmental pollutants (such as polycyclic aromatic hydrocarbons) mediating oxidative stress and free radicals scavenging in the body were also summarized.

  12. 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 with a...... with a narrow definition of a (false-)positive test. The aim of this paper was to illustrate how the narrow definition affected the measured adverse effects of HPV DNA screening compared with cytology screening....

  13. How Can Humanities Interventions Promote Progress in the Environmental Sciences?

    Directory of Open Access Journals (Sweden)

    Sally L. Kitch

    2017-10-01

    Full Text Available Environmental humanists make compelling arguments about the importance of the environmental humanities (EH for discovering new ways to conceptualize and address the urgent challenges of the environmental crisis now confronting the planet. Many environmental scientists in a variety of fields are also committed to incorporating socio-cultural analyses in their work. Despite such intentions and rhetoric, however, and some humanists’ eagerness to incorporate science into their own work, “radical interdisciplinarity [across the humanities and sciences] is ... rare ... and does not have the impact one would hope for” (Holm et al. 2013, p. 32. This article discusses reasons for the gap between transdisciplinary intentions and the work being done in the environmental sciences. The article also describes a project designed to address that gap. Entitled “From Innovation to Progress: Addressing Hazards of the Sustainability Sciences”, the project encourages humanities interventions in problem definition, before any solution or action is chosen. Progress offers strategies for promoting expanded stakeholder engagement, enhancing understanding of power struggles and inequities that underlie problems and over-determine solutions, and designing multiple future scenarios based on alternative values, cultural practices and beliefs, and perspectives on power distribution and entitlement.

  14. 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

  15. 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

  16. Caffeine and human DNA metabolism: the magic and the mystery

    Energy Technology Data Exchange (ETDEWEB)

    Kaufmann, William K.; Heffernan, Timothy P.; Beaulieu, Lea M.; Doherty, Sharon; Frank, Alexandra R.; Zhou Yingchun; Bryant, Miriam F.; Zhou Tong; Luche, Douglas D.; Nikolaishvili-Feinberg, Nana; Simpson, Dennis A.; Cordeiro-Stone, Marila

    2003-11-27

    The ability of caffeine to reverse cell cycle checkpoint function and enhance genotoxicity after DNA damage was examined in telomerase-expressing human fibroblasts. Caffeine reversed the ATM-dependent S and G2 checkpoint responses to DNA damage induced by ionizing radiation (IR), as well as the ATR- and Chk1-dependent S checkpoint response to ultraviolet radiation (UVC). Remarkably, under conditions in which IR-induced G2 delay was reversed by caffeine, IR-induced G1 arrest was not. Incubation in caffeine did not increase the percentage of cells entering the S phase 6-8 h after irradiation; ATM-dependent phosphorylation of p53 and transactivation of p21{sup Cip1/Waf1} post-IR were resistant to caffeine. Caffeine alone induced a concentration- and time-dependent inhibition of DNA synthesis. It inhibited the entry of human fibroblasts into S phase by 70-80% regardless of the presence or absence of wildtype ATM or p53. Caffeine also enhanced the inhibition of cell proliferation induced by UVC in XP variant fibroblasts. This effect was reversed by expression of DNA polymerase {eta}, indicating that translesion synthesis of UVC-induced pyrimidine dimers by DNA pol {eta} protects human fibroblasts against UVC genotoxic effects even when other DNA repair functions are compromised by caffeine.

  17. Low-Dose Formaldehyde Delays DNA Damage Recognition and DNA Excision Repair in Human Cells

    Science.gov (United States)

    Luch, Andreas; Frey, Flurina C. Clement; Meier, Regula; Fei, Jia; Naegeli, Hanspeter

    2014-01-01

    Objective Formaldehyde is still widely employed as a universal crosslinking agent, preservative and disinfectant, despite its proven carcinogenicity in occupationally exposed workers. Therefore, it is of paramount importance to understand the possible impact of low-dose formaldehyde exposures in the general population. Due to the concomitant occurrence of multiple indoor and outdoor toxicants, we tested how formaldehyde, at micromolar concentrations, interferes with general DNA damage recognition and excision processes that remove some of the most frequently inflicted DNA lesions. Methodology/Principal Findings The overall mobility of the DNA damage sensors UV-DDB (ultraviolet-damaged DNA-binding) and XPC (xeroderma pigmentosum group C) was analyzed by assessing real-time protein dynamics in the nucleus of cultured human cells exposed to non-cytotoxic (formaldehyde concentrations. The DNA lesion-specific recruitment of these damage sensors was tested by monitoring their accumulation at local irradiation spots. DNA repair activity was determined in host-cell reactivation assays and, more directly, by measuring the excision of DNA lesions from chromosomes. Taken together, these assays demonstrated that formaldehyde obstructs the rapid nuclear trafficking of DNA damage sensors and, consequently, slows down their relocation to DNA damage sites thus delaying the excision repair of target lesions. A concentration-dependent effect relationship established a threshold concentration of as low as 25 micromolar for the inhibition of DNA excision repair. Conclusions/Significance A main implication of the retarded repair activity is that low-dose formaldehyde may exert an adjuvant role in carcinogenesis by impeding the excision of multiple mutagenic base lesions. In view of this generally disruptive effect on DNA repair, we propose that formaldehyde exposures in the general population should be further decreased to help reducing cancer risks. PMID:24722772

  18. 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.

  19. Focal Segmental Glomerulosclerosis Associated with Chronic Progressive External Ophthalmoplegia and Mitochondrial DNA A3243G Mutation.

    Science.gov (United States)

    Narumi, Kaori; Mishima, Eikan; Akiyama, Yukako; Matsuhashi, Tetsuro; Nakamichi, Takashi; Kisu, Kiyomi; Nishiyama, Shuhei; Ikenouchi, Hajime; Kikuchi, Akio; Izumi, Rumiko; Miyazaki, Mariko; Abe, Takaaki; Sato, Hiroshi; Ito, Sadayoshi

    2017-11-30

    Focal segmental glomerulosclerosis (FSGS) is caused by various etiologies, with mitochondrial dysfunction being one of the causes. FSGS is known to be associated with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), which is a subclass of mitochondrial disease. However, it has rarely been reported in other mitochondrial disease subclasses. Here, we reported a 20-year-old man diagnosed with FSGS associated with chronic progressive external ophthalmoplegia (CPEO) due to mitochondrial DNA (mtDNA) 3243A>G mutation. He presented with left ptosis, short stature, mild sensorineural deafness, and cardiac conduction block. A renal biopsy sample showed segmental sclerosis and adhesions between capillaries and Bowman's capsule, indicating FSGS. Electron microscopy demonstrated abnormal aggregated mitochondria in podocytes, and the basement membrane and epithelial cells of Bowman's capsule. Skeletal muscle biopsy also showed accumulation of abnormal mitochondria. mtDNA analysis identified heteroplasmic mtDNA 3243A>G mutation with no large-scale deletions. From these findings, we diagnosed the case as CPEO with multi-organ involvement including FSGS. Our report demonstrates that CPEO, as well as MELAS, can be associated with FSGS. Because mitochondrial disease presents with a variety of clinical symptoms, atypical cases with non-classical manifestations are observed. Thus, mitochondrial disease should be considered as an underlying cause of FSGS with systemic manifestations even with atypical phenotypes. © 2017 S. Karger AG, Basel.

  20. Multiple Functions of Long Non-Coding RNAs in Oxidative Stress, DNA Damage Response and Cancer Progression.

    Science.gov (United States)

    Tehrani, Sadra Samavarchi; Karimian, Ansar; Parsian, Hadi; Majidinia, Maryam; Yousefi, Bahman

    2017-06-13

    In addition to aberrant alternation of transcriptome, it is now suggested that dysregulation of the non-coding transcripts, particularly long non-coding RNAs (lncRNAs), which comprise the majority of the genome, is contributed to cancer initiation and progression. As the result of recent huge efforts, the possible roles of numerous lncRNAs in the human cancers were characterized, as well as various strategies with inhibitory effects to target these transcripts on the transformed cells. Moreover, DNA damage response (DDR) pathway is a complex regulatory network responsible for the identification of disruptions in DNA structure, integrity and stability- it is reported to be associated with the up-regulation and down-regulation of lncRNAs. This review explores the involvement of the various lncRNAs in different human cancers, afterwards discusses the association of the lncRNAs expression with the DDR and oxidative stress, which are implicated in a myriad pathophysiological and physiological intra- and extracellular damages. J. Cell. Biochem. 9999: 1-14, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  1. Comprehensive analysis of genome-wide DNA methylation across human polycystic ovary syndrome ovary granulosa cell.

    Science.gov (United States)

    Xu, Jiawei; Bao, Xiao; Peng, Zhaofeng; Wang, Linlin; Du, Linqing; Niu, Wenbin; Sun, Yingpu

    2016-05-10

    Polycystic ovary syndrome (PCOS) affects approximately 7% of the reproductive-age women. A growing body of evidence indicated that epigenetic mechanisms contributed to the development of PCOS. The role of DNA modification in human PCOS ovary granulosa cell is still unknown in PCOS progression. Global DNA methylation and hydroxymethylation were detected between PCOS' and controls' granulosa cell. Genome-wide DNA methylation was profiled to investigate the putative function of DNA methylaiton. Selected genes expressions were analyzed between PCOS' and controls' granulosa cell. Our results showed that the granulosa cell global DNA methylation of PCOS patients was significant higher than the controls'. The global DNA hydroxymethylation showed low level and no statistical difference between PCOS and control. 6936 differentially methylated CpG sites were identified between control and PCOS-obesity. 12245 differential methylated CpG sites were detected between control and PCOS-nonobesity group. 5202 methylated CpG sites were significantly differential between PCOS-obesity and PCOS-nonobesity group. Our results showed that DNA methylation not hydroxymethylation altered genome-wide in PCOS granulosa cell. The different methylation genes were enriched in development protein, transcription factor activity, alternative splicing, sequence-specific DNA binding and embryonic morphogenesis. YWHAQ, NCF2, DHRS9 and SCNA were up-regulation in PCOS-obesity patients with no significance different between control and PCOS-nonobesity patients, which may be activated by lower DNA methylaiton. Global and genome-wide DNA methylation alteration may contribute to different genes expression and PCOS clinical pathology.

  2. Alternative mechanisms of telomere lengthening: Permissive mutations, DNA repair proteins and tumorigenic progression

    Energy Technology Data Exchange (ETDEWEB)

    Gocha, April Renee Sandy; Harris, Julia [Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States); Groden, Joanna, E-mail: joanna.groden@osumc.edu [Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University College of Medicine, Columbus, OH 43210 (United States)

    2013-03-15

    Highlights: ► Neoplastic cells maintain telomeres by telomerase or ALT. ► Genetic mutations in p53, ATRX, DAXX or H3F3A may activate ALT. ► Many DNA repair proteins are involved in ALT. ► Tumor progression is favored by telomerase expression. - Abstract: Telomeres protect chromosome termini to maintain genomic stability and regulate cellular lifespan. Maintenance of telomere length is required for neoplastic cells after the acquisition of mutations that deregulate cell cycle control and increase cellular proliferation, and can occur through expression of the enzyme telomerase or in a telomerase-independent manner termed alternative lengthening of telomeres (ALT). The precise mechanisms that govern the activation of ALT or telomerase in tumor cells are unknown, although cellular origin may favor one or the other mechanisms. ALT pathways are incompletely understood to date; however, recent publications have increasingly broadened our understanding of how ALT is activated, how it proceeds, and how it influences tumor growth. Specific mutational events influence ALT activation, as mutations in genes that suppress recombination and/or alterations in the regulation of telomerase expression are associated with ALT. Once engaged, ALT uses DNA repair proteins to maintain telomeres in the absence of telomerase; experiments that manipulate the expression of specific proteins in cells using ALT are illuminating some of its mechanisms. Furthermore, ALT may influence tumor growth, as experimental and clinical data suggest that telomerase expression may favor tumor progression. This review summarizes recent findings in mammalian cells and models, as well as clinical data, that identify the genetic mutations permissive to ALT, the DNA repair proteins involved in ALT mechanisms and the importance of telomere maintenance mechanisms for tumor progression. A comprehensive understanding of the mechanisms that permit tumor cell immortalization will be important for identifying

  3. Progress towards human primordial germ cell specification in vitro.

    Science.gov (United States)

    Canovas, S; Campos, R; Aguilar, E; Cibelli, J B

    2017-01-01

    Primordial germ cells (PGCs) have long been considered the link between one generation and the next. PGC specification begins in the early embryo as a result of a highly orchestrated combination of transcriptional and epigenetic mechanisms. Understanding the molecular events that lead to proper PGC development will facilitate the development of new treatments for human infertility as well as species conservation. This article describes the latest, most relevant findings about the mechanisms of PGC formation, emphasizing human PGC. It also discusses our own laboratory's progress in using transdifferentiation protocols to derive human PGCs (hPGCs). Our preliminary results arose from our pursuit of a sequential hPGC induction strategy that starts with the repression of lineage-specific factors in the somatic cell, followed by the reactivation of germ cell-related genes using specific master regulators, which can indeed reactivate germ cell-specific genes in somatic cells. While it is still premature to assume that fully functional human gametes can be obtained in a dish, our results, together with those recently published by others, provide strong evidence that generating their precursors, PGCs, is within reach. © The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  4. Prolonged incubation of processed human spermatozoa will increase DNA fragmentation.

    Science.gov (United States)

    Nabi, A; Khalili, M A; Halvaei, I; Roodbari, F

    2014-05-01

    One of the causes of failure in ART is sperm DNA fragmentation which may be associated with long period of spermatozoa incubation at 37 °C. The objective was to evaluate the rate of sperm DNA fragmentation using the sperm chromatin dispersion (SCD) test after swim-up at different time intervals prior to use. In this prospective study, 21 normozoospermic specimens were analysed. The samples were incubated at 37 °C after preparation by direct swim-up. DNA fragmentation was assessed at different time intervals (0, 1, 2 and 3 h) using SCD test. Spermatozoa with no DNA fragmentation showed large- or medium-sized halos, and sperm cells with DNA fragmentation showed either a small halo or no halo. The rates of normal morphology and progressive motility after sperm processing were 72.33 ± 2.53% and 90 ± 1.02%, respectively. The rate of sperm DNA fragmentation was significantly higher after 2 h (8.81 ± 0.93%, P = 0.004) and 3 h (10.76 ± 0.89%, P fragmentation. Therefore, sperm samples intended for ART procedures should be used within 2 h of incubation at 37 °C. © 2013 Blackwell Verlag GmbH.

  5. Correlation between Gene Expression and Osteoarthritis Progression in Human

    Directory of Open Access Journals (Sweden)

    Leilei Zhong

    2016-07-01

    Full Text Available Osteoarthritis (OA is a multifactorial disease characterized by gradual degradation of joint cartilage. This study aimed to quantify major pathogenetic factors during OA progression in human cartilage. Cartilage specimens were isolated from OA patients and scored 0–5 according to the Osteoarthritis Research Society International (OARSI guidelines. Protein and gene expressions were measured by immunohistochemistry and qPCR, respectively. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL assays were used to detect apoptotic cells. Cartilage degeneration in OA is a gradual progress accompanied with gradual loss of collagen type II and a gradual decrease in mRNA expression of SOX9, ACAN and COL2A1. Expression of WNT antagonists DKK1 and FRZB was lost, while hypertrophic markers (RUNX2, COL10A1 and IHH increased during OA progression. Moreover, DKK1 and FRZB negatively correlated with OA grading, while RUNX2 and IHH showed a significantly positive correlation with OA grading. The number of apoptotic cells was increased with the severity of OA. Taken together, our results suggested that genetic profiling of the gene expression could be used as markers for staging OA at the molecular level. This helps to understand the molecular pathology of OA and may lead to the development of therapies based on OA stage.

  6. 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.

  7. Mouse DNA contamination in human tissue tested for XMRV

    Directory of Open Access Journals (Sweden)

    Uiprasertkul Mongkol

    2010-12-01

    Full Text Available Abstract Background We used a PCR-based approach to study the prevalence of genetic sequences related to a gammaretrovirus, xenotropic murine leukemia virus-related virus, XMRV, in human prostate cancer. This virus has been identified in the US in prostate cancer patients and in those with chronic fatigue syndrome. However, with the exception of two patients in Germany, XMRV has not been identified in prostate cancer tissue in Europe. Most putative associations of new or old human retroviruses with diseases have turned out to be due to contamination. We have looked for XMRV sequences in DNA extracted from formalin-fixed paraffin- embedded prostate tissues. To control for contamination, PCR assays to detect either mouse mitochondrial DNA (mtDNA or intracisternal A particle (IAP long terminal repeat DNA were run on all samples, owing to their very high copy number in mouse cells. Results In general agreement with the US prevalence, XMRV-like sequences were found in 4.8% of prostate cancers. However, these were also positive, as were 21.5% of XMRV-negative cases, for IAP sequences, and many, but not all were positive for mtDNA sequences. Conclusions These results show that contamination with mouse DNA is widespread and detectable by the highly sensitive IAP assay, but not always with less sensitive assays, such as murine mtDNA PCR. This study highlights the ubiquitous presence of mouse DNA in laboratory specimens and offers a means of rigorous validation for future studies of murine retroviruses in human disease.

  8. 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.

  9. 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...

  10. 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.

  11. Acute inactivation of the replicative helicase in human cells triggers MCM8-9-dependent DNA synthesis

    DEFF Research Database (Denmark)

    Natsume, Toyoaki; Nishimura, Kohei; Minocherhomji, Sheroy

    2017-01-01

    DNA replication fork progression can be disrupted at difficult to replicate loci in the human genome, which has the potential to challenge chromosome integrity. This replication fork disruption can lead to the dissociation of the replisome and the formation of DNA damage. To model the events....... This RAD51/MCM8-9 axis is distinct from the recently described RAD52-dependent DNA synthesis pathway that operates in early mitosis at common fragile sites. We propose that stalled replication forks can be restarted in S phase via homologous recombination using MCM8-9 as an alternative replicative helicase....... stemming from replisome dissociation during DNA replication perturbation, we used a degron-based system for inducible proteolysis of a subunit of the replicative helicase. We show that MCM2-depleted cells activate a DNA damage response pathway and generate replication-associated DNA double-strand breaks...

  12. Progressive GAA.TTC repeat expansion in human cell lines.

    Science.gov (United States)

    Ditch, Scott; Sammarco, Mimi C; Banerjee, Ayan; Grabczyk, Ed

    2009-10-01

    Trinucleotide repeat expansion is the genetic basis for a sizeable group of inherited neurological and neuromuscular disorders. Friedreich ataxia (FRDA) is a relentlessly progressive neurodegenerative disorder caused by GAA.TTC repeat expansion in the first intron of the FXN gene. The expanded repeat reduces FXN mRNA expression and the length of the repeat tract is proportional to disease severity. Somatic expansion of the GAA.TTC repeat sequence in disease-relevant tissues is thought to contribute to the progression of disease severity during patient aging. Previous models of GAA.TTC instability have not been able to produce substantial levels of expansion within an experimentally useful time frame, which has limited our understanding of the molecular basis for this expansion. Here, we present a novel model for studying GAA.TTC expansion in human cells. In our model system, uninterrupted GAA.TTC repeat sequences display high levels of genomic instability, with an overall tendency towards progressive expansion. Using this model, we characterize the relationship between repeat length and expansion. We identify the interval between 88 and 176 repeats as being an important length threshold where expansion rates dramatically increase. We show that expansion levels are affected by both the purity and orientation of the repeat tract within the genomic context. We further demonstrate that GAA.TTC expansion in our model is independent of cell division. Using unique reporter constructs, we identify transcription through the repeat tract as a major contributor to GAA.TTC expansion. Our findings provide novel insight into the mechanisms responsible for GAA.TTC expansion in human cells.

  13. Inferring chronological age from DNA methylation patterns of human teeth.

    Science.gov (United States)

    Giuliani, Cristina; Cilli, Elisabetta; Bacalini, Maria Giulia; Pirazzini, Chiara; Sazzini, Marco; Gruppioni, Giorgio; Franceschi, Claudio; Garagnani, Paolo; Luiselli, Donata

    2016-04-01

    Current methods to determine chronological age from modern and ancient remains rely on both morphological and molecular approaches. However, low accuracy and the lack of standardized protocols make the development of alternative methods for the estimation of individual's age even more urgent for several research fields, such as biological anthropology, biodemography, forensics, evolutionary genetics, and ancient DNA studies. Therefore, the aim of this study is to identify genomic regions whose DNA methylation level correlates with age in modern teeth. We used MALDI-TOF mass spectrometry to analyze DNA methylation levels of specific CpGs located in the ELOVL2, FHL2, and PENK genes. We considered methylation data from cementum, dentin and pulp of 21 modern teeth (from 17 to 77 years old) to construct a mathematical model able to exploit DNA methylation values to predict age of the individuals. The median difference between the real age and that estimated using DNA methylation values is 1.20 years (SD = 1.9) if DNA is recovered from both cementum and pulp of the same modern teeth, 2.25 years (SD = 2.5) if DNA is recovered from dental pulp, 2.45 years (SD = 3.3) if DNA is extracted from cementum and 7.07 years (SD = 7.0) when DNA is recovered from dentin only. We propose for the first time the evaluation of DNA methylation at ELOVL2, FHL2, and PENK genes as a powerful tool to predict age in modern teeth for anthropological applications. Future studies are needed to apply this method also to historical and relatively ancient human teeth. © 2015 Wiley Periodicals, Inc.

  14. Field contamination of skeletonized human remains with exogenous DNA.

    Science.gov (United States)

    Edson, Suni M; Christensen, Alexander F

    2013-01-01

    The Armed Forces DNA Identification Laboratory reports the mitochondrial DNA (mtDNA) sequences of over 800 skeletal samples a year for the Joint POW/MIA Accounting Command-Central Identification Laboratory. These sequences are generated from degraded skeletal remains that are presumed to belong to U.S. service members missing from past military conflicts. In the laboratory, it is possible to control for contamination of remains; however, in the field, it can be difficult to prevent modern DNA from being transferred to skeletal elements and being carried forward through the analysis process. Four such cases are described here along with the controls in place in the laboratory to eliminate the possibility of the exogenous DNA being reported as authentic. In each case, the controls implemented by the laboratories prevented the false reporting of contaminant exogenous DNA from remains that were either faunal or human, but lacked endogenous DNA. © 2012 American Academy of Forensic Sciences Published 2012. This article is a U.S. Government work and is in the public domain in the U.S.A.

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

    Directory of Open Access Journals (Sweden)

    Chengchao Wu

    2017-02-01

    Full Text Available 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.

  16. [Deletions of the mitochondrial DNA associated to chronic progressive external ophthalmoplegia with ragged-red fibers in 2 Brazilian patients].

    Science.gov (United States)

    Carod-Artal, Francisco J; López-Gallardo, Ester; Solano, Abelardo; Dahmani, Yahya; Ruiz-Pesini, Eduardo; Montoya, Julio

    2006-04-01

    Our purpose was to report the neurological manifestations and molecular-genetic analysis of mitochondrial DNA associated with chronic progressive external ophthalmoplegia (CPEO) and raged red fibers (RRFs). Two patients, a male and a female (32 and 28 year-old, respectively), were studied due to progressive palpebral ptosis associated with RRFs in muscle biopsy. Both patients were subjected to neurological, histochemical and enzymatic analysis of muscular biopsy, analysis of cerebro-spinal fluid, and molecular analysis of mitochondrial DNA. Symptoms started at ages 24 and 17 years. Initial symptoms were palpebral ptosis, progressive limitation of vertical and horizontal gaze, fatigue and exercise intolerance, and weakness of proximal muscles. Brain MRIs were normal in both patients. Both patients had deletions of muscle mitochondrial DNA with similar size (5,425 and 5,112 base pairs) and location. CPEO with RRFs is usually associated with huge deletions in mitochondrial DNA. Fatigue and proximal muscle weakness can be found during the follow-up.

  17. Lobaplatin arrests cell cycle progression in human hepatocellular carcinoma cells

    Directory of Open Access Journals (Sweden)

    Chen Chang-Jie

    2010-10-01

    Full Text Available Abstract Background Hepatocellular carcinoma (HCC still is a big burden for China. In recent years, the third-generation platinum compounds have been proposed as potential active agents for HCC. However, more experimental and clinical data are warranted to support the proposal. In the present study, the effect of lobaplatin was assessed in five HCC cell lines and the underlying molecular mechanisms in terms of cell cycle kinetics were explored. Methods Cytotoxicity of lobaplatin to human HCC cell lines was examined using MTT cell proliferation assay. Cell cycle distribution was determined by flow cytometry. Expression of cell cycle-regulated genes was examined at both the mRNA (RT-PCR and protein (Western blot levels. The phosphorylation status of cyclin-dependent kinases (CDKs and retinoblastoma (Rb protein was also examined using Western blot analysis. Results Lobaplatin inhibited proliferation of human HCC cells in a dose-dependent manner. For the most sensitive SMMC-7721 cells, lobaplatin arrested cell cycle progression in G1 and G2/M phases time-dependently which might be associated with the down-regulation of cyclin B, CDK1, CDC25C, phosphorylated CDK1 (pCDK1, pCDK4, Rb, E2F, and pRb, and the up-regulation of p53, p21, and p27. Conclusion Cytotoxicity of lobaplatin in human HCC cells might be due to its ability to arrest cell cycle progression which would contribute to the potential use of lobaplatin for the management of HCC.

  18. The Role of DNA Methylation in the Development and Progression of Lung Adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Keith M. Kerr

    2007-01-01

    Full Text Available Lung cancer, caused by smoking in ∼87% of cases, is the leading cause of cancer death in the United States and Western Europe. Adenocarcinoma is now the most common type of lung cancer in men and women in the United States, and the histological subtype most frequently seen in never-smokers and former smokers. The increasing frequency of adenocarcinoma, which occurs more peripherally in the lung, is thought to be at least partially related to modifications in cigarette manufacturing that have led to a change in the depth of smoke inhalation. The rising incidence of lung adenocarcinoma and its lethal nature underline the importance of understanding the development and progression of this disease. Alterations in DNA methylation are recognized as key epigenetic changes in cancer, contributing to chromosomal instability through global hypomethylation, and aberrant gene expression through alterations in the methylation levels at promoter CpG islands. The identification of sequential changes in DNA methylation during progression and metastasis of lung adenocarcinoma, and the elucidation of their interplay with genetic changes, will broaden our molecular understanding of this disease, providing insights that may be applicable to the development of targeted drugs, as well as powerful markers for early detection and patient classification.

  19. Rapidly progressive periodontal disease associated with human immunodeficiency virus.

    Science.gov (United States)

    Al-Hezaimi, Khalid; Javed, Fawad; Ali, Tazeen Saeed; Al-Askar, Mansour; Al-Rasheed, Abdulaziz

    2012-03-01

    Severe periodontal inflammation with generalized dental plaque accumulation, spontaneous and severe gingival bleeding, fungal infection, and interdental papillae necrosis are presented in a patient infected with human immunodeficiency virus (HIV). Bite-wing radiographs revealed a generalized horizontal alveolar bone loss of 7-8 millimetres in both arches. Erythematous patches were noted on the gingival mucosa in both jaws. DNA testing was performed to indentify the periodontopathogens. The patient had no signs or symptoms of acquired immunodeficiency syndrome. This case-report presents the massive periodontal destruction that occurred in a patient infected with HIV. Therefore, it is highly recommended that patients infected with HIV should be regularly monitored to aid in early detection and to provide proper management of periodontal inflammatory conditions to minimize its destruction.

  20. 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

  1. The DNA methylome of human peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Li, Yingrui; Zhu, Jingde; Tian, Geng

    2010-01-01

    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......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...

  2. The fractal based analysis of human face and DNA variations during aging.

    Science.gov (United States)

    Namazi, Hamidreza; Akrami, Amin; Hussaini, Jamal; Silva, Osmar N; Wong, Albert; Kulish, Vladimir V

    2017-01-16

    Human DNA is the main unit that shapes human characteristics and features such as behavior. Thus, it is expected that changes in DNA (DNA mutation) influence human characteristics and features. Face is one of the human features which is unique and also dependent on his gen. In this paper, for the first time we analyze the variations of human DNA and face simultaneously. We do this job by analyzing the fractal dimension of DNA walk and face during human aging. The results of this study show the human DNA and face get more complex by aging. These complexities are mapped on fractal exponents of DNA walk and human face. The method discussed in this paper can be further developed in order to investigate the direct influence of DNA mutation on the face variations during aging, and accordingly making a model between human face fractality and the complexity of DNA walk.

  3. DNA synthesis in human bone marrow is circadian stage dependent.

    Science.gov (United States)

    Smaaland, R; Laerum, O D; Lote, K; Sletvold, O; Sothern, R B; Bjerknes, R

    1991-06-15

    Fraction of human bone marrow (BM) cells in DNA synthesis has been studied by sampling BM from the sternum or the iliac crests every 4 hours during one 24-hour period in 16 healthy male volunteers. Three of the subjects underwent the sampling procedure twice, resulting in 19 24-hour profiles. The percentage of cells in DNA synthesis measured by flow cytometry demonstrated a large variation along the circadian time scale for each 24-hour profile, with a range of variation from 29% to 339% from lowest to highest value. Seventeen profiles (89.5%) had the highest DNA synthesis during waking hours between 08:00 hours and 20:00 hours, and the lowest percentage of cells in DNA synthesis between 00:00 hours and 04:00 hours. The mean value of the lowest DNA synthesis for each 19 24-hour period was 8.7% +/- 0.6%, while the mean value of the highest DNA synthesis was 17.6% +/- 0.6%, ie, a twofold difference. There was no difference in DNA synthesis between winter and summer. A significantly higher DNA synthesis was demonstrated for samples obtained from sternum as compared with the iliac crests, but the same circadian pattern was demonstrated for both localizations. By taking circadian stage-dependent variations in DNA synthesis into account it may be possible to reduce BM sensitivity to cytotoxic chemotherapy, to increase the effect of hematopoietic growth factors as well as increase the fraction of proliferating cells with careful selection of time of day for harvesting BM cells for auto- or allografting.

  4. 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....

  5. 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.

  6. Expression of thioredoxin during progression of hamster and human cholangiocarcinoma.

    Science.gov (United States)

    Yoon, Byung-Il; Kim, Yeong-Hun; Yi, Jung-Yeon; Kang, Min-Soo; Jang, Ja-June; Joo, Kyoung-Hwan; Kim, Yongbaek; McHugh Law, J; Kim, Dae-Yong

    2010-01-01

    Thioredoxin (Trx) is a multifunctional redox protein that has growth-promoting and anti-apoptotic effects on cells and protects cells from endogenous and exogenous free radicals. Recently, altered expression of Trx has been reported in various cancers. In the present study, we investigated altered expression of Trx at the precancerous and carcinogenic phases during cholangiocarcinogenesis in a hamster cholangiocarcinoma (ChC) model, using semiquantitative immunohistochemical and Western blot analyses. Moreover, to determine if the results correlated well with those in human ChCs, we carried out a comparative immunohistochemical study for Trx in tissue-arrayed human ChCs with different grades of tumor cell differentiation. Trx was found highly expressed in the cytoplasm of dysplastic bile ducts with highly abnormal growth patterns and ChCs irrespective of tumor type or tumor cell differentiation. Overexpression of Trx at the precancerous and carcinogenic phases was further supported by significant elevation of Trx protein in Western blotting. The results from the hamster ChCs were in good agreement with those from human ChCs. Our results strongly suggested that the redox regulatory function of Trx plays an important role in bile duct cell transformation and tumor progression during cholangiocarcinogenesis.

  7. Hairpin structures formed by alpha satellite DNA of human centromeres are cleaved by human topoisomerase IIα

    Science.gov (United States)

    Jonstrup, Anette Thyssen; Thomsen, Tina; Wang, Yong; Knudsen, Birgitta R.; Koch, Jørn; Andersen, Anni H.

    2008-01-01

    Although centromere function has been conserved through evolution, apparently no interspecies consensus DNA sequence exists. Instead, centromere DNA may be interconnected through the formation of certain DNA structures creating topological binding sites for centromeric proteins. DNA topoisomerase II is a protein, which is located at centromeres, and enzymatic topoisomerase II activity correlates with centromere activity in human cells. It is therefore possible that topoisomerase II recognizes and interacts with the alpha satellite DNA of human centromeres through an interaction with potential DNA structures formed solely at active centromeres. In the present study, human topoisomerase IIα-mediated cleavage at centromeric DNA sequences was examined in vitro. The investigation has revealed that the enzyme recognizes and cleaves a specific hairpin structure formed by alpha satellite DNA. The topoisomerase introduces a single-stranded break at the hairpin loop in a reaction, where DNA ligation is partly uncoupled from the cleavage reaction. A mutational analysis has revealed, which features of the hairpin are required for topoisomerease IIα-mediated cleavage. Based on this a model is discussed, where topoisomerase II interacts with two hairpins as a mediator of centromere cohesion. PMID:18824478

  8. Relationship of DNA repair processes to mutagenesis and carcinogenesis in mammalian cells. Progress report, August 1, 1977-October 31, 1980

    Energy Technology Data Exchange (ETDEWEB)

    Evans, H.H.

    1980-10-01

    The objective of this research is to determine the role of DNA repair in mutagenesis and carcinogenesis in mammalian cells. More specifically, mutant strains will be selected which are deficient in various DNA repair pathways. These strains will be studied with regard to (1) the nature of the defect in repair, and (2) the mutability and transformability of the defective cells by various agents as compared to the wild type parental cells. The results to date include progress in the following areas: (1) determination of optimum conditions for growth and maintenance of cells and for quantitative measurement of various cellular parameters; (2) investigation of the effect of holding mutagenized cells for various periods in a density inhibited state on survival and on mutation and transformation frequencies; (3) examination of the repair capabilities of BHK cells, as compared to repair-proficient and repair-deficient human cells and excision-deficient mouse cells, as measured by the reactivation of Herpes simplex virus (HSV) treated with radiation and ethylmethane sulfonate (EMS); (4) initiation of host cell reactivation viral sucide enrichment and screening of survivors of the enrichment for sensitivity to ionizing radiation; and (5) investigation of the toxicity, mutagenicity, and carcinogenicity of various metabolites of 4-nitroquinoline-1-oxide (4-NQO). (ERB)

  9. Human cytomegalovirus-mediated immunomodulation: Effects on glioblastoma progression.

    Science.gov (United States)

    Foster, Haidn; Ulasov, Ilya V; Cobbs, Charles S

    2017-08-01

    The presence of human cytomegalovirus (HCMV) and glioblastoma multiforme (GBM), first established in 2002, has developed into an area of considerable interest and controversy. Numerous studies have found evidence of possible HCMV infection of GBM tumor cells as well as myriad onco- and immunomodulatory properties exhibited by HCMV antigens and transcripts, while recent reports have failed to detect HCMV particles in GBM and question the virus' role in tumor progression. This review highlights the known immunomodulatory properties of HCMV, independent of GBM infection status, that help drive the virus from peripheral blood into the vital tissues and subsequently dampen local immune response, assisting GBM tumors in evading immune surveillance and contributing to the disease's poor prognosis. Emerging antiviral approaches to treating GBM, including antiviral drugs and immunotherapies directed against HCMV, are also examined. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Hypomorphic PCNA mutation underlies a human DNA repair disorder.

    Science.gov (United States)

    Baple, Emma L; Chambers, Helen; Cross, Harold E; Fawcett, Heather; Nakazawa, Yuka; Chioza, Barry A; Harlalka, Gaurav V; Mansour, Sahar; Sreekantan-Nair, Ajith; Patton, Michael A; Muggenthaler, Martina; Rich, Phillip; Wagner, Karin; Coblentz, Roselyn; Stein, Constance K; Last, James I; Taylor, A Malcolm R; Jackson, Andrew P; Ogi, Tomoo; Lehmann, Alan R; Green, Catherine M; Crosby, Andrew H

    2014-07-01

    Numerous human disorders, including Cockayne syndrome, UV-sensitive syndrome, xeroderma pigmentosum, and trichothiodystrophy, result from the mutation of genes encoding molecules important for nucleotide excision repair. Here, we describe a syndrome in which the cardinal clinical features include short stature, hearing loss, premature aging, telangiectasia, neurodegeneration, and photosensitivity, resulting from a homozygous missense (p.Ser228Ile) sequence alteration of the proliferating cell nuclear antigen (PCNA). PCNA is a highly conserved sliding clamp protein essential for DNA replication and repair. Due to this fundamental role, mutations in PCNA that profoundly impair protein function would be incompatible with life. Interestingly, while the p.Ser228Ile alteration appeared to have no effect on protein levels or DNA replication, patient cells exhibited marked abnormalities in response to UV irradiation, displaying substantial reductions in both UV survival and RNA synthesis recovery. The p.Ser228Ile change also profoundly altered PCNA's interaction with Flap endonuclease 1 and DNA Ligase 1, DNA metabolism enzymes. Together, our findings detail a mutation of PCNA in humans associated with a neurodegenerative phenotype, displaying clinical and molecular features common to other DNA repair disorders, which we showed to be attributable to a hypomorphic amino acid alteration.

  11. Structure and mechanism of human DNA polymerase [eta

    Energy Technology Data Exchange (ETDEWEB)

    Biertümpfel, Christian; Zhao, Ye; Kondo, Yuji; Ramón-Maiques, Santiago; Gregory, Mark; Lee, Jae Young; Masutani, Chikahide; Lehmann, Alan R.; Hanaoka, Fumio; Yang, Wei (Sussex); (NIH); (Gakushuin); (Osaka)

    2010-11-03

    The variant form of the human syndrome xeroderma pigmentosum (XPV) is caused by a deficiency in DNA polymerase {eta} (Pol{eta}), a DNA polymerase that enables replication through ultraviolet-induced pyrimidine dimers. Here we report high-resolution crystal structures of human Pol{eta} at four consecutive steps during DNA synthesis through cis-syn cyclobutane thymine dimers. Pol{eta} acts like a 'molecular splint' to stabilize damaged DNA in a normal B-form conformation. An enlarged active site accommodates the thymine dimer with excellent stereochemistry for two-metal ion catalysis. Two residues conserved among Pol{eta} orthologues form specific hydrogen bonds with the lesion and the incoming nucleotide to assist translesion synthesis. On the basis of the structures, eight Pol{eta} missense mutations causing XPV can be rationalized as undermining the molecular splint or perturbing the active-site alignment. The structures also provide an insight into the role of Pol{eta} in replicating through D loop and DNA fragile sites.

  12. [Does scientific-technological progress endanger human dignity?].

    Science.gov (United States)

    Forschner, Maximilian

    2002-01-01

    1) The goal of modern science, to which also today's medicine is obliged, is the unlimited advance of knowledge and ability. It objectifies, neutralises and levels all of its objects to parameters which can be analysed, calculated, manipulated and performed; it abstracts from all personal mentality, experience and achievements in our surroundings and from all goals and personal daily appraisals of ourselves and the world around us. 2) The goal of scientific knowledge, its progress and its translation into technology is in today s understanding the abolition of distress, disease and suffering as well as the increase of ways and means to make human existence more endurable, comfortable, richer in experience and longer. As such a functionally defined good, science is bound to the laws and dynamics of the (national and global) market. 3) The term 'human dignity' as it is used in article 1 of the German constitution is based on the idea of self-purpose, respectively self-worth, which a human has a right to for his own sake, irrespective of other goods or purposes. This term finds its binding interpretation in the system of basic rights (R. Zippelius). Here, an elemental legal right of the individual is addressed, which cannot be put to disposition for any benefit or damage to the state or the society, one last taboo which protects every individual 'without consideration of the person' from interference and complete instrumentation by others. 4) Man as an object of modern, scientific research is 'objectified' like any other object. The drive to knowledge is limitless. The general and scientific relations between the progress of knowledge and ability and the goods and market values mentioned above offer no protection of man as a person from complete objectification and instrumentation. In the context of a society-dominated naturalistic concept of man, in which man is considered no more than a complex system of needs and interests, human dignity does in fact seem to be

  13. DNA damage responses in human induced pluripotent stem cells and embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Olga Momcilovic

    2010-10-01

    Full Text Available Induced pluripotent stem (iPS cells have the capability to undergo self-renewal and differentiation into all somatic cell types. Since they can be produced through somatic cell reprogramming, which uses a defined set of transcription factors, iPS cells represent important sources of patient-specific cells for clinical applications. However, before these cells can be used in therapeutic designs, it is essential to understand their genetic stability.Here, we describe DNA damage responses in human iPS cells. We observe hypersensitivity to DNA damaging agents resulting in rapid induction of apoptosis after γ-irradiation. Expression of pluripotency factors does not appear to be diminished after irradiation in iPS cells. Following irradiation, iPS cells activate checkpoint signaling, evidenced by phosphorylation of ATM, NBS1, CHEK2, and TP53, localization of ATM to the double strand breaks (DSB, and localization of TP53 to the nucleus of NANOG-positive cells. We demonstrate that iPS cells temporary arrest cell cycle progression in the G(2 phase of the cell cycle, displaying a lack of the G(1/S cell cycle arrest similar to human embryonic stem (ES cells. Furthermore, both cell types remove DSB within six hours of γ-irradiation, form RAD51 foci and exhibit sister chromatid exchanges suggesting homologous recombination repair. Finally, we report elevated expression of genes involved in DNA damage signaling, checkpoint function, and repair of various types of DNA lesions in ES and iPS cells relative to their differentiated counterparts.High degrees of similarity in DNA damage responses between ES and iPS cells were found. Even though reprogramming did not alter checkpoint signaling following DNA damage, dramatic changes in cell cycle structure, including a high percentage of cells in the S phase, increased radiosensitivity and loss of DNA damage-induced G(1/S cell cycle arrest, were observed in stem cells generated by induced pluripotency.

  14. Caffeine and human DNA metabolism: the magic and the mystery

    OpenAIRE

    Kaufmann, William K; Heffernan, Timothy P.; Beaulieu, Lea M.; Doherty, Sharon; Frank, Alexandra R.; Zhou, Yingchun; Bryant, Miriam F.; Zhou, Tong; Luche, Douglas D.; Nikolaishvili-Feinberg, Nana; Simpson, Dennis A.; Cordeiro-Stone, Marila

    2003-01-01

    The ability of caffeine to reverse cell cycle checkpoint function and enhance genotoxicity after DNA damage was examined in telomerase-expressing human fibroblasts. Caffeine reversed the ATM-dependent S and G2 checkpoint responses to DNA damage induced by ionizing radiation (IR), as well as the ATR- and Chk1-dependent S checkpoint response to ultraviolet radiation (UVC). Remarkably, under conditions in which IR-induced G2 delay was reversed by caffeine, IR-induced G1 arrest was not. Incubatio...

  15. Sequential inflammatory processes define human progression from M. tuberculosis infection to tuberculosis disease.

    Directory of Open Access Journals (Sweden)

    Thomas J Scriba

    2017-11-01

    Full Text Available Our understanding of mechanisms underlying progression from Mycobacterium tuberculosis infection to pulmonary tuberculosis disease in humans remains limited. To define such mechanisms, we followed M. tuberculosis-infected adolescents longitudinally. Blood samples from forty-four adolescents who ultimately developed tuberculosis disease (“progressors” were compared with those from 106 matched controls, who remained healthy during two years of follow up. We performed longitudinal whole blood transcriptomic analyses by RNA sequencing and plasma proteome analyses using multiplexed slow off-rate modified DNA aptamers. Tuberculosis progression was associated with sequential modulation of immunological processes. Type I/II interferon signalling and complement cascade were elevated 18 months before tuberculosis disease diagnosis, while changes in myeloid inflammation, lymphoid, monocyte and neutrophil gene modules occurred more proximally to tuberculosis disease. Analysis of gene expression in purified T cells also revealed early suppression of Th17 responses in progressors, relative to M. tuberculosis-infected controls. This was confirmed in an independent adult cohort who received BCG re-vaccination; transcript expression of interferon response genes in blood prior to BCG administration was associated with suppression of IL-17 expression by BCG-specific CD4 T cells 3 weeks post-vaccination. Our findings provide a timeline to the different immunological stages of disease progression which comprise sequential inflammatory dynamics and immune alterations that precede disease manifestations and diagnosis of tuberculosis disease. These findings have important implications for developing diagnostics, vaccination and host-directed therapies for tuberculosis.Clincialtrials.gov, NCT01119521.

  16. Sequential inflammatory processes define human progression from M. tuberculosis infection to tuberculosis disease.

    Science.gov (United States)

    Scriba, Thomas J; Penn-Nicholson, Adam; Shankar, Smitha; Hraha, Tom; Thompson, Ethan G; Sterling, David; Nemes, Elisa; Darboe, Fatoumatta; Suliman, Sara; Amon, Lynn M; Mahomed, Hassan; Erasmus, Mzwandile; Whatney, Wendy; Johnson, John L; Boom, W Henry; Hatherill, Mark; Valvo, Joe; De Groote, Mary Ann; Ochsner, Urs A; Aderem, Alan; Hanekom, Willem A; Zak, Daniel E

    2017-11-01

    Our understanding of mechanisms underlying progression from Mycobacterium tuberculosis infection to pulmonary tuberculosis disease in humans remains limited. To define such mechanisms, we followed M. tuberculosis-infected adolescents longitudinally. Blood samples from forty-four adolescents who ultimately developed tuberculosis disease (“progressors”) were compared with those from 106 matched controls, who remained healthy during two years of follow up. We performed longitudinal whole blood transcriptomic analyses by RNA sequencing and plasma proteome analyses using multiplexed slow off-rate modified DNA aptamers. Tuberculosis progression was associated with sequential modulation of immunological processes. Type I/II interferon signalling and complement cascade were elevated 18 months before tuberculosis disease diagnosis, while changes in myeloid inflammation, lymphoid, monocyte and neutrophil gene modules occurred more proximally to tuberculosis disease. Analysis of gene expression in purified T cells also revealed early suppression of Th17 responses in progressors, relative to M. tuberculosis-infected controls. This was confirmed in an independent adult cohort who received BCG re-vaccination; transcript expression of interferon response genes in blood prior to BCG administration was associated with suppression of IL-17 expression by BCG-specific CD4 T cells 3 weeks post-vaccination. Our findings provide a timeline to the different immunological stages of disease progression which comprise sequential inflammatory dynamics and immune alterations that precede disease manifestations and diagnosis of tuberculosis disease. These findings have important implications for developing diagnostics, vaccination and host-directed therapies for tuberculosis. Clincialtrials.gov, NCT01119521.

  17. DNA Damage, Cell Cycle Arrest, and Apoptosis Induction Caused by Lead in Human Leukemia Cells.

    Science.gov (United States)

    Yedjou, Clement G; Tchounwou, Hervey M; Tchounwou, Paul B

    2015-12-22

    In recent years, the industrial use of lead has been significantly reduced from paints and ceramic products, caulking, and pipe solder. Despite this progress, lead exposure continues to be a significant public health concern. The main goal of this research was to determine the in vitro mechanisms of lead nitrate [Pb(NO₃)₂] to induce DNA damage, apoptosis, and cell cycle arrest in human leukemia (HL-60) cells. To reach our goal, HL-60 cells were treated with different concentrations of Pb(NO₃)₂ for 24 h. Live cells and necrotic death cells were measured by the propidium idiode (PI) assay using the cellometer vision. Cell apoptosis was measured by the flow cytometry and DNA laddering. Cell cycle analysis was evaluated by the flow cytometry. The result of the PI demonstrated a significant (p cell death in Pb(NO₃)₂-treated cells, indicative of membrane rupture by Pb(NO₃)₂ compared to the control. Data generated from the comet assay indicated a concentration-dependent increase in DNA damage, showing a significant increase (p cells (apoptotic cells) compared to the control. The flow cytometry assessment also indicated Pb(NO₃)₂ exposure caused cell cycle arrest at the G₀/G₁ checkpoint. The result of DNA laddering assay showed presence of DNA smear in the agarose gel with little presence of DNA fragments in the treated cells compared to the control. In summary, Pb(NO₃)₂ inhibits HL-60 cells proliferation by not only inducing DNA damage and cell cycle arrest at the G₀/G₁ checkpoint but also triggering the apoptosis through caspase-3 activation and nucleosomal DNA fragmentation accompanied by secondary necrosis. We believe that our study provides a new insight into the mechanisms of Pb(NO₃)₂ exposure and its associated adverse health effects.

  18. Relationship of DNA repair processes to mutagenesis and carcinogenesis in mammalian cells. Progress report, November 1, 1979-October 31, 1980

    Energy Technology Data Exchange (ETDEWEB)

    Evans, H.H.

    1980-10-01

    The objective of this research is to determine the role of DNA repair in mutagenesis and carcinogenesis in mammalian cells. Use of the host-cell reactivation viral suicide enrichment procedure was initiated in the isolation of repair-deficient mutants. Lightly mutagenized BHK cells were infected with irradiated Herpes simplex virus (HSV); several radiation-sensitive strains were isolated among the survivors of the infection. The characterization of these strains is progressing and the enrichments are continuing. That alterations in the frequency of mutation of C3H/10T 1/2 cells, occurring as a result of holding the cells in a confluent state following treatment with ethylmethane sulfonate, parallel the alterations in the frequency of neoplastic transformation was found. The repair capabilities of BHK cells were found to be intermediate in comparison to repair-proficient and -deficient human cells with regard to the reactivation of HSV treated with various inactivating agents. The effect of confluency and of low serum levels on DNA synthesis, as well as the response to the cytotoxic effects of MNNG and acriflavin were determined in BHK cells in preparation for the investigation of the role of DNA repair in mutagenesis and transformation. It was also found that C3H/10T 1/2 cells partially recover from the toxic effects of 4-nitroquinoline-1-oxide if they are held in a confluent state for 6 to 22 hrs following treatment. Addition of catalase did not alleviate the toxic effects of 4-NQO. The cells contain a relatively high endogenous level of this enzyme. (ERB)

  19. Bi-directional routing of DNA mismatch repair protein human exonuclease 1 to replication foci and DNA double strand breaks

    DEFF Research Database (Denmark)

    Liberti, Sascha E; Andersen, Sofie Dabros; Wang, Jing

    2011-01-01

    Human exonuclease 1 (hEXO1) is implicated in DNA metabolism, including replication, recombination and repair, substantiated by its interactions with PCNA, DNA helicases BLM and WRN, and several DNA mismatch repair (MMR) proteins. We investigated the sub-nuclear localization of hEXO1 during S-phas...

  20. CD4 cell count and HIV DNA level are independent predictors of disease progression after primary HIV type 1 infection in untreated patients.

    Science.gov (United States)

    Goujard, Cécile; Bonarek, Mojgan; Meyer, Laurence; Bonnet, Fabrice; Chaix, Marie-Laure; Deveau, Christiane; Sinet, Martine; Galimand, Julie; Delfraissy, Jean-François; Venet, Alain; Rouzioux, Christine; Morlat, Philippe

    2006-03-01

    Treatment initiation at the time of primary human immunodeficiency virus (HIV) type 1 (HIV-1) infection has become less frequent in recent years. In the French prospective PRIMO Cohort, in which patients are enrolled at the time of primary HIV-1 infection, 30% of the 552 patients recruited during 1996-2004 did not start receiving antiretroviral treatment during the first 3 months after diagnosis. We analyzed the patients' clinical and immunological outcomes and examined potential predictors of disease progression. Progression was defined as the occurrence of an acquired immunodeficiency syndrome (AIDS)-related clinical event or a CD4 cell count <350 cells/mm3. Fifty-six (34%) of the untreated patients experienced immunological progression during a median duration of follow-up of 24 months, and 1 of these patients had an AIDS-related event. The estimated risks of progression were 25%, 34%, and 42% at 1, 2, and 3 years after enrollment, respectively. Compared with patients who did not have progression, those with progression had significantly lower CD4 cell counts at diagnosis (455 vs. 738 cells/mm3), higher plasma HIV RNA levels (4.9 vs. 4.5 log10 copies/mL), and higher HIV DNA levels (3.3 vs. 3.0 log(10) copies/10(6) peripheral blood mononuclear cells [PBMCs]). All 3 parameters were significantly associated with progression in univariate analysis. In multivariate analysis, only the CD4 cell count and HIV DNA level were independently predictive of disease progression (relative hazard for CD4 cell count, 1.84 per decrease of 100 cells/mm3; relative hazard for HIV DNA level, 2.73 per increase of 1 log(10) copies/10(6) PBMCs). Both a low initial CD4 cell count and a high HIV DNA level are predictive of rapid progression of untreated primary HIV-1 infection. Affected patients may therefore benefit from close clinical and laboratory monitoring and/or early administration of treatment.

  1. DNA structure in human RNA polymerase II promoters

    DEFF Research Database (Denmark)

    Pedersen, Anders Gorm; Baldi, Pierre; Chauvin, Yves

    1998-01-01

    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...... in a region upstream of the transcriptional start point and significantly higher downstream. Investigation of the sequence composition in the two regions shows that the bendability profile originates from the sequential structure of the DNA, rather than the general nucleotide composition...

  2. Cell Cycle Progression of Human Cells Cultured in Rotating Bioreactor

    Science.gov (United States)

    Parks, Kelsey

    2009-01-01

    Space flight has been shown to alter the astronauts immune systems. Because immune performance is complex and reflects the influence of multiple organ systems within the host, scientists sought to understand the potential impact of microgravity alone on the cellular mechanisms critical to immunity. Lymphocytes and their differentiated immature form, lymphoblasts, play an important and integral role in the body's defense system. T cells, one of the three major types of lymphocytes, play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptors. Reported studies have shown that spaceflight can affect the expression of cell surface markers. Cell surface markers play an important role in the ability of cells to interact and to pass signals between different cells of the same phenotype and cells of different phenotypes. Recent evidence suggests that cell-cycle regulators are essential for T-cell function. To trigger an effective immune response, lymphocytes must proliferate. The objective of this project is to investigate the changes in growth of human cells cultured in rotating bioreactors and to measure the growth rate and the cell cycle distribution for different human cell types. Human lymphocytes and lymphoblasts will be cultured in a bioreactor to simulate aspects of microgravity. The bioreactor is a cylindrical culture vessel that incorporates the aspects of clinostatic rotation of a solid fluid body around a horizontal axis at a constant speed, and compensates gravity by rotation and places cells within the fluid body into a sustained free-fall. Cell cycle progression and cell proliferation of the lymphocytes will be measured for a number of days. In addition, RNA from the cells will be isolated for expression of genes related in cell cycle regulations.

  3. Human identification and analysis of DNA in bones.

    Science.gov (United States)

    Iwamura, Edna Sadayo Miazato; Soares-Vieira, José Arnaldo; Muñoz, Daniel Romero

    2004-12-01

    The introduction of molecular biology techniques, especially of DNA analysis, for human identification is a recent advance in legal medicine. Substantial effort has continuously been made in an attempt to identify cadavers and human remains after wars, socio-political problems and mass disasters. In addition, because of the social dynamics of large cities, there are always cases of missing people, as well as unidentified cadavers and human remains that are found. In the last few years, there has also been an increase in requests for exhumation of human remains in order to determine genetic relationships in civil suits and court action. The authors provide an extensive review of the literature regarding the use of this new methodology for human identification of ancient or recent bones.

  4. The blood DNA virome in 8,000 humans.

    Directory of Open Access Journals (Sweden)

    Ahmed Moustafa

    2017-03-01

    Full Text Available The characterization of the blood virome is important for the safety of blood-derived transfusion products, and for the identification of emerging pathogens. We explored non-human sequence data from whole-genome sequencing of blood from 8,240 individuals, none of whom were ascertained for any infectious disease. Viral sequences were extracted from the pool of sequence reads that did not map to the human reference genome. Analyses sifted through close to 1 Petabyte of sequence data and performed 0.5 trillion similarity searches. With a lower bound for identification of 2 viral genomes/100,000 cells, we mapped sequences to 94 different viruses, including sequences from 19 human DNA viruses, proviruses and RNA viruses (herpesviruses, anelloviruses, papillomaviruses, three polyomaviruses, adenovirus, HIV, HTLV, hepatitis B, hepatitis C, parvovirus B19, and influenza virus in 42% of the study participants. Of possible relevance to transfusion medicine, we identified Merkel cell polyomavirus in 49 individuals, papillomavirus in blood of 13 individuals, parvovirus B19 in 6 individuals, and the presence of herpesvirus 8 in 3 individuals. The presence of DNA sequences from two RNA viruses was unexpected: Hepatitis C virus is revealing of an integration event, while the influenza virus sequence resulted from immunization with a DNA vaccine. Age, sex and ancestry contributed significantly to the prevalence of infection. The remaining 75 viruses mostly reflect extensive contamination of commercial reagents and from the environment. These technical problems represent a major challenge for the identification of novel human pathogens. Increasing availability of human whole-genome sequences will contribute substantial amounts of data on the composition of the normal and pathogenic human blood virome. Distinguishing contaminants from real human viruses is challenging.

  5. The blood DNA virome in 8,000 humans.

    Science.gov (United States)

    Moustafa, Ahmed; Xie, Chao; Kirkness, Ewen; Biggs, William; Wong, Emily; Turpaz, Yaron; Bloom, Kenneth; Delwart, Eric; Nelson, Karen E; Venter, J Craig; Telenti, Amalio

    2017-03-01

    The characterization of the blood virome is important for the safety of blood-derived transfusion products, and for the identification of emerging pathogens. We explored non-human sequence data from whole-genome sequencing of blood from 8,240 individuals, none of whom were ascertained for any infectious disease. Viral sequences were extracted from the pool of sequence reads that did not map to the human reference genome. Analyses sifted through close to 1 Petabyte of sequence data and performed 0.5 trillion similarity searches. With a lower bound for identification of 2 viral genomes/100,000 cells, we mapped sequences to 94 different viruses, including sequences from 19 human DNA viruses, proviruses and RNA viruses (herpesviruses, anelloviruses, papillomaviruses, three polyomaviruses, adenovirus, HIV, HTLV, hepatitis B, hepatitis C, parvovirus B19, and influenza virus) in 42% of the study participants. Of possible relevance to transfusion medicine, we identified Merkel cell polyomavirus in 49 individuals, papillomavirus in blood of 13 individuals, parvovirus B19 in 6 individuals, and the presence of herpesvirus 8 in 3 individuals. The presence of DNA sequences from two RNA viruses was unexpected: Hepatitis C virus is revealing of an integration event, while the influenza virus sequence resulted from immunization with a DNA vaccine. Age, sex and ancestry contributed significantly to the prevalence of infection. The remaining 75 viruses mostly reflect extensive contamination of commercial reagents and from the environment. These technical problems represent a major challenge for the identification of novel human pathogens. Increasing availability of human whole-genome sequences will contribute substantial amounts of data on the composition of the normal and pathogenic human blood virome. Distinguishing contaminants from real human viruses is challenging.

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

    Science.gov (United States)

    Ammerpohl, Ole; Bens, Susanne; Appari, Mahesh; Werner, Ralf; Korn, Bernhard; Drop, Stenvert L S; Verheijen, Frans; van der Zwan, Yvonne; Bunch, Trevor; Hughes, Ieuan; Cools, Martine; Riepe, Felix G; Hiort, Olaf; Siebert, Reiner; Holterhus, Paul-Martin

    2013-01-01

    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.

  7. 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.

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

    Science.gov (United States)

    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; Koenig, Christoph; Liddle, Jennifer; Niblett, David; Otto, Thomas; Pettett, Roger; Seemann, Stefanie; Thompson, Christian; West, Tony; Rogers, Jane; Olek, Alex; Berlin, Kurt; Beck, Stephan

    2011-01-01

    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 methylation and a core region surrounding the transcriptional start site as informative surrogate for promoter methylation. We find 17% of the 873 analyzed genes differentially methylated in their 5′-untranslated regions (5′-UTR) and about one third of the differentially methylated 5′-UTRs to be inversely correlated with transcription. While our study was controlled for factors reported to affect DNA methylation such as sex and age, we did not find any significant attributable effects. Our data suggest DNA methylation to be ontogenetically more stable than previously thought. PMID:17072317

  9. 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%). © 2010 American Academy of Forensic Sciences.

  10. Human papillomavirus DNA detection in sperm using polymerase chain reaction.

    Science.gov (United States)

    Olatunbosun, O; Deneer, H; Pierson, R

    2001-03-01

    To detect human papillomavirus (HPV) in semen and find if sperm washing removes HPV DNA. Amplification by nested polymerase chain reaction (PCR) was used to detect viral DNA sequences in semen samples from 85 volunteers. Forty-five men had historical or clinical evidence of genital HPV infection (study group) and 40 were healthy, clinically HPV-negative semen donors. We detected HPV DNA in the sperm cells of 24 of 45 subjects (53%) with past or current HPV infections in contrast to three of 40 healthy subjects (8%) (P HPV in 21 of 32 subjects (66%) with identifiable lesions and six of 53 (11%) without them (P sperm cells with HPV reduced cellular HPV DNA below detectable levels in only two cases. HPV is present in sperm cells from infected and apparently healthy subjects, and sperm washing does not eliminate the risk of HPV transmission to recipients. We suggest that HPV DNA testing should be done on the semen of prospective donors, and those with positive tests should be excluded from donation.

  11. [Heteroplasmy in human mtDNA control region].

    Science.gov (United States)

    Cao, Yang; Wan, Li-Hua; Gu, Lin-Gang; Huang, Ying-Xue; Xiu, Cong-Xian; Hu, Shu-Hui; Mi, Can

    2006-06-01

    To observe the length heteroplasmy and point heteroplasmy in human mtDNA control region. The peripheral blood, buccal cell, and single hair shaft from 50 individuals and 16 family members, related in their maternallineage were analyzed by direct sequencing, and clones from 20 individuals whose mtDNA sequences have a T-C transition at 16189 nt were sequenced. No point heteroplasmy were observed in peripheral blood, buccal cell, single hair shaft from the same individual, neither in maternally related individuals. Length heteroplasmy was observed in those individuals with a homopolymeric tract and the different clones from the same individual has different proportions of length variants, but the hair shafts from the same individual were very similar to the measurements made from blood DNA. No length heteroplasmy was observed between different tissues from the same individual. mtDNA sequences have a characteristic of high consistency and genetic stability, mtDNA sequencing is a suitable tool for forensic applications such as individual identification.

  12. Analysis of human accelerated DNA regions using archaic hominin genomes.

    Directory of Open Access Journals (Sweden)

    Hernán A Burbano

    Full Text Available 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.

  13. 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

  14. Slowly Progressive Encephalopathy with Hearing Loss Due to a Mutation in the mtDNA tRNALeu(CUN) Gene

    Science.gov (United States)

    Çoku, Jorida; Shanske, Sara; Mehrazin, Mahsa; Tanji, Kurenai; Naini, Ali; Emmanuele, Valentina; Patterson, Marc; Hirano, Michio; DiMauro, Salvatore

    2014-01-01

    Pathogenic mutations in the tRNALeu(UCN) gene of mitochondrial DNA (mtDNA) have been invariably accompanied by skeletal myopathy with or without chronic progressive external ophthalmoplegia (CPEO). We report a young woman with a heteroplasmic m.12276G>A mutation in tRNALeu(UCN), who had childhood-onset and slowly progressive encephalopathy with ataxia, cognitive impairment, and hearing loss. Sequencing of the 22 tRNA mitochondrial genes is indicated in all unusual neurological syndromes, even in the absence of maternal inheritance. PMID:20022607

  15. A Role of hIPI3 in DNA Replication Licensing in Human Cells.

    Science.gov (United States)

    Huang, Yining; Amin, Aftab; Qin, Yan; Wang, Ziyi; Jiang, Huadong; Liang, Lu; Shi, Linjing; Liang, Chun

    2016-01-01

    The yeast Ipi3p is required for DNA replication and cell viability in Sacharomyces cerevisiae. It is an essential component of the Rix1 complex (Rix1p/Ipi2p-Ipi1p-Ipi3p) that is required for the processing of 35S pre-rRNA in pre-60S ribosomal particles and for the initiation of DNA replication. The human IPI3 homolog is WDR18 (WD repeat domain 18), which shares significant homology with yIpi3p. Here we report that knockdown of hIPI3 resulted in substantial defects in the chromatin association of the MCM complex, DNA replication, cell cycle progression and cell proliferation. Importantly, hIPI3 silencing did not result in a reduction of the protein level of hCDC6, hMCM7, or the ectopically expressed GFP protein, indicating that protein synthesis was not defective in the same time frame of the DNA replication and cell cycle defects. Furthermore, the mRNA and protein levels of hIPI3 fluctuate in the cell cycle, with the highest levels from M phase to early G1 phase, similar to other pre-replicative (pre-RC) proteins. Moreover, hIPI3 interacts with other replication-initiation proteins, co-localizes with hMCM7 in the nucleus, and is important for the nuclear localization of hMCM7. We also found that hIPI3 preferentially binds to the origins of DNA replication including those at the c-Myc, Lamin-B2 and β-Globin loci. These results indicate that hIPI3 is involved in human DNA replication licensing independent of its role in ribosome biogenesis.

  16. Nonextensive statistical approach to non-coding human DNA

    Science.gov (United States)

    Oikonomou, Th.; Provata, A.; Tirnakli, U.

    2008-04-01

    We use q-exponential distributions, which maximize the nonextensive entropy Sq (defined as Sq≡(1-∑ipiq)/(q-1)), to study the size distributions of non-coding DNA (including introns and intergenic regions) in all human chromosomes. We show that the value of the exponent q describing the non-coding size distributions is similar for all chromosomes and varies between 2≤q≤2.3 with the exception of chromosomes X and Y.

  17. Dynamic protein interaction modules in human hepatocellular carcinoma progression.

    Science.gov (United States)

    Yu, Hui; Lin, Chen-Ching; Li, Yuan-Yuan; Zhao, Zhongming

    2013-01-01

    Gene expression profiles have been frequently integrated with the human protein interactome to uncover functional modules under specific conditions like disease state. Beyond traditional differential expression analysis, differential co-expression analysis has emerged as a robust approach to reveal condition-specific network modules, with successful applications in a few human disease studies. Hepatocellular carcinoma (HCC), which is often interrelated with the Hepatitis C virus, typically develops through multiple stages. A comprehensive investigation of HCC progression-specific differential co-expression modules may advance our understanding of HCC's pathophysiological mechanisms. Compared with differentially expressed genes, differentially co-expressed genes were found more likely enriched with Hepatitis C virus binding proteins and cancer-mutated genes, and they were clustered more densely in the human reference protein interaction network. These observations indicated that a differential co-expression approach could outperform the standard differential expression network analysis in searching for disease-related modules. We then proposed a differential co-expression network approach to uncover network modules involved in HCC development. Specifically, we discovered subnetworks that enriched differentially co-expressed gene pairs in each HCC transition stage, and further resolved modules with coherent co-expression change patterns over all HCC developmental stages. Our identified network modules were enriched with HCC-related genes and implicated in cancer-related biological functions. In particular, APC and YWHAZ were highlighted as two most remarkable genes in the network modules, and their dynamic interaction partnership was resolved in HCC development. We demonstrated that integration of differential co-expression with the protein interactome could outperform the traditional differential expression approach in discovering network modules of human diseases

  18. 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.

    2014-01-01

    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. PMID:24753594

  19. Nuclear and cytoplasmic LIMK1 enhances human breast cancer progression

    Directory of Open Access Journals (Sweden)

    Gutierrez-Hartmann Arthur

    2011-06-01

    Full Text Available Abstract Background LIM kinase 1 (LIMK1 is expressed in both cytoplasmic and nuclear compartments, and is a key regulator of cytoskeletal organization involved in cell migration and proliferation. LIMK1 levels are increased in several human cancers, with LIMK1 over-expression in prostate and breast cancer cells leading to tumor progression. While it has been presumed that the mechanism by which LIMK1 promotes cancer progression is via its cytoplasmic effects, the role of nuclear vs cytoplasmic LIMK1 in the tumorigenic process has not been examined. Results To determine if cytoplasmic or nuclear LIMK1 expression correlated with breast cancer, we performed immunohistochemical (IHC analysis of breast tissue microarrays (TMAs, The IHC analysis of breast TMAs revealed that 76% of malignant breast tissue samples strongly expressed LIMK1 in the cytoplasm, with 52% of these specimens also expressing nuclear LIMK1. Only 48% of benign breast samples displayed strong cytoplasmic LIMK1 expression and 27% of these expressed nuclear LIMK1. To investigate the respective roles of cytoplamsic and nuclear LIMK1 in breast cancer progression, we targeted GFP-LIMK1 to cytoplasmic and nuclear subcellular compartments by fusing nuclear export signals (NESs or nuclear localization sequences (NLS, respectively, to the amino-terminus of GFP-LIMK1. Stable pools of MDA-MB-231 cells were generated by retroviral transduction, and fluorescence microscopy revealed that GFP alone (control and GFP-LIMK1 were each expressed in both the cytoplasm and nucleus of MDA-MB-231 cells, whereas NLS-GFP-LIMK1 was expressed in the nucleus and NES-GFP-LIMK1 was expressed in the cytoplasm. Western blot analyses revealed equal expression of GFP-LIMK1 and NES-GFP-LIMK1, with NLS-GFP-LIMK1 expression being less but equal to endogenous LIMK1. Also, Western blotting revealed increased levels of phospho-cofilin, phospho-FAK, phospho-paxillin, phospho-Src, phospho-AKT, and phospho-Erk1/2 in cells

  20. Prediction of cancer progression in a group of 73 gastric cancer patients by circulating cell-free DNA.

    Science.gov (United States)

    Pu, Wang-Yang; Zhang, Rong; Xiao, Li; Wu, Yong-You; Gong, Wei; Lv, Xiao-Dong; Zhong, Feng-Yun; Zhuang, Zhi-Xiang; Bai, Xu-Ming; Li, Kai; Xing, Chun-Gen

    2016-12-09

    Circulating cell-free DNA (ccf-DNA) in plasma may contain both specific and non-specific of tumor markers. The concentration and integrity of ccf-DNA may be clinical useful for detecting and predicting cancer progression. Plasma samples from 40 healthy controls and 73 patients with gastric cancers (two stage 0, 17 stage I, 11 stage II, 33 stage III, and 10 stage IV according to American Joint Committee on Cancer stage) were assessed respectively. qPCR targeting the Alu repeats was performed using two different sets of primers amplifying the long and short segments. DNA integrity was calculated as a ratio of the long to the short fragments of Alu repeats. Plasma DNA concentration was significantly higher in patients with stage III and IV gastric cancers than in healthy controls (p = 0.028 and 0.029 respectively). The receiver operating characteristic (ROC) curve for discriminating patients with stage III and IV gastric cancers from healthy controls had an area under the curve (AUC) of 0.744 (95% CI, 0.64 to 0.85). Circulating cell-free DNA concentration increased within 21 days following surgery and dropped by 3 months after surgery. Concentration of ccf-DNA is a promising molecular marker for assessing gastric cancer progression. Current Controlled Trials ChiCTR-DDT-12002848 , 8 October 2012.

  1. The study of human Y chromosome variation through ancient DNA.

    Science.gov (United States)

    Kivisild, Toomas

    2017-05-01

    High throughput sequencing methods have completely transformed the study of human Y chromosome variation by offering a genome-scale view on genetic variation retrieved from ancient human remains in context of a growing number of high coverage whole Y chromosome sequence data from living populations from across the world. The ancient Y chromosome sequences are providing us the first exciting glimpses into the past variation of male-specific compartment of the genome and the opportunity to evaluate models based on previously made inferences from patterns of genetic variation in living populations. Analyses of the ancient Y chromosome sequences are challenging not only because of issues generally related to ancient DNA work, such as DNA damage-induced mutations and low content of endogenous DNA in most human remains, but also because of specific properties of the Y chromosome, such as its highly repetitive nature and high homology with the X chromosome. Shotgun sequencing of uniquely mapping regions of the Y chromosomes to sufficiently high coverage is still challenging and costly in poorly preserved samples. To increase the coverage of specific target SNPs capture-based methods have been developed and used in recent years to generate Y chromosome sequence data from hundreds of prehistoric skeletal remains. Besides the prospects of testing directly as how much genetic change in a given time period has accompanied changes in material culture the sequencing of ancient Y chromosomes allows us also to better understand the rate at which mutations accumulate and get fixed over time. This review considers genome-scale evidence on ancient Y chromosome diversity that has recently started to accumulate in geographic areas favourable to DNA preservation. More specifically the review focuses on examples of regional continuity and change of the Y chromosome haplogroups in North Eurasia and in the New World.

  2. Retinal mitochondrial DNA mismatch repair in the development of diabetic retinopathy, and its continued progression after termination of hyperglycemia.

    Science.gov (United States)

    Mishra, Manish; Kowluru, Renu A

    2014-09-23

    Mitochondrial DNA (mtDNA) is damaged in the retina in diabetes, and mitochondria copy numbers are decreased. The displacement-loop (D-loop) of the mtDNA, the region with transcription/replication elements, experiences more damage than other regions of mtDNA. Our aim was to examine the role of DNA mismatch repair (MMR) in mitochondria homeostasis in diabetic retinopathy, and in its continued progression after cessation of hyperglycemia. Effect of hyperglycemia on sequence variants in the D-loop region was investigated in retinal endothelial cells and in the retina from streptozotocin-induced diabetic rats using mismatch-specific surveyor nuclease. The role of MMR machinery in mtDNA damage and mitochondrial respiration was investigated in retinal endothelial cells overexpressing Mlh1, an MMR enzyme mainly associated with mtDNA polymerase gamma, or Msh2 (associated with nuclear polymerase beta). Hyperglycemia increased sequence variants in the D-loop region. While overexpression of Mlh1 in endothelial cells ameliorated glucose-induced increase in D-loop sequence variants, decrease in respiration rate and increase in apoptosis, overexpression of Msh2 did not protect the mitochondria damage. Termination of hyperglycemia failed to reverse decrease in MMR enzymes and increase in D-loop sequence variants. Due to a compromised MMR system, the sequence variants in the D-loop region were not repaired, and that resulted in impaired mtDNA transcription. Mitochondria become dysfunctional, and they continued to be dysfunctional even after hyperglycemia was terminated, contributing to the development, and progression of diabetic retinopathy. Thus, strategies targeting mitochondrial MMR machinery could help maintain mitochondria homeostasis, and inhibit the development of diabetic retinopathy and its continued progression. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

  3. 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.

  4. DNA methylation variation of human-specific Alu repeats

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    Bakshi, Arundhati; Herke, Scott W.; Batzer, Mark A.; Kim, Joomyeong

    2016-01-01

    ABSTRACT DNA methylation is the major repression mechanism for human retrotransposons, such as the Alu family. Here, we have determined the methylation levels associated with 5238 loci belonging to 2 Alu subfamilies, AluYa5 and AluYb8, using high-throughput targeted repeat element bisulfite sequencing (HT-TREBS). The results indicate that ∼90% of loci are repressed by high methylation levels. Of the remaining loci, many of the hypomethylated elements are found near gene promoters and show high levels of DNA methylation variation. We have characterized this variation in the context of tumorigenesis and interindividual differences. Comparison of a primary breast tumor and its matched normal tissue revealed early DNA methylation changes in ∼1% of AluYb8 elements in response to tumorigenesis. Simultaneously, AluYa5/Yb8 elements proximal to promoters also showed differences in methylation of up to one order of magnitude, even between normal individuals. Overall, the current study demonstrates that early loss of methylation occurs during tumorigenesis in a subset of young Alu elements, suggesting their potential clinical relevance. However, approaches such as deep-bisulfite-sequencing of individual loci using HT-TREBS are required to distinguish clinically relevant loci from the background observed for AluYa5/Yb8 elements in general with regard to high levels of interindividual variation in DNA methylation. PMID:26890526

  5. Multivalent human papillomavirus l1 DNA vaccination utilizing electroporation.

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    Kihyuck Kwak

    Full Text Available Naked DNA vaccines can be manufactured simply and are stable at ambient temperature, but require improved delivery technologies to boost immunogenicity. Here we explore in vivo electroporation for multivalent codon-optimized human papillomavirus (HPV L1 and L2 DNA vaccination.Balb/c mice were vaccinated three times at two week intervals with a fusion protein comprising L2 residues ∼11-88 of 8 different HPV types (11-88×8 or its DNA expression vector, DNA constructs expressing L1 only or L1+L2 of a single HPV type, or as a mixture of several high-risk HPV types and administered utilizing electroporation, i.m. injection or gene gun. Serum was collected two weeks and 3 months after the last vaccination. Sera from immunized mice were tested for in-vitro neutralization titer, and protective efficacy upon passive transfer to naive mice and vaginal HPV challenge. Heterotypic interactions between L1 proteins of HPV6, HPV16 and HPV18 in 293TT cells were tested by co-precipitation using type-specific monoclonal antibodies.Electroporation with L2 multimer DNA did not elicit detectable antibody titer, whereas DNA expressing L1 or L1+L2 induced L1-specific, type-restricted neutralizing antibodies, with titers approaching those induced by Gardasil. Co-expression of L2 neither augmented L1-specific responses nor induced L2-specific antibodies. Delivery of HPV L1 DNA via in vivo electroporation produces a stronger antibody response compared to i.m. injection or i.d. ballistic delivery via gene gun. Reduced neutralizing antibody titers were observed for certain types when vaccinating with a mixture of L1 (or L1+L2 vectors of multiple HPV types, likely resulting from heterotypic L1 interactions observed in co-immunoprecipitation studies. High titers were restored by vaccinating with individual constructs at different sites, or partially recovered by co-expression of L2, such that durable protective antibody titers were achieved for each type

  6. The DNA methylome of human peripheral blood mononuclear cells.

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    Yingrui Li

    2010-11-01

    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.

  7. Duplex Alu Screening for Degraded DNA of Skeletal Human Remains

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    Fabian Haß

    2017-10-01

    Full Text Available The human-specific Alu elements, belonging to the class of Short INterspersed Elements (SINEs, have been shown to be a powerful tool for population genetic studies. An earlier study in this department showed that it was possible to analyze Alu presence/absence in 3000-year-old skeletal human remains from the Bronze Age Lichtenstein cave in Lower Saxony, Germany. We developed duplex Alu screening PCRs with flanking primers for two Alu elements, each combined with a single internal Alu primer. By adding an internal primer, the approximately 400–500 bp presence signals of Alu elements can be detected within a range of less than 200 bp. Thus, our PCR approach is suited for highly fragmented ancient DNA samples, whereas NGS analyses frequently are unable to handle repetitive elements. With this analysis system, we examined remains of 12 individuals from the Lichtenstein cave with different degrees of DNA degradation. The duplex PCRs showed fully informative amplification results for all of the chosen Alu loci in eight of the 12 samples. Our analysis system showed that Alu presence/absence analysis is possible in samples with different degrees of DNA degradation and it reduces the amount of valuable skeletal material needed by a factor of four, as compared with a singleplex approach.

  8. Genome-Wide Analysis of DNA Methylation in Human Amnion

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    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.

  9. Interaction of the retinoblastoma protein with Orc1 and its recruitment to human origins of DNA replication.

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    Ramiro Mendoza-Maldonado

    Full Text Available BACKGROUND: The retinoblastoma protein (Rb is a crucial regulator of cell cycle progression by binding with E2F transcription factor and repressing the expression of a variety of genes required for the G1-S phase transition. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that Rb and E2F1 directly participate in the control of initiation of DNA replication in human HeLa, U2OS and T98G cells by specifically binding to origins of DNA replication in a cell cycle regulated manner. We show that, both in vitro and inside the cells, the largest subunit of the origin recognition complex (Orc1 specifically binds hypo-phosphorylated Rb and that this interaction is competitive with the binding of Rb to E2F1. The displacement of Rb-bound Orc1 by E2F1 at origins of DNA replication marks the progression of the G1 phase of the cell cycle toward the G1-S border. CONCLUSIONS/SIGNIFICANCE: The participation of Rb and E2F1 in the formation of the multiprotein complex that binds origins of DNA replication in mammalian cells appears to represent an effective mechanism to couple the expression of genes required for cell cycle progression to the activation of DNA replication.

  10. 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.

  11. [Influence of the treatment of chronic hepatitis B on HBV DNA suppression and prevention of disease progression].

    Science.gov (United States)

    Pawłowska, Małgorzata

    2008-01-01

    Appropriate treatment for chronic hepatitis B to present disease progression and clinical complications requires an accurate knowledge of the natural history of this disorder. The natural course of chronic HBV infections now perceive as consisting of 4 phases: immune tolerance, immune clearance, inactive carrier state and reactivation. In patients who acquire the disease in early life complications of CHB continue to develop because of the prolonged insidious damage to the liver. Measurement of serum HBV DNA is the most important factor to evaluate disease activity, assess the efficacy of antiviral therapy and predict treatment outcomes. It was shown that higher HBV DNA levels are associated with increased risk of progression to cirrhosis and HCC. Long-term suppression of viral replication to very low levels with nucleoside (nucleotide) analogues has been shown to decrease or delay liver disease progression and even partially reverse preexisting fibrosis and cirrhosis.

  12. Progresses in DNA-based heterologous prime-boost immunization strategies.

    Science.gov (United States)

    Jackson, Ronald J; Boyle, David B; Ranasinghe, Charani

    2014-01-01

    Although recombinant DNA and recombinant viral vectors expressing HIV antigens have yielded positive outcomes in animal models, these vaccines have not been effectively translated to humans. Despite this, there is still a high level of optimism that poxviral-based vaccine strategies could offer the best hope for developing an effective vaccine against not only HIV-1 but also other chronic diseases where good-quality T and B cell immunity is needed for protection. In this chapter we discuss step by step (1) how recombinant poxviral vectors co-expressing HIV antigens and promising mucosal/systemic adjuvants (e.g., IL-13Rα2) are constructed, (2) how these vectors can be used in alternative heterologous prime-boost immunization strategies, (3) how systemic and mucosal samples are prepared for analysis, followed by (4) two immunological assays: multicolor intracellular cytokine staining and tetramer/homing maker analysis that are used to evaluate effective systemic and mucosal T cell immunity.

  13. DNA-binding polarity of human replication protein A positions nucleases in nucleotide excision repair

    NARCIS (Netherlands)

    W.L. de Laat (Wouter); E. Appeldoorn (Esther); K. Sugasawa (Kaoru); E.P.W.C. Weterings (Eric); J.H.J. Hoeijmakers (Jan); N.G.J. Jaspers (Nicolaas)

    1998-01-01

    textabstractThe human single-stranded DNA-binding replication A protein (RPA) is involved in various DNA-processing events. By comparing the affinity of hRPA for artificial DNA hairpin structures with 3'- or 5'-protruding single-stranded arms, we found that hRPA binds ssDNA with a

  14. The E-cadherin repressor slug and progression of human extrahepatic hilar cholangiocarcinoma

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    Wang Xin-sheng

    2010-07-01

    Full Text Available Abstract Objectives This study explored the expression and function of Slug in human extrahepatic hilar cholangiocarcinoma (EHC to identify its role in tumor progression. Methods The expression of Snail and Slug mRNA in 52 human tissue samples of EHC was investigated. The mRNA of Snail and Slug were quantified using reverse transcriptase-PCR, and correlations with E-cadherin expression and clinicopathological factors were investigated. We then investigated transfection of Slug cDNA in endogenous E-cadherin-positive human EHC FRH0201 cells, selectively induced the loss of E-cadherin protein expression, and then small interfering RNA (siRNA for inhibition of Slug expression in endogenous Slug-positive human EHC QBC939 cells, selectively induced the loss of Slug protein expression. A Boyden chamber transwell assay was used for invasion. Results Slug mRNA was overexpressed in 18 cases (34.6% of EHC compared with adjacent noncancerous tissue. E-Cadherin protein expression determined in the same 52 cases by immunohistochemistry was significantly down-regulated in those cases with Slug mRNA overexpression (P = 0.0001. The tumor and nontumor ratio of Slug mRNA was correlated with nodal metastasis(p = 0.0102, distant metastasis (p = 0.0001and Survival time(p = 0.0443. However, Snail mRNA correlated with neither E-cadherin expression nor tumor invasiveness. By inhibiting Slug expression by RNA interference, we found that reduced Slug levels upregulated E-cadherin and decreased invasion in QBC939 cell. When the QBC939 cells was infected with Slug cDNA,, significant E-cadherin was downregulated and increased invasion in QBC939 cell. Conclusions The results suggested that Slug expression plays an important role in both the regulation of E-cadherin expression and in the acquisition of invasive potential in human EHC. Slug is possibly a potential target for an antitumor therapy blocking the functions of invasion and metastasis in human EHCs.

  15. Hydroxytyrosol Protects against Oxidative DNA Damage in Human Breast Cells

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    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.

  16. DNA replication stress: from molecular mechanisms to human disease.

    Science.gov (United States)

    Muñoz, Sergio; Méndez, Juan

    2017-02-01

    The genome of proliferating cells must be precisely duplicated in each cell division cycle. Chromosomal replication entails risks such as the possibility of introducing breaks and/or mutations in the genome. Hence, DNA replication requires the coordinated action of multiple proteins and regulatory factors, whose deregulation causes severe developmental diseases and predisposes to cancer. In recent years, the concept of "replicative stress" (RS) has attracted much attention as it impinges directly on genomic stability and offers a promising new avenue to design anticancer therapies. In this review, we summarize recent progress in three areas: (1) endogenous and exogenous factors that contribute to RS, (2) molecular mechanisms that mediate the cellular responses to RS, and (3) the large list of diseases that are directly or indirectly linked to RS.

  17. Isolating human DNA repair genes using rodent-cell mutants

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, L.H.; Weber, C.A.; Brookman, K.W.; Salazar, E.P.; Stewart, S.A.; Mitchell, D.L.

    1987-03-23

    The DNA repair systems of rodent and human cells appear to be at least as complex genetically as those in lower eukaryotes and bacteria. The use of mutant lines of rodent cells as a means of identifying human repair genes by functional complementation offers a new approach toward studying the role of repair in mutagenesis and carcinogenesis. In each of six cases examined using hybrid cells, specific human chromosomes have been identified that correct CHO cell mutations affecting repair of damage from uv or ionizing radiations. This finding suggests that both the repair genes and proteins may be virtually interchangeable between rodent and human cells. Using cosmid vectors, human repair genes that map to chromosome 19 have cloned as functional sequences: ERCC2 and XRCC1. ERCC1 was found to have homology with the yeast excision repair gene RAD10. Transformants of repair-deficient cell lines carrying the corresponding human gene show efficient correction of repair capacity by all criteria examined. 39 refs., 1 fig., 1 tab.

  18. 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.

  19. A novel DNA sequence database for analyzing human demographic history.

    Science.gov (United States)

    Wall, Jeffrey D; Cox, Murray P; Mendez, Fernando L; Woerner, August; Severson, Tesa; Hammer, Michael F

    2008-08-01

    While there are now extensive databases of human genomic sequences from both private and public efforts to catalog human nucleotide variation, there are very few large-scale surveys designed for the purpose of analyzing human population history. Demographic inference from patterns of SNP variation in current large public databases is complicated by ascertainment biases associated with SNP discovery and the ways that populations and regions of the genome are sampled. Here, we present results from a resequencing survey of 40 independent intergenic regions on the autosomes and X chromosome comprising ~210 kb from each of 90 humans from six geographically diverse populations (i.e., a total of ~18.9 Mb). Unlike other public DNA sequence databases, we include multiple indigenous populations that serve as important reservoirs of human genetic diversity, such as the San of Namibia, the Biaka of the Central African Republic, and Melanesians from Papua New Guinea. In fact, only 20% of the SNPs that we find are contained in the HapMap database. We identify several key differences in patterns of variability in our database compared with other large public databases, including higher levels of nucleotide diversity within populations, greater levels of differentiation between populations, and significant differences in the frequency spectrum. Because variants at loci included in this database are less likely to be subject to ascertainment biases or linked to sites under selection, these data will be more useful for accurately reconstructing past changes in size and structure of human populations.

  20. DNA synthesis determines the binding mode of the human mitochondrial single-stranded DNA-binding protein.

    Science.gov (United States)

    Morin, José A; Cerrón, Fernando; Jarillo, Javier; Beltran-Heredia, Elena; Ciesielski, Grzegorz L; Arias-Gonzalez, J Ricardo; Kaguni, Laurie S; Cao, Francisco J; Ibarra, Borja

    2017-07-07

    Single-stranded DNA-binding proteins (SSBs) play a key role in genome maintenance, binding and organizing single-stranded DNA (ssDNA) intermediates. Multimeric SSBs, such as the human mitochondrial SSB (HmtSSB), present multiple sites to interact with ssDNA, which has been shown in vitro to enable them to bind a variable number of single-stranded nucleotides depending on the salt and protein concentration. It has long been suggested that different binding modes might be used selectively for different functions. To study this possibility, we used optical tweezers to determine and compare the structure and energetics of long, individual HmtSSB-DNA complexes assembled on preformed ssDNA and on ssDNA generated gradually during 'in situ' DNA synthesis. We show that HmtSSB binds to preformed ssDNA in two major modes, depending on salt and protein concentration. However, when protein binding was coupled to strand-displacement DNA synthesis, only one of the two binding modes was observed under all experimental conditions. Our results reveal a key role for the gradual generation of ssDNA in modulating the binding mode of a multimeric SSB protein and consequently, in generating the appropriate nucleoprotein structure for DNA synthetic reactions required for genome maintenance. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. The relationship between DNA replication and human genome organization.

    Science.gov (United States)

    Necsulea, Anamaria; Guillet, Claire; Cadoret, Jean-Charles; Prioleau, Marie-Noëlle; Duret, Laurent

    2009-04-01

    Assessment of the impact of DNA replication on genome architecture in Eukaryotes has long been hampered by the scarcity of experimental data. Recent work, relying on computational predictions of origins of replication, suggested that replication might be a major determinant of gene organization in human (Huvet et al. 2007. Human gene organization driven by the coordination of replication and transcription. Genome Res. 17:1278-1285). Here, we address this question by analyzing the first large-scale data set of experimentally determined origins of replication in human: 283 origins identified in HeLa cells, in 1% of the genome covered by ENCODE regions (Cadoret et al. 2008. Genome-wide studies highlight indirect links between human replication origins and gene regulation. Proc Natl Acad Sci USA. 105:15837-15842). We show that origins of replication are not randomly distributed as they display significant overlap with promoter regions and CpG islands. The hypothesis of a selective pressure to avoid frontal collisions between replication and transcription polymerases is not supported by experimental data as we find no evidence for gene orientation bias in the proximity of origins of replication. The lack of a significant orientation bias remains manifest even when considering only genes expressed at a high rate, or in a wide number of tissues, and is not affected by the regional replication timing. Gene expression breadth does not appear to be correlated with the distance from the origins of replication. We conclude that the impact of DNA replication on human genome organization is considerably weaker than previously proposed.

  2. A soliton mechanism for DNA melting: Progress report, November 1, 1988--October 31, 1989

    Energy Technology Data Exchange (ETDEWEB)

    Sobell, H.M.

    1989-06-01

    A key feature of the soliton mechanism for DNA melting that has been proposed is the existence of an activated metastable DNA conformational state called ..beta..-DNA. Being metastable, this DNA conformation must be ''pinned'' or stabilized by an intercalating drug molecule such as actinomycin D in order to be observed. We have synthesized a series dodecamer oligonucleotide fragments, and have succeeded in forming crystalline complexes with actinomycin D. Their x-ray determination should allow atomic resolution and clarify how actinomycin binds to DNA. This will allow us to prove (or disprove) the actinomycin-DNA model we have proposed.

  3. SULFs in human neoplasia: implication as progression and prognosis factors

    Directory of Open Access Journals (Sweden)

    Schved Jean-François

    2011-05-01

    and in renal carcinoma compared to corresponding normal tissues. Furthermore, we found that high SULF1 expression was associated with a poor prognosis in lung adenocarcinoma. Finally, SULF1 and SULF2 were simultaneously overexpressed in 6 cancer types: brain, breast, head and neck, renal, skin and testicular cancers. Conclusions SULF1 and SULF2 are overexpressed in various human cancer types and can be associated to progression and prognosis. Targeting SULF1 and/or SULF2 could be interesting strategies to develop novel cancer therapies.

  4. The DNA methylation profile of activated human natural killer cells.

    Science.gov (United States)

    Wiencke, John K; Butler, Rondi; Hsuang, George; Eliot, Melissa; Kim, Stephanie; Sepulveda, Manuel A; Siegel, Derick; Houseman, E Andres; Kelsey, Karl T

    2016-05-03

    Natural killer (NK) cells are now recognized to exhibit characteristics akin to cells of the adaptive immune system. The generation of adaptive memory is linked to epigenetic reprogramming including alterations in DNA methylation. The study herein found reproducible genome wide DNA methylation changes associated with human NK cell activation. Activation led predominately to CpG hypomethylation (81% of significant loci). Bioinformatics analysis confirmed that non-coding and gene-associated differentially methylated sites (DMS) are enriched for immune related functions (i.e., immune cell activation). Known DNA methylation-regulated immune loci were also identified in activated NK cells (e.g., TNFA, LTA, IL13, CSF2). Twenty-one loci were designated high priority and further investigated as potential markers of NK activation. BHLHE40 was identified as a viable candidate for which a droplet digital PCR assay for demethylation was developed. The assay revealed high demethylation in activated NK cells and low demethylation in naïve NK, T- and B-cells. We conclude the NK cell methylome is plastic with potential for remodeling. The differentially methylated region signature of activated NKs revealed similarities with T cell activation, but also provided unique biomarker candidates of NK activation, which could be useful in epigenome-wide association studies to interrogate the role of NK subtypes in global methylation changes associated with exposures and/or disease states.

  5. Random-breakage mapping method applied to human DNA sequences

    Science.gov (United States)

    Lobrich, M.; Rydberg, B.; Cooper, P. K.; Chatterjee, A. (Principal Investigator)

    1996-01-01

    The random-breakage mapping method [Game et al. (1990) Nucleic Acids Res., 18, 4453-4461] was applied to DNA sequences in human fibroblasts. The methodology involves NotI restriction endonuclease digestion of DNA from irradiated calls, followed by pulsed-field gel electrophoresis, Southern blotting and hybridization with DNA probes recognizing the single copy sequences of interest. The Southern blots show a band for the unbroken restriction fragments and a smear below this band due to radiation induced random breaks. This smear pattern contains two discontinuities in intensity at positions that correspond to the distance of the hybridization site to each end of the restriction fragment. By analyzing the positions of those discontinuities we confirmed the previously mapped position of the probe DXS1327 within a NotI fragment on the X chromosome, thus demonstrating the validity of the technique. We were also able to position the probes D21S1 and D21S15 with respect to the ends of their corresponding NotI fragments on chromosome 21. A third chromosome 21 probe, D21S11, has previously been reported to be close to D21S1, although an uncertainty about a second possible location existed. Since both probes D21S1 and D21S11 hybridized to a single NotI fragment and yielded a similar smear pattern, this uncertainty is removed by the random-breakage mapping method.

  6. Extensive human DNA contamination in extracts from ancient dog bones and teeth.

    Science.gov (United States)

    Malmström, Helena; Storå, Jan; Dalén, Love; Holmlund, Gunilla; Götherström, Anders

    2005-10-01

    Ancient DNA (aDNA) sequences, especially those of human origin, are notoriously difficult to analyze due to molecular damage and exogenous DNA contamination. Relatively few systematic studies have focused on this problem. Here we investigate the extent and origin of human DNA contamination in the most frequently used sources for aDNA studies, that is, bones and teeth from museum collections. To distinguish contaminant DNA from authentic DNA we extracted DNA from dog (Canis familiaris) specimens. We monitored the presence of a 148-bp human-specific and a 152-bp dog-specific mitochondrial DNA (mtDNA) fragment in DNA extracts as well as in negative controls. The total number of human and dog template molecules were quantified using real-time polymerase chain reaction (PCR), and the sequences were characterized by amplicon cloning and sequencing. Although standard precautions to avoid contamination were taken, we found that all samples from the 29 dog specimens contained human DNA, often at levels exceeding the amount of authentic ancient dog DNA. The level of contaminating human DNA was also significantly higher in the dog extracts than in the negative controls, and an experimental setup indicated that this was not caused by the carrier effect. This suggests that the contaminating human DNA mainly originated from the dog bones rather than from laboratory procedures. When cloned, fragments within a contaminated PCR product generally displayed several different sequences, although one haplotype was often found in majority. This leads us to believe that recognized criteria for authenticating aDNA cannot separate contamination from ancient human DNA the way they are presently used.

  7. BLM–DNA2–RPA–MRN and EXO1–BLM–RPA–MRN constitute two DNA end resection machineries for human DNA break repair

    Science.gov (United States)

    Nimonkar, Amitabh V.; Genschel, Jochen; Kinoshita, Eri; Polaczek, Piotr; Campbell, Judith L.; Wyman, Claire; Modrich, Paul; Kowalczykowski, Stephen C.

    2011-01-01

    Repair of dsDNA breaks requires processing to produce 3′-terminated ssDNA. We biochemically reconstituted DNA end resection using purified human proteins: Bloom helicase (BLM); DNA2 helicase/nuclease; Exonuclease 1 (EXO1); the complex comprising MRE11, RAD50, and NBS1 (MRN); and Replication protein A (RPA). Resection occurs via two routes. In one, BLM and DNA2 physically and specifically interact to resect DNA in a process that is ATP-dependent and requires BLM helicase and DNA2 nuclease functions. RPA is essential for both DNA unwinding by BLM and enforcing 5′ → 3′ resection polarity by DNA2. MRN accelerates processing by recruiting BLM to the end. In the other, EXO1 resects the DNA and is stimulated by BLM, MRN, and RPA. BLM increases the affinity of EXO1 for ends, and MRN recruits and enhances the processivity of EXO1. Our results establish two of the core machineries that initiate recombinational DNA repair in human cells. PMID:21325134

  8. 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

  9. Domain within the helicase subunit Mcm4 integrates multiple kinase signals to control DNA replication initiation and fork progression.

    Science.gov (United States)

    Sheu, Yi-Jun; Kinney, Justin B; Lengronne, Armelle; Pasero, Philippe; Stillman, Bruce

    2014-05-06

    Eukaryotic DNA synthesis initiates from multiple replication origins and progresses through bidirectional replication forks to ensure efficient duplication of the genome. Temporal control of initiation from origins and regulation of replication fork functions are important aspects for maintaining genome stability. Multiple kinase-signaling pathways are involved in these processes. The Dbf4-dependent Cdc7 kinase (DDK), cyclin-dependent kinase (CDK), and Mec1, the yeast Ataxia telangiectasia mutated/Ataxia telangiectasia mutated Rad3-related checkpoint regulator, all target the structurally disordered N-terminal serine/threonine-rich domain (NSD) of mini-chromosome maintenance subunit 4 (Mcm4), a subunit of the mini-chromosome maintenance (MCM) replicative helicase complex. Using whole-genome replication profile analysis and single-molecule DNA fiber analysis, we show that under replication stress the temporal pattern of origin activation and DNA replication fork progression are altered in cells with mutations within two separate segments of the Mcm4 NSD. The proximal segment of the NSD residing next to the DDK-docking domain mediates repression of late-origin firing by checkpoint signals because in its absence late origins become active despite an elevated DNA damage-checkpoint response. In contrast, the distal segment of the NSD at the N terminus plays no role in the temporal pattern of origin firing but has a strong influence on replication fork progression and on checkpoint signaling. Both fork progression and checkpoint response are regulated by the phosphorylation of the canonical CDK sites at the distal NSD. Together, our data suggest that the eukaryotic MCM helicase contains an intrinsic regulatory domain that integrates multiple signals to coordinate origin activation and replication fork progression under stress conditions.

  10. Repair of DNA treated with. gamma. -irradiation and chemical carcinogens. Progress report, 1980-1983

    Energy Technology Data Exchange (ETDEWEB)

    Goldthwait, D.A.

    1984-02-01

    We have studied in vitro DNA repair with the isolation and characterization of DNA glycosylases active in the removable of 3-methyladenine and the problem of repair of DNA in chromatin. The second area of focus has been on transposable elements and carcinogen action. The work on DNA adducts with ..beta..-propiolactone was done to define potential new substrates useful in a search for new glycosylases.

  11. Boosting of DNA Vaccine-Elicited Gamma Interferon Responses in Humans by Exposure to Malaria Parasites

    National Research Council Canada - National Science Library

    Wang, Ruobing; Richie, Thomas L; Baraceros, Maria F; Rahardjo, Nancy; Gay, Tanya; Banania, Jo-Glenna; Charoenvit, Yupin; Epstein, Judith E; Luke, Thomas; Freilich, Daniel A; Norman, Jon; Hoffman, Stephen L

    2004-01-01

    A mixture of DNA plasmids expressing five Plasmodium falciparum pre-erythrocyte-stage antigens was administered with or without a DNA plasmid encoding human granulocyte-macrophage colony-stimulating factor (hGM-CSF...

  12. Natural mummification of the human gut preserves bacteriophage DNA.

    Science.gov (United States)

    Santiago-Rodriguez, Tasha M; Fornaciari, Gino; Luciani, Stefania; Dowd, Scot E; Toranzos, Gary A; Marota, Isolina; Cano, Raul J

    2016-01-01

    The natural mummification process of the human gut represents a unique opportunity to study the resulting microbial community structure and composition. While results are providing insights into the preservation of bacteria, fungi, pathogenic eukaryotes and eukaryotic viruses, no studies have demonstrated that the process of natural mummification also results in the preservation of bacteriophage DNA. We characterized the gut microbiome of three pre-Columbian Andean mummies, namely FI3, FI9 and FI12, and found sequences homologous to viruses. From the sequences attributable to viruses, 50.4% (mummy FI3), 1.0% (mummy FI9) and 84.4% (mummy FI12) were homologous to bacteriophages. Sequences corresponding to the Siphoviridae, Myoviridae, Podoviridae and Microviridae families were identified. Predicted putative bacterial hosts corresponded mainly to the Firmicutes and Proteobacteria, and included Bacillus, Staphylococcus, Clostridium, Escherichia, Vibrio, Klebsiella, Pseudomonas and Yersinia. Predicted functional categories associated with bacteriophages showed a representation of structural, replication, integration and entry and lysis genes. The present study suggests that the natural mummification of the human gut results in the preservation of bacteriophage DNA, representing an opportunity to elucidate the ancient phageome and to hypothesize possible mechanisms of preservation. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. High expression of DNA methyltransferases in primary human medulloblastoma.

    Science.gov (United States)

    Pócza, T; Krenács, T; Turányi, E; Csáthy, J; Jakab, Z; Hauser, P

    2016-01-01

    Epigenetic alterations have been implicated in cancer development. DNA methylation modulates gene expression, which is catalyzed by DNA methyltransferases (DNMTs). The objective of our study was to evaluate expression of DNMTs in medulloblastoma and analyze its correlation with clinical features. Nuclear expression of DNMT1, DNMT3A and DNMT3B was analyzed in human primary medulloblastoma of 44 patients using immunohistochemistry. Correlation of expression of DNMT levels with classical histological subtypes, novel molecular subgroups and survival of patients was analyzed. Elevated expression of DNMT1, DNMT3A and DNMT3B was observed in 63.64%, 68.18% and 72.73% of all cases, respectively. None of them showed a correlation with classical histology or survival. Concerning molecular subtypes, significantly higher expression of DNMT1 was observed in the SHH group compared to non-SHH samples (p = 0.02), but without significant difference in DNMT3A or DNMT3B levels between any subtypes. In conclusion, DNMT1, DNMT3A and DNMT3B are highly expressed in human medulloblastoma samples, suggesting that promoter hypermethylation may play a role in medulloblastoma development. Demethylation of tumor suppressor gene promoters may be considered as a possible future target in therapy of medulloblastoma.

  14. [Human herpesvirus-8 DNA in patients with certain demyelinating disorders].

    Science.gov (United States)

    Olut, Ali Ilgin; Ozünlü, Haluk; Tan, Ersin; Kocagöz, Tanal

    2005-04-01

    Infectious etiology of the demyelinating diseases is an intensive matter of research. Among the suspected pathogens, herpesviruses had attracted particular attention because of their capacity to remain latent in nervous tissues, axonal transportation of some members within neurons, relapsing-remitting characteristic of the infections, and capability of inducing demyelination both in human host and animal models. Human herpesvirus-8 (HHV-8) is the least studied of this group even some of the HHV-8 related disorders such as HIV associated Castleman's disease, some lymphomas, monoclonal gammopathy of uncertain significance (MGUS), may be seen in patients with demyelinating conditions. The aim of this study was the investigation of a probable relationship between HHV-8 infection and certain demyelinating diseases. For this purpose, the presence of HHV-8 DNA has been investigated by polymerase chain reaction in the blood samples of 14 multiple sclerosis (MS), six chronic inflammatory demyelinizing polyneuropathy (CIDP), three Guillain-Barre syndrome (GBS), and one Miller-Fisher syndrome patients, together with 24 age- and sex-matched healthy subjects as control. As a result, one of MS, two of CIDP and all of the GBS patients were found HHV-8 DNA positive, whereas all the subjects in control group were negative. Although the interpretation of the results of this study does not seem to be possible owing to the limited number of patients, it emphasizes the need for larger scale, detailed studies on this subject since no other report dealing with this matter has been encountered in the literature.

  15. Human factors in aviation maintenance, phase five : progress report.

    Science.gov (United States)

    1996-01-01

    The fifth phase of research on human factors in aviation maintenance continued to look at the human's role in the aviation maintenance system via investigations, demonstrations, and evaluations of the research program outputs. This report describes t...

  16. DNA repair proteins in cells of the human immune system; Le proteine della riparazione del DNA in cellule del sistema immunitario umano

    Energy Technology Data Exchange (ETDEWEB)

    Frasca, D.; Barattini, P.; Guidi, F.; Scarpaci, S. [ENEA, Sez. Tossicologia e Scienze Biomediche, Rome (Italy); Doria, G. [Rome Univ. Tor Vergata, Rome (Italy). Cattedra di Immunologia

    2001-02-01

    Human longevity depends on the efficiency of DNA repair mechanisms. In irradiated cells of the human immune system, the principal repair mechanism involves the DNA-Pk protein complex. [Italian] La durata della vita dipende dalla efficienza di meccanismi di riparazione del DNA. Nelle cellule del sistema immunitario umano danneggiate il principale meccanismo di riparazione coinvolge il complesso proteico DNA-PK.

  17. Progressive outer retinal necrosis: manifestation of human immunodeficiency virus infection

    Science.gov (United States)

    Lo, Phey Feng; Lim, Rongxuan; Antonakis, Serafeim N; Almeida, Goncalo C

    2015-01-01

    We present the case of a 54-year-old man who developed progressive outer retinal necrosis (PORN) as an initial manifestation of HIV infection without any significant risk factors for infection with HIV. PORN is usually found as a manifestation of known AIDS late in the disease. Our patient presented with transient visual loss followed by decrease in visual acuity and facial rash. Subsequent investigation revealed anterior chamber tap positive for varicella zoster virus (VZV), as well as HIV positivity, with an initial CD4 count of 48 cells/µL. Systemic and intravitreal antivirals against VZV, and highly active antiretroviral therapy against HIV were started, which halted further progression of retinal necrosis. This case highlights the importance of suspecting PORN where there is a rapidly progressive retinitis, and also testing the patient for HIV, so appropriate treatment can be started. PMID:25948844

  18. Progressive outer retinal necrosis: manifestation of human immunodeficiency virus infection.

    Science.gov (United States)

    Lo, Phey Feng; Lim, Rongxuan; Antonakis, Serafeim N; Almeida, Goncalo C

    2015-05-06

    We present the case of a 54-year-old man who developed progressive outer retinal necrosis (PORN) as an initial manifestation of HIV infection without any significant risk factors for infection with HIV. PORN is usually found as a manifestation of known AIDS late in the disease. Our patient presented with transient visual loss followed by decrease in visual acuity and facial rash. Subsequent investigation revealed anterior chamber tap positive for varicella zoster virus (VZV), as well as HIV positivity, with an initial CD4 count of 48 cells/µL. Systemic and intravitreal antivirals against VZV, and highly active antiretroviral therapy against HIV were started, which halted further progression of retinal necrosis. This case highlights the importance of suspecting PORN where there is a rapidly progressive retinitis, and also testing the patient for HIV, so appropriate treatment can be started. 2015 BMJ Publishing Group Ltd.

  19. The Modified Human DNA Repair Enzyme O6-Methylguanine-DNA Methyltransferase Is a Negative Regulator of Estrogen Receptor-Mediated Transcription upon Alkylation DNA Damage

    OpenAIRE

    Teo, Alvin K. C.; Oh, Hue Kian; Ali, Rahmen B.; Li, Benjamin F. L.

    2001-01-01

    Cell proliferation requires precise control to prevent mutations from replication of (unrepaired) damaged DNA in cells exposed spontaneously to mutagens. Here we show that the modified human DNA repair enzyme O6-methylguanine-DNA methyltransferase (R-MGMT), formed from the suicidal repair of the mutagenic O6-alkylguanine (6RG) lesions by MGMT in the cells exposed to alkylating carcinogens, functions in such control by preventing the estrogen receptor (ER) from transcription activation that me...

  20. Frequency and phenotypic implications of mitochondrial DNA mutations in human squamous cell cancers of the head and neck.

    Science.gov (United States)

    Zhou, Shaoyu; Kachhap, Sushant; Sun, Wenyue; Wu, Guojun; Chuang, Alice; Poeta, Luana; Grumbine, Lawson; Mithani, Suhail K; Chatterjee, Aditi; Koch, Wayne; Westra, William H; Maitra, Anirban; Glazer, Chad; Carducci, Michael; Sidransky, David; McFate, Thomas; Verma, Ajay; Califano, Joseph A

    2007-05-01

    Mitochondrial genomic mutations are found in a variety of human cancers; however, the frequency of mitochondrial DNA (mtDNA) mutations in coding regions remains poorly defined, and the functional effects of mitochondrial mutations found in primary human cancers are not well described. Using MitoChip, we sequenced the whole mitochondrial genome in 83 head and neck squamous cell carcinomas. Forty-one of 83 (49%) tumors contained mtDNA mutations. Mutations occurred within noncoding (D-loop) and coding regions. A nonrandom distribution of mutations was found throughout the mitochondrial enzyme complex components. Sequencing of margins with dysplasia demonstrated an identical nonconservative mitochondrial mutation (A76T in ND4L) as the tumor, suggesting a role of mtDNA mutation in tumor progression. Analysis of p53 status showed that mtDNA mutations correlated positively with p53 mutations (P < 0.002). To characterize biological function of the mtDNA mutations, we cloned NADH dehydrogenase subunit 2 (ND2) mutants based on primary tumor mutations. Expression of the nuclear-transcribed, mitochondrial-targeted ND2 mutants resulted in increased anchorage-dependent and -independent growth, which was accompanied by increased reactive oxygen species production and an aerobic glycolytic metabolic phenotype with hypoxia-inducible factor (HIF)-1alpha induction that is reversible by ascorbate. Cancer-specific mitochondrial mutations may contribute to development of a malignant phenotype by direct genotoxic effects from increased reactive oxygen species production as well as induction of aerobic glycolysis and growth promotion.

  1. Single Cell Analysis of Human RAD18-Dependent DNA Post-Replication Repair by Alkaline Bromodeoxyuridine Comet Assay

    Science.gov (United States)

    Mórocz, Mónika; Gali, Himabindu; Raskó, István; Downes, C. Stephen; Haracska, Lajos

    2013-01-01

    Damage to DNA can block replication progression resulting in gaps in the newly synthesized DNA. Cells utilize a number of post-replication repair (PRR) mechanisms such as the RAD18 controlled translesion synthesis or template switching to overcome the discontinuities formed opposite the DNA lesions and to complete DNA replication. Gaining more insights into the role of PRR genes promotes better understanding of DNA damage tolerance and of how their malfunction can lead to increased genome instability and cancer. However, a simple and efficient method to characterise gene specific PRR deficiencies at a single cell level has not been developed. Here we describe the so named BrdU comet PRR assay to test the contribution of human RAD18 to PRR at a single cell level, by which we kinetically characterized the consequences of the deletion of human RAD18 on the replication of UV-damaged DNA. Moreover, we demonstrate the capability of our method to evaluate PRR at a single cell level in unsynchronized cell population. PMID:23936422

  2. A human lymph node in vitro--challenges and progress.

    Science.gov (United States)

    Giese, Christoph; Demmler, Christian D; Ammer, Richard; Hartmann, Stefan; Lubitz, Annika; Miller, Lilja; Müller, Riccarda; Marx, Uwe

    2006-10-01

    Extracorporeal human lymphatic organs are expected to be excellent tools in the study of human molecular and cellular bases of the immunologic balance and tissue harmony. A rational approach and process to design a device and a procedure to recreate the human lymph node environment in vitro is described with emphasis on T-cell activation. Based on this approach, a bioreactor and a process supporting self-assembly of human lymphatic tissues due to proper emulation of human architecture and homeostasis could be developed.

  3. TERRA, hnRNP A1, and DNA-PKcs Interactions at Human Telomeres.

    Science.gov (United States)

    Le, Phuong N; Maranon, David G; Altina, Noelia H; Battaglia, Christine L R; Bailey, Susan M

    2013-01-01

    Maintenance of telomeres, repetitive elements at eukaryotic chromosomal termini, and the end-capping structure and function they provide, are imperative for preserving genome integrity and stability. The discovery that telomeres are transcribed into telomere repeat containing RNA (TERRA) has revolutionized our view of this repetitive, rather unappreciated region of the genome. We have previously shown that the non-homologous end-joining, shelterin associated DNA dependent protein kinase catalytic subunit (DNA-PKcs) participates in mammalian telomeric end-capping, exclusively at telomeres created by leading-strand synthesis. Here, we explore potential roles of DNA-PKcs and its phosphorylation target heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) in the localization of TERRA at human telomeres. Evaluation of co-localized foci utilizing RNA-FISH and three-dimensional (3D) reconstruction strategies provided evidence that both inhibition of DNA-PKcs kinase activity and siRNA depletion of hnRNP A1 result in accumulation of TERRA at individual telomeres; depletion of hnRNP A1 also resulted in increased frequencies of fragile telomeres. These observations are consistent with previous demonstrations that decreased levels of the nonsense RNA-mediated decay factors SMG1 and UPF1 increase TERRA at telomeres and interfere with replication of leading-strand telomeres. We propose that hTR mediated stimulation of DNA-PKcs and subsequent phosphorylation of hnRNP A1 influences the cell cycle dependent distribution of TERRA at telomeres by contributing to the removal of TERRA from telomeres, an action important for progression of S-phase, and thereby facilitating efficient telomere replication and end-capping.

  4. FXR silencing in human colon cancer by DNA methylation and KRAS signaling.

    Science.gov (United States)

    Bailey, Ann M; Zhan, Le; Maru, Dipen; Shureiqi, Imad; Pickering, Curtis R; Kiriakova, Galina; Izzo, Julie; He, Nan; Wei, Caimiao; Baladandayuthapani, Veerabhadran; Liang, Han; Kopetz, Scott; Powis, Garth; Guo, Grace L

    2014-01-01

    Farnesoid X receptor (FXR) is a bile acid nuclear receptor described through mouse knockout studies as a tumor suppressor for the development of colon adenocarcinomas. This study investigates the regulation of FXR in the development of human colon cancer. We used immunohistochemistry of FXR in normal tissue (n = 238), polyps (n = 32), and adenocarcinomas, staged I-IV (n = 43, 39, 68, and 9), of the colon; RT-quantitative PCR, reverse-phase protein array, and Western blot analysis in 15 colon cancer cell lines; NR1H4 promoter methylation and mRNA expression in colon cancer samples from The Cancer Genome Atlas; DNA methyltransferase inhibition; methyl-DNA immunoprecipitation (MeDIP); bisulfite sequencing; and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) knockdown assessment to investigate FXR regulation in colon cancer development. Immunohistochemistry and quantitative RT-PCR revealed that expression and function of FXR was reduced in precancerous lesions and silenced in a majority of stage I-IV tumors. FXR expression negatively correlated with phosphatidylinositol-4, 5-bisphosphate 3 kinase signaling and the epithelial-to-mesenchymal transition. The NR1H4 promoter is methylated in ~12% colon cancer The Cancer Genome Atlas samples, and methylation patterns segregate with tumor subtypes. Inhibition of DNA methylation and KRAS silencing both increased FXR expression. FXR expression is decreased early in human colon cancer progression, and both DNA methylation and KRAS signaling may be contributing factors to FXR silencing. FXR potentially suppresses epithelial-to-mesenchymal transition and other oncogenic signaling cascades, and restoration of FXR activity, by blocking silencing mechanisms or increasing residual FXR activity, represents promising therapeutic options for the treatment of colon cancer.

  5. Analysis of molecular changes during human melanocytic tumor progression

    NARCIS (Netherlands)

    Wit, Nicole Johanna Wilhelmina de

    2005-01-01

    Melanoma is one of the most aggressive types of cancer, due to its potency to disseminate early in tumor progression. The incidence is still rising, even though the rate of change has leveled off in the last decade. As melanoma cells are relatively insensitive to classical systemic therapies, like

  6. Epigenomic maintenance through dietary intervention can facilitate DNA repair process to slow down the progress of premature aging.

    Science.gov (United States)

    Ghosh, Shampa; Sinha, Jitendra Kumar; Raghunath, Manchala

    2016-09-01

    DNA damage caused by various sources remains one of the most researched topics in the area of aging and neurodegeneration. Increased DNA damage causes premature aging. Aging is plastic and is characterised by the decline in the ability of a cell/organism to maintain genomic stability. Lifespan can be modulated by various interventions like calorie restriction, a balanced diet of macro and micronutrients or supplementation with nutrients/nutrient formulations such as Amalaki rasayana, docosahexaenoic acid, resveratrol, curcumin, etc. Increased levels of DNA damage in the form of double stranded and single stranded breaks are associated with decreased longevity in animal models like WNIN/Ob obese rats. Erroneous DNA repair can result in accumulation of DNA damage products, which in turn result in premature aging disorders such as Hutchinson-Gilford progeria syndrome. Epigenomic studies of the aging process have opened a completely new arena for research and development of drugs and therapeutic agents. We propose here that agents or interventions that can maintain epigenomic stability and facilitate the DNA repair process can slow down the progress of premature aging, if not completely prevent it. © 2016 IUBMB Life, 68(9):717-721, 2016. © 2016 International Union of Biochemistry and Molecular Biology.

  7. Human Navigational Performance in a Complex Network with Progressive Disruptions

    CERN Document Server

    Ramesh, Amitash; Iyengar, Sudarshan; Sekhar, Vinod

    2012-01-01

    The current paper is an investigation towards understanding the navigational performance of humans on a network when the "landmark" nodes are blocked. We observe that humans learn to cope up, despite the continued introduction of blockages in the network. The experiment proposed involves the task of navigating on a word network based on a puzzle called the wordmorph. We introduce blockages in the network and report an incremental improvement in performance with respect to time. We explain this phenomenon by analyzing the evolution of the knowledge in the human participants of the underlying network as more and more landmarks are removed. We hypothesize that humans learn the bare essentials to navigate unless we introduce blockages in the network which would whence enforce upon them the need to explore newer ways of navigating. We draw a parallel to human problem solving and postulate that obstacles are catalysts for humans to innovate techniques to solve a restricted variant of a familiar problem.

  8. 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.

  9. Ultrasensitive fluorescence detection of DNA. Progress report, February 1, 1991--January 31, 1992

    Energy Technology Data Exchange (ETDEWEB)

    Mathies, R.A.; Glazer, A.N.

    1992-12-01

    We have shown that a number of polycationic highly fluorescent dyes form complexes with double-stranded DNA (dsDNA) which are stable to electrophoresis and have characterized in detail such dsDNA complexes with TOTO (1,1{prime}-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene]-quinolinium tetraiodide) and oxazole yellow dimer (YOYO; an analogue of TOTO with a benzo-1,3-oxazole in place of the benzo-1,3-thiazole). TOTO and YOYO are virtually non-fluorescent in solution, but form highly fluorescent complexes with dsDNA, up to a maximum dye to DNA bp ratio of 1:4, with >1000-fold fluorescence enhancement. We have developed an assay using YOYO for the quantitation of single-stranded and dsDNA in solution applicable over a range of DNA concentrations from 0.5 to 100 ng per ml. The fluorescent dsDNA-dye complexes allow detection of dsDNA on agarose and acrylamide gels with picogram sensitivity. We have applied these complexes in multiplex mapping experiments for accurate sizing and quantitation of restriction fragments. We have shown that in gel shift experiments the stable dsDNA-dye complexes can be used to detect heteroduplex-Muts complexes with a sensitivity comparable to radioisotopic detection.

  10. Progress on the HUPO Draft Human Proteome: 2017 Metrics of the Human Proteome Project.

    Science.gov (United States)

    Omenn, Gilbert S; Lane, Lydie; Lundberg, Emma K; Overall, Christopher M; Deutsch, Eric W

    2017-12-01

    The Human Proteome Organization (HUPO) Human Proteome Project (HPP) continues to make progress on its two overall goals: (1) completing the protein parts list, with an annual update of the HUPO draft human proteome, and (2) making proteomics an integrated complement to genomics and transcriptomics throughout biomedical and life sciences research. neXtProt version 2017-01-23 has 17 008 confident protein identifications (Protein Existence [PE] level 1) that are compliant with the HPP Guidelines v2.1 ( https://hupo.org/Guidelines ), up from 13 664 in 2012-12 and 16 518 in 2016-04. Remaining to be found by mass spectrometry and other methods are 2579 "missing proteins" (PE2+3+4), down from 2949 in 2016. PeptideAtlas 2017-01 has 15 173 canonical proteins, accounting for nearly all of the 15 290 PE1 proteins based on MS data. These resources have extensive data on PTMs, single amino acid variants, and splice isoforms. The Human Protein Atlas v16 has 10 492 highly curated protein entries with tissue and subcellular spatial localization of proteins and transcript expression. Organ-specific popular protein lists have been generated for broad use in quantitative targeted proteomics using SRM-MS or DIA-SWATH-MS studies of biology and disease.

  11. Early in vitro differentiation of mouse definitive endoderm is not correlated with progressive maturation of nuclear DNA methylation patterns.

    Directory of Open Access Journals (Sweden)

    Jian Tajbakhsh

    Full Text Available The genome organization in pluripotent cells undergoing the first steps of differentiation is highly relevant to the reprogramming process in differentiation. Considering this fact, chromatin texture patterns that identify cells at the very early stage of lineage commitment could serve as valuable tools in the selection of optimal cell phenotypes for regenerative medicine applications. Here we report on the first-time use of high-resolution three-dimensional fluorescence imaging and comprehensive topological cell-by-cell analyses with a novel image-cytometrical approach towards the identification of in situ global nuclear DNA methylation patterns in early endodermal differentiation of mouse ES cells (up to day 6, and the correlations of these patterns with a set of putative markers for pluripotency and endodermal commitment, and the epithelial and mesenchymal character of cells. Utilizing this in vitro cell system as a model for assessing the relationship between differentiation and nuclear DNA methylation patterns, we found that differentiating cell populations display an increasing number of cells with a gain in DNA methylation load: first within their euchromatin, then extending into heterochromatic areas of the nucleus, which also results in significant changes of methylcytosine/global DNA codistribution patterns. We were also able to co-visualize and quantify the concomitant stochastic marker expression on a per-cell basis, for which we did not measure any correlation to methylcytosine loads or distribution patterns. We observe that the progression of global DNA methylation is not correlated with the standard transcription factors associated with endodermal development. Further studies are needed to determine whether the progression of global methylation could represent a useful signature of cellular differentiation. This concept of tracking epigenetic progression may prove useful in the selection of cell phenotypes for future regenerative

  12. DNA Aptamer Raised Against AGEs Blocks the Progression of Experimental Diabetic Nephropathy

    Science.gov (United States)

    Kaida, Yusuke; Fukami, Kei; Matsui, Takanori; Higashimoto, Yuichiro; Nishino, Yuri; Obara, Nana; Nakayama, Yosuke; Ando, Ryotaro; Toyonaga, Maki; Ueda, Seiji; Takeuchi, Masayoshi; Inoue, Hiroyoshi; Okuda, Seiya

    2013-01-01

    Advanced glycation end products (AGEs) and their receptor (RAGE) play a role in diabetic nephropathy. We screened DNA aptamer directed against AGEs (AGEs-aptamer) in vitro and examined its effects on renal injury in KKAy/Ta mice, an animal model of type 2 diabetes. Eight-week-old male KKAy/Ta or C57BL/6J mice received continuous intraperitoneal infusion of AGEs- or control-aptamer for 8 weeks. AGEs-aptamer was detected and its level was increased in the kidney for at least 7 days. The elimination half-lives of AGEs-aptamer in the kidney were about 7 days. Compared with those in C57BL/6J mice, glomerular AGEs levels were significantly increased in KKAy/Ta mice, which were blocked by AGEs-aptamer. Urinary albumin and 8-hydroxy-2′-deoxy-guanosine levels were increased, and glomerular hypertrophy and enhanced extracellular matrix accumulation were observed in KKAy/Ta mice, all of which were prevented by AGEs-aptamer. Moreover, AGEs-aptamer significantly reduced gene expression of RAGE, monocyte chemoattractant protein-1, connective tissue growth factor, and type IV collagen both in the kidney of KKAy/Ta mice and in AGE-exposed human cultured mesangial cells. Our present data suggest that continuous administration of AGEs-aptamer could protect against experimental diabetic nephropathy by blocking the AGEs-RAGE axis and may be a feasible and promising therapeutic strategy for the treatment of diabetic nephropathy. PMID:23630304

  13. Human cytomegalovirus infection contributes to glioma disease progression via upregulating endocan expression.

    Science.gov (United States)

    Xing, Yan; Wang, Yisong; Wang, Shijie; Wang, Xin; Fan, Dongying; Zhou, Dabiao; An, Jing

    2016-11-01

    The etiology of malignant glioma remains unclear. To examine the association between glioma and human cytomegalovirus (HCMV) infection and the possible mechanism through which HCMV contributes to malignant glioma, we investigated the expression of HCMV components and an angiogenesis marker, endocan, in 79 glioma specimens and 8 control brain samples. HCMV pp65 protein and DNA were detected in 65.8% (52 of 79) and 54.4% (43 of 79) of glioma specimens, respectively. The positive rate and expression levels of pp65 were significantly correlated with the glioma grades. The endocan expression was detected in 78.5% (62 of 79) of glioma specimens, and elevated endocan immunoreactivity was also significantly associated with high-grade glioma. The pp65 was predominantly detected and colocalized with endocan in the cytoplasm of tumor cells. Importantly, there was a significant positive correlation in detection rates between those 2 proteins. In control samples, neither HCMV pp65 nor endocan expression was detected. Moreover, the serum endocan levels in glioma patients were markedly higher than that in healthy subjects. In in vitro study, HCMV infection induced the expression of interleukin 6 and tumor necrosis factor-α in human glioblastoma U87 MG (U87) cells and human umbilical vein endothelial cells (HUVECs). Furthermore, elevated endocan levels were also observed in HCMV-infected U87 cells and HUVECs and antiviral treatment with ganciclovir reduced the endocan expression. These results suggest HCMV infection leads to glioma progression through an upregulation of endocan and the secretion of inflammatory cytokines. Thus, anti-HCMV treatment may represent a potentially novel therapeutic strategy for glioma. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  14. A novel heteroplasmic tRNA(Leu(CUN)) mtDNA point mutation associated with chronic progressive external ophthalmoplegia.

    Science.gov (United States)

    Cardaioli, Elena; Da Pozzo, Paola; Radi, Elena; Dotti, Maria Teresa; Federico, Antonio

    2005-02-18

    We have sequenced all mitochondrial tRNA genes from a patient with chronic progressive external ophthalmoplegia (CPEO) and mitochondrial myopathy, who had no detectable large mtDNA deletions. Direct sequencing failed to detect previously reported mutations and showed a heteroplasmic mutation at nucleotide 12,276 in the tRNA(Leu(CUN)) gene, in the dihydrouridine stem, which is highly conserved through the species during evolution. RFLP analyses confirmed that 18% of muscle mtDNA harbored the mutation, while it was absent from DNA of fibroblasts and lymphocytes of the proband and in 110 patients with other encephalomyopathies. To date, besides large and single nucleotide deletions, several point mutations on mitochondrial tRNA genes have been reported in CPEO patients, but only three were in the gene coding for tRNA(Leu(CUN)).

  15. ISFG: recommendations regarding the use of non-human (animal) DNA in forensic genetic investigations.

    Science.gov (United States)

    Linacre, A; Gusmão, L; Hecht, W; Hellmann, A P; Mayr, W R; Parson, W; Prinz, M; Schneider, P M; Morling, N

    2011-11-01

    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 that is integral to a forensic science investigation and are not relevant to the breeding of animals for commercial purposes. This DNA commission was formed out of discussions at the International Society for Forensic Genetics 23rd Congress in Buenos Aires to outline recommendations on the use of non-human DNA in a forensic science investigation. Due to the scope of non-human DNA typing that is possible, the remit of this commission is confined to animal DNA typing only. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

  16. p27Kip1represses the Pitx2-mediated expression of p21Cip1and regulates DNA replication during cell cycle progression.

    Science.gov (United States)

    Gallastegui, E; Biçer, A; Orlando, S; Besson, A; Pujol, M J; Bachs, O

    2017-01-19

    The tumor suppressor p21 regulates cell cycle progression and peaks at mid/late G 1 . However, the mechanisms regulating its expression during cell cycle are poorly understood. We found that embryonic fibroblasts from p27 null mice at early passages progress slowly through the cell cycle. These cells present an elevated basal expression of p21 suggesting that p27 participates to its repression. Mechanistically, we found that p27 represses the expression of Pitx2 (an activator of p21 expression) by associating with the ASE-regulatory region of this gene together with an E2F4 repressive complex. Furthermore, we found that Pitx2 binds to the p21 promoter and induces its transcription. Finally, silencing Pitx2 or p21 in proliferating cells accelerates DNA replication and cell cycle progression. Collectively, these results demonstrate an unprecedented connection between p27, Pitx2 and p21 relevant for the regulation of cell cycle progression and cancer and for understanding human pathologies associated with p27 germline mutations.

  17. DNA Microarray Detection of 18 Important Human Blood Protozoan Species.

    Science.gov (United States)

    Chen, Mu-Xin; Ai, Lin; Chen, Jun-Hu; Feng, Xin-Yu; Chen, Shao-Hong; Cai, Yu-Chun; Lu, Yan; Zhou, Xiao-Nong; Chen, Jia-Xu; Hu, Wei

    2016-12-01

    Accurate detection of blood protozoa from clinical samples is important for diagnosis, treatment and control of related diseases. In this preliminary study, a novel DNA microarray system was assessed for the detection of Plasmodium, Leishmania, Trypanosoma, Toxoplasma gondii and Babesia in humans, animals, and vectors, in comparison with microscopy and PCR data. Developing a rapid, simple, and convenient detection method for protozoan detection is an urgent need. The microarray assay simultaneously identified 18 species of common blood protozoa based on the differences in respective target genes. A total of 20 specific primer pairs and 107 microarray probes were selected according to conserved regions which were designed to identify 18 species in 5 blood protozoan genera. The positive detection rate of the microarray assay was 91.78% (402/438). Sensitivity and specificity for blood protozoan detection ranged from 82.4% (95%CI: 65.9% ~ 98.8%) to 100.0% and 95.1% (95%CI: 93.2% ~ 97.0%) to 100.0%, respectively. Positive predictive value (PPV) and negative predictive value (NPV) ranged from 20.0% (95%CI: 2.5% ~ 37.5%) to 100.0% and 96.8% (95%CI: 95.0% ~ 98.6%) to 100.0%, respectively. Youden index varied from 0.82 to 0.98. The detection limit of the DNA microarrays ranged from 200 to 500 copies/reaction, similar to PCR findings. The concordance rate between microarray data and DNA sequencing results was 100%. Overall, the newly developed microarray platform provides a convenient, highly accurate, and reliable clinical assay for the determination of blood protozoan species.

  18. Comparative Molecular Dynamics Studies of Human DNA Polymerase η

    Science.gov (United States)

    2015-01-01

    High-energy ultraviolet radiation damages DNA through the formation of cyclobutane pyrimidine dimers, which stall replication. When the lesion is a thymine–thymine dimer (TTD), human DNA polymerase η (Pol η) assists in resuming the replication process by inserting nucleotides opposite the damaged site. We performed extensive molecular dynamics (MD) simulations to investigate the structural and dynamical effects of four different Pol η complexes with or without a TTD and with either dATP or dGTP as the incoming base. No major differences in the overall structures and equilibrium dynamics were detected among the four systems, suggesting that the specificity of this enzyme is due predominantly to differences in local interactions in the binding regions. Analysis of the hydrogen-bonding interactions between the enzyme and the DNA and dNTP provided molecular-level insights. Specifically, the TTD was observed to engage in more hydrogen-bonding interactions with the enzyme than its undamaged counterpart of two normal thymines. The resulting greater rigidity and specific orientation of the TTD are consistent with the experimental observation of higher processivity and overall efficiency at TTD sites than at analogous sites with two normal thymines. The similarities between the systems containing dATP and dGTP are consistent with the experimental observation of relatively low fidelity with respect to the incoming base. Moreover, Q38 and R61, two strictly conserved amino acids across the Pol η family, were found to exhibit persistent hydrogen-bonding interactions with the TTD and cation-π interactions with the free base, respectively. Thus, these simulations provide molecular level insights into the basis for the selectivity and efficiency of this enzyme, as well as the roles of the two most strictly conserved residues. PMID:26562587

  19. Quantification and presence of human ancient DNA in burial place ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-10-19

    Oct 19, 2009 ... useful for the improved ancient DNA extraction in anthropology and archeology. Key words: Ancient DNA, fossil bone, RT-PCR. INTRODUCTION. Ancient DNA research, defined as the retrieval and analysis of DNA sequences from various degraded biological source materials, has promoted many.

  20. Dynamic control of strand excision during human DNA mismatch repair.

    Science.gov (United States)

    Jeon, Yongmoon; Kim, Daehyung; Martín-López, Juana V; Lee, Ryanggeun; Oh, Jungsic; Hanne, Jeungphill; Fishel, Richard; Lee, Jong-Bong

    2016-03-22

    Mismatch repair (MMR) is activated by evolutionarily conserved MutS homologs (MSH) and MutL homologs (MLH/PMS). MSH recognizes mismatched nucleotides and form extremely stable sliding clamps that may be bound by MLH/PMS to ultimately authorize strand-specific excision starting at a distant 3'- or 5'-DNA scission. The mechanical processes associated with a complete MMR reaction remain enigmatic. The purified human (Homo sapien or Hs) 5'-MMR excision reaction requires the HsMSH2-HsMSH6 heterodimer, the 5' → 3' exonuclease HsEXOI, and the single-stranded binding heterotrimer HsRPA. The HsMLH1-HsPMS2 heterodimer substantially influences 5'-MMR excision in cell extracts but is not required in the purified system. Using real-time single-molecule imaging, we show that HsRPA or Escherichia coli EcSSB restricts HsEXOI excision activity on nicked or gapped DNA. HsMSH2-HsMSH6 activates HsEXOI by overcoming HsRPA/EcSSB inhibition and exploits multiple dynamic sliding clamps to increase tract length. Conversely, HsMLH1-HsPMS2 regulates tract length by controlling the number of excision complexes, providing a link to 5' MMR.

  1. CASA derived human sperm abnormalities: correlation with chromatin packing and DNA fragmentation

    National Research Council Canada - National Science Library

    Sivanarayana, T; Krishna, Ch Ravi; Prakash, G Jaya; Krishna, K Murali; Madan, K; Rani, B Sireesha; Sudhakar, G; Raju, G A. Rama

    2012-01-01

    The present study was undertaken to evaluate the effects of morphokinetic abnormalities of human spermatozoa on chromatin packing and DNA integrity and possible beneficial effects of sperm selection...

  2. Frequent detection of high human papillomavirus DNA loads in oral potentially malignant disorders.

    Science.gov (United States)

    Pierangeli, A; Cannella, F; Scagnolari, C; Gentile, M; Sciandra, I; Antonelli, G; Ciolfi, C; Russo, C; Palaia, G; Romeo, U; Polimeni, A

    2016-01-01

    Human papillomavirus (HPV) is estimated to be the cause of 40--80% of the squamous cell carcinoma of the oropharynx but only of a small fraction of the oral cavity cancers. The prevalence of oral HPV infection has significantly increased in the last decade, raising concerns about the role of HPV in progression of oral potentially malignant disorders (OPMD) toward squamous cell carcinomas. We sought to study HPV infection in patients with oral lesions, and in control individuals, using non-invasive and site-specific oral brushing and sensitive molecular methods. HPV DNA positivity and viral loads were evaluated in relation to patient data and clinical diagnosis. We enrolled 116 individuals attending Dental Clinics: 62 patients with benign oral lesions (e.g. fibromas, papillomatosis, ulcers) or OPMD (e.g. lichen, leukoplakia) and 54 controls. Oral cells were collected with Cytobrush and HPV-DNA was detected with quantitative real-time PCR for the more common high-risk (HR) and low-risk (LR) genotypes. HPV detection rate, percentage of HR HPVs and HPV-DNA loads (namely HPV16 and in particular, HPV18) were significantly higher in patients than in controls. Lichen planus cases had the highest HPV-positive rate (75.0%), hairy leukoplakia the lowest (33.3%). This study detected unexpectedly high rates of HPV infection in cells of the oral mucosa. The elevated HR HPV loads found in OPMD suggest the effectiveness of quantitative PCR in testing oral lesions. Prospective studies are needed to establish whether elevated viral loads represent a clinically useful marker of the risk of malignant progression. Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  3. 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.

  4. Nonperiodic activity of the human anaphase-promoting complex-Cdh1 ubiquitin ligase results in continuous DNA synthesis uncoupled from mitosis

    DEFF Research Database (Denmark)

    Lukas, C; Kramer, E R; Peters, J M

    2000-01-01

    , in Saccharomyces cerevisiae and Drosophila spp., triggers exit from mitosis and during G(1) prevents unscheduled DNA replication. In this study we investigated the importance of periodic oscillation of the APC-Cdh1 activity for the cell cycle progression in human cells. We show that conditional interference...

  5. Progress in HIV-1 antibody research using humanized mice.

    Science.gov (United States)

    Gruell, Henning; Klein, Florian

    2017-05-01

    Recent discoveries of highly potent broadly HIV-1 neutralizing antibodies provide new opportunities to successfully prevent, treat, and potentially cure HIV-1 infection. To test their activity in vivo, humanized mice have been shown to be a powerful model and were used to investigate antibody-mediated prevention and therapy approaches. In this review, we will summarize recent findings in humanized mice that have informed on the potential use of broadly neutralizing antibodies targeting HIV-1 in humans. Humanized mouse models have been used to demonstrate the antiviral efficacy of HIV-1 neutralizing antibodies in vivo. It has been shown that a combination of antibodies can suppress viremia below the limit of detection and targets the HIV-1 reservoir. Moreover, passively administered antibodies and vector-mediated antibody production protect humanized mice from HIV-1 infection. Finally, immunization studies in knock-in/transgenic mice carrying human antibody gene segments have informed on potential vaccination strategies to induce broad and potent HIV-1 neutralizing antibodies. Humanized mouse models are of great value for HIV-1 research. They represent a highly versatile in vivo system to investigate novel approaches for HIV-1 prevention and therapy and expedite the critical translation from basic findings to clinical application.

  6. Human extraocular muscles in mitochondrial diseases: comparing chronic progressive external ophthalmoplegia with Leber's hereditary optic neuropathy.

    Science.gov (United States)

    Carta, A; Carelli, V; D'Adda, T; Ross-Cisneros, F N; Sadun, A A

    2005-07-01

    To compare the ultrastructural aspects of human extraocular muscles in two types of mitochondrial disease: chronic progressive external ophthalmoplegia (CPEO) and Leber's hereditary optic neuropathy (LHON). Muscle samples of the medial rectus obtained from surgery in a sporadic case of CPEO associated with deleted mitochondrial DNA, and post mortem in a case of 3460/ND1 LHON were processed for electron microscopy (EM). The medial rectus from an autoptic time to fixation matched control was used to exclude postmortem artefacts. The CPEO specimen revealed focal areas of disruption and abnormalities of mitochondria in some muscle fibres, creating a "mosaic-like" pattern. In the LHON specimen a diffuse increase in both number and size of mitochondria (mean diameter 0.85 mum v 0.65 mum of control, pCPEO and LHON reveals marked differences. A "mosaic-like" pattern caused by a selective damage of muscle fibres was evident in CPEO, whereas a diffuse increase in mitochondria with preservation of myofibrils characterised the LHON case. These ultrastructural changes may relate to the different expression of the two diseases, resulting in ophthalmoplegia in CPEO and normal eye movements in LHON.

  7. Human DNA Extraction by Two Extraction Methods for Forensic Typification from Human Feces on FTA Paper

    Directory of Open Access Journals (Sweden)

    Shirleny Monserrat Sandoval-Arias

    2014-11-01

    Full Text Available The identification of suspects in criminal investigations has been facilitated since DNA test are executed on different samples. The application of this technology for forensic typification from human fecal samples still presents complications therefore this research evaluated two DNA extraction protocols with modifications to determine that of major efficiency. Organic extractions and extractions using the commercial kit “IQTM DNA Casework Sample Kit for Maxwell ® 16” on FTA portions of 4cm2 and 1cm2 impregnated with feces from the same individual were done to accomplish the objective. In all the assays the results were useful, however; the best forensic typification (by the electropherogram characteristics was obtained by using the commercial kit in an area of 1 cm2 of FTA paper impregnated in a 1:4 dilution.

  8. Identification of DNA methylation changes associated with human gastric cancer

    Directory of Open Access Journals (Sweden)

    Park Jung-Hoon

    2011-12-01

    Full Text Available Abstract Background Epigenetic alteration of gene expression is a common event in human cancer. DNA methylation is a well-known epigenetic process, but verifying the exact nature of epigenetic changes associated with cancer remains difficult. Methods We profiled the methylome of human gastric cancer tissue at 50-bp resolution using a methylated DNA enrichment technique (methylated CpG island recovery assay in combination with a genome analyzer and a new normalization algorithm. Results We were able to gain a comprehensive view of promoters with various CpG densities, including CpG Islands (CGIs, transcript bodies, and various repeat classes. We found that gastric cancer was associated with hypermethylation of 5' CGIs and the 5'-end of coding exons as well as hypomethylation of repeat elements, such as short interspersed nuclear elements and the composite element SVA. Hypermethylation of 5' CGIs was significantly correlated with downregulation of associated genes, such as those in the HOX and histone gene families. We also discovered long-range epigenetic silencing (LRES regions in gastric cancer tissue and identified several hypermethylated genes (MDM2, DYRK2, and LYZ within these regions. The methylation status of CGIs and gene annotation elements in metastatic lymph nodes was intermediate between normal and cancerous tissue, indicating that methylation of specific genes is gradually increased in cancerous tissue. Conclusions Our findings will provide valuable data for future analysis of CpG methylation patterns, useful markers for the diagnosis of stomach cancer, as well as a new analysis method for clinical epigenomics investigations.

  9. Optimized DNA electroporation for primary human T cell engineering.

    Science.gov (United States)

    Zhang, Zhang; Qiu, Shunfang; Zhang, Xiaopeng; Chen, Wei

    2018-01-30

    Effective gene-delivery systems for primary human T cell engineering are useful tools for both basic research and clinical immunotherapy applications. Pseudovirus-based systems and electro-transfection are the most popular strategies for genetic material transduction. Compared with viral-particle-mediated approaches, electro-transfection is theoretically safer, because it does not promote transgene integration into the host genome. Additionally, the simplicity and speed of the procedure increases the attractiveness of electroporation. Here, we developed and optimized an electro-transfection method for the production of engineered chimeric antigen receptor (CAR)-T cells. Stimulation of T cells had the greatest effect on their transfection, with stimulation of cells for up to 3 days substantially improving transfection efficiency. Additionally, the strength of the external electric field, input cell number, and the initial amount of DNA significantly affected transfection performance. The voltage applied during electroporation affected plasmid permeation and was negatively correlated with the number of viable cells after electroporation. Moreover, higher plasmid concentration increased the proportion of positively transfected cells, but decreased cell viability, and for single-activated cells, higher cell density enhanced their viability. We evaluated the effects of two clinically relevant factors, serum supplementation in the culture medium and cryopreservation immediately after the isolation of peripheral blood lymphocytes. Our findings showed that our protocol performed well using xeno-free cultured, fresh T cells, with application resulting in a lower but acceptable transfection efficiency of cells cultured with fetal bovine serum or thawed cells. Furthermore, we described an optimized procedure to generate CAR-T cells within 6 days and that exhibited cytotoxicity toward targeted cells. Our investigation of DNA electro-transfection for the use in human primary

  10. The human mitochondrial single-stranded DNA-binding protein displays distinct kinetics and thermodynamics of DNA binding and exchange.

    Science.gov (United States)

    Qian, Yufeng; Johnson, Kenneth A

    2017-08-04

    The human mitochondrial ssDNA-binding protein (mtSSB) is a homotetrameric protein, involved in mtDNA replication and maintenance. Although mtSSB is structurally similar to SSB from Escherichia coli (EcoSSB), it lacks the C-terminal disordered domain, and little is known about the biophysics of mtSSB-ssDNA interactions. Here, we characterized the kinetics and thermodynamics of mtSSB binding to ssDNA by equilibrium titrations and stopped-flow kinetic measurements. We show that the mtSSB tetramer can bind to ssDNA in two distinct binding modes: (SSB) 30 and (SSB) 60 , defined by DNA binding site sizes of 30 and 60 nucleotides, respectively. We found that the binding mode is modulated by magnesium ion and NaCl concentration, but unlike EcoSSB, the mtSSB does not show negative intersubunit cooperativity. Global fitting of both the equilibrium and kinetic data afforded estimates for the rate and equilibrium constants governing the formation of (SSB) 60 and (SSB) 30 complexes and for the transitions between the two binding modes. We found that the mtSSB tetramer binds to ssDNA with a rate constant near the diffusion limit (2 × 10 9 m -1 s -1 ) and that longer DNA (≥60 nucleotides) rapidly wraps around all four monomers, as revealed by FRET assays. We also show that the mtSSB tetramer can directly transfer from one ssDNA molecule to another via an intermediate with two DNA molecules bound to the mtSSB. In conclusion, our results indicate that human mtSSB shares many physicochemical properties with EcoSSB and that the differences may be explained by the lack of an acidic, disordered C-terminal tail in human mtSSB protein. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Human factors in aviation maintenance, phase two : progress report.

    Science.gov (United States)

    1993-04-01

    In this second phase of research on Human Factors in Aviation Maintenance, the emphasis has evolved from problem definition to development of demonstrations and prototypes. These demonstrations include a computer-based training simulation for trouble...

  12. Human microbiota-associated swine: current progress and future opportunities.

    Science.gov (United States)

    Wang, Mei; Donovan, Sharon M

    2015-01-01

    Gnotobiotic (GN) rodent models have provided insight into the contributions of the gut microbiota to host health and preventing disease. However, rodent models are limited by several important physiological and metabolic differences from humans, and many rodent models do not dependably replicate the clinical manifestations of human diseases. Due to the high degree of similarity in anatomy, physiology, immunology and brain growth, the domestic pig (Sus scrofa) is considered a clinically relevant model to study factors influencing human gastrointestinal, immune, and brain development. Gnotobiotic piglet models have been developed and shown to recapitulate key aspects of GN rodent models. Human microbiota-associated (HMA) piglets have been established using inocula from infants, children, and adults. The gut microbiota of recipient HMA piglets was more similar to that of the human donor than that of conventionally reared piglets harboring a pig microbiota. Moreover, Bifidobacterium and Bacteroides, two predominant bacterial groups of infant gut, were successfully established in the HMA piglets. Thus, the HMA pig model has the potential to be a valuable model for investigating how the gut microbiota composition changes in response to environmental factors, such as age, diet, vaccination, antibiotic use and infection. The HMA also represents a robust model for screening the efficacy of pre- and probiotic interventions. Lastly, HMA piglets can be an ideal model with which to elucidate microbe-host interactions in human health and disease due to the similarities to humans in anatomy, physiology, developmental maturity at birth, and the pathophysiology of many human diseases. © The Author 2015. Published by Oxford University Press on behalf of the Institute for Laboratory Animal Research. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  13. Bioengineering of a human whole tooth: progress and challenge

    OpenAIRE

    Yanding Zhang; YiPing Chen

    2014-01-01

    A major challenge in stem cell-based bioengineering of an implantable human tooth is to identify appropriate sources of postnatal stem cells that are odontogenic competent as the epithelial component due to the lack of enamel epithelial cells in adult teeth. In a recent issue (2013, 2:6) of Cell Regeneration, Cai and colleagues reported that epithelial sheets derived from human induced pluripotent stem cells (iPSCs) can functionally substitute for tooth germ epithelium to regenerate tooth-lik...

  14. Progress integrating medical humanities into medical education: a global overview.

    Science.gov (United States)

    Pfeiffer, Stefani; Chen, Yuchia; Tsai, Duujian

    2016-09-01

    The article reviews the most recent developments in integrating humanities into medical education. Global implications and future trends are illustrated. The main concern of medical humanities education is teaching professionalism; one important aspect that has emerged is the goal of nurturing emotion through reflexivity. Relating effectively to all stakeholders and being sensitive to inequitable power dynamics are essential for professional social accountability in modern medical contexts. Mediating doctors' understanding of the clinical encounter through creative arts and narrative is part of most recent pedagogic innovations aimed at motivating learners to become empowered, engaged and caring clinicians. Scenario-based and discursive-oriented evaluations of such activities should be aligned with the medical humanities' problem-based learning curriculum. Medical humanities education fosters professional reflexivity that is important for achieving patient-centered care. Countering insufficient empathy with reflective professionalism is an urgent challenge in medical education; to answer this need, creative arts and narrative understanding have emerged as crucial tools of medical humanities education. To ensure competent professional identity formation in the era of translational medicine, medical humanities programs have adopted scenario-based assessments through inclusion of different voices and emphasizing personal reflection and social critique.

  15. Twin-based DNA methylation analysis takes the center stage of studies of human complex diseases

    DEFF Research Database (Denmark)

    Zhang, Dongfeng; Li, Shuxia; Tan, Qihua

    2012-01-01

    The etiology of complex diseases is characterized by the interaction between the genome and environmental conditions and the interface of epigenetics may be a central mechanism. Current technologies already allow us high-throughput profiling of epigenetic patterns at genome level. However, our...... and complex traits. In the era of functional genomics, the valuable samples of twins are helping to bridge the gap between gene activity and environmental conditions through epigenetic mechanisms unlimited to DNA sequence variations. We review the recent progresses in using twins to study disease...... understanding of the epigenetic processes remains limited. Twins are special samples in genetic studies due to their genetic similarity and rearing-environment sharing. In the past decades, twins have made a great contribution in dissecting the genetic and environmental contributions to human diseases...

  16. Immortality, but not oncogenic transformation, of primary human cells leads to epigenetic reprogramming of DNA methylation and gene expression.

    Science.gov (United States)

    Gordon, Katrina; Clouaire, Thomas; Bao, Xun X; Kemp, Sadie E; Xenophontos, Maria; de Las Heras, Jose Ignacio; Stancheva, Irina

    2014-04-01

    Tumourigenic transformation of normal cells into cancer typically involves several steps resulting in acquisition of unlimited growth potential, evasion of apoptosis and non-responsiveness to growth inhibitory signals. Both genetic and epigenetic changes can contribute to cancer development and progression. Given the vast genetic heterogeneity of human cancers and difficulty to monitor cancer-initiating events in vivo, the precise relationship between acquisition of genetic mutations and the temporal progression of epigenetic alterations in transformed cells is largely unclear. Here, we use an in vitro model system to investigate the contribution of cellular immortality and oncogenic transformation of primary human cells to epigenetic reprogramming of DNA methylation and gene expression. Our data demonstrate that extension of replicative life span of the cells is sufficient to induce accumulation of DNA methylation at gene promoters and large-scale changes in gene expression in a time-dependent manner. In contrast, continuous expression of cooperating oncogenes in immortalized cells, although essential for anchorage-independent growth and evasion of apoptosis, does not affect de novo DNA methylation at promoters and induces subtle expression changes. Taken together, these observations imply that cellular immortality promotes epigenetic adaptation to highly proliferative state, whereas transforming oncogenes confer additional properties to transformed human cells.

  17. Progress report on research on human genetics in Iceland

    Energy Technology Data Exchange (ETDEWEB)

    None

    1980-10-31

    Records of the Icelandic population are being used to investigate the possible inheritance of disabilities and diseases as well as other characteristics and the effect of environment on man. The progress report of research covers the period from 1977 to 1980. The investigation was begun in 1965 by the Genetical Committee of the University of Iceland and the materials used are demographic records from the year 1840 to present and various medical information. The records are being computerized and linked together to make them effective for use in hereditary studies.

  18. Research on human genetics in Iceland. Progress report

    Energy Technology Data Exchange (ETDEWEB)

    None

    1980-10-31

    Records of the Icelandic Population are being used to investigate the possible inheritance of disabilities and diseases as well as other characters and the effect of environment on man. The progress report of research covers the period 1977 to 1980. The investigation was begun in 1965 by the Genetical Committee of the University of Iceland and the materials used are demographic records from the year 1840 to present and various medical information. The records are being computerized and linked together to make them effective for use in hereditary studies.

  19. [Research Progress on the Detection Method of DNA Methylation and Its Application in Forensic Science].

    Science.gov (United States)

    Nie, Y C; Yu, L J; Guan, H; Zhao, Y; Rong, H B; Jiang, B W; Zhang, T

    2017-06-01

    As an important part of epigenetic marker, DNA methylation involves in the gene regulation and attracts a wide spread attention in biological auxology, geratology and oncology fields. In forensic science, because of the relative stable, heritable, abundant, and age-related characteristics, DNA methylation is considered to be a useful complement to the classic genetic markers for age-prediction, tissue-identification, and monozygotic twins' discrimination. Various methods for DNA methylation detection have been validated based on methylation sensitive restriction endonuclease, bisulfite modification and methylation-CpG binding protein. In recent years, it is reported that the third generation sequencing method can be used to detect DNA methylation. This paper aims to make a review on the detection method of DNA methylation and its applications in forensic science. Copyright© by the Editorial Department of Journal of Forensic Medicine.

  20. Comparison of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens.

    Science.gov (United States)

    Ariefdjohan, Merlin W; Savaiano, Dennis A; Nakatsu, Cindy H

    2010-05-22

    The influence of diet on intestinal microflora has been investigated mainly using conventional microbiological approaches. Although these studies have advanced knowledge on human intestinal microflora, it is imperative that new methods are applied to facilitate scientific progress. Culture-independent molecular fingerprinting method of Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis (PCR-DGGE) has been used to study microbial communities in a variety of environmental samples. However, these protocols must be optimized prior to their application in order to enhance the quality and accuracy of downstream analyses. In this study, the relative efficacy of four commercial DNA extraction kits (Mobio Ultra Clean(R) Fecal DNA Isolation Kit, M; QIAamp DNA Stool Mini Kit, Q; FastDNA SPIN Kit, FSp; FastDNA SPIN Kit for Soil, FSo) were evaluated. Further, PCR-DGGE technique was also assessed for its feasibility in detecting differences in human intestinal bacterial fingerprint profiles. Total DNA was extracted from varying weights of human fecal specimens using four different kits, followed by PCR amplification of bacterial 16S rRNA genes, and DGGE separation of the amplicons. Regardless of kit, maximum DNA yield was obtained using 10 to 50 mg (wet wt) of fecal specimens and similar DGGE profiles were obtained. However, kits FSp and FSo extracted significantly larger amounts of DNA per g dry fecal specimens and produced more bands on their DGGE profiles than kits M and Q due to their use of bead-containing lysing matrix and vigorous shaking step. DGGE of 16S rRNA gene PCR products was suitable for capturing the profiles of human intestinal microbial community and enabled rapid comparative assessment of inter- and intra-subject differences. We conclude that extraction kits that incorporated bead-containing lysing matrix and vigorous shaking produced high quality DNA from human fecal specimens (10 to 50 mg, wet wt) that can be resolved as bacterial

  1. Identification and cloning of endogenous retroviral sequences present in human DNA.

    OpenAIRE

    Martin, M. A.; Bryan, T; Rasheed, S; Khan, A S

    1981-01-01

    Using nonstringent annealing conditions and a 2.75-kilobase segment of cloned African green monkey DNA that specifically hybridizes to the proviruses of AKR ecotropic murine leukemia virus (MuLV) and baboon endogenous virus (BaEV) as a probe, we detected related sequences in three different preparations of human brain DNA fragments. The blot-hybridization pattern obtained with cleaved human DNA was similar to that previously reported for the interaction of MuLV cDNA and cleaved mouse DNA and ...

  2. Single-stranded DNA catenation mediated by human EVL and a type I topoisomerase

    OpenAIRE

    Takaku, Motoki; Takahashi, Daisuke; Machida, Shinichi; Ueno, Hiroyuki; Hosoya, Noriko; Ikawa, Shukuko; Miyagawa, Kiyoshi; Shibata, Takehiko; Kurumizaka, Hitoshi

    2010-01-01

    The human Ena/Vasp-like (EVL) protein is considered to be a bifunctional protein, involved in both actin remodeling and homologous recombination. In the present study, we found that human EVL forms heat-stable multimers of circular single-stranded DNA (ssDNA) molecules in the presence of a type I topoisomerase in vitro. An electron microscopic analysis revealed that the heat-stable ssDNA multimers formed by EVL and topoisomerase were ssDNA catemers. The ssDNA catenation did not occur when eit...

  3. 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......, which fall within haplogroups X and C, consistent with Northeastern Native populations today. In addition we have sexed the male using a novel-sexing assay and confirmed the authenticity of his Y chromosome with the presence of the Native American specific Y-QM3 single nucleotide polymorphism (SNP......, 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...

  4. Characterization of noncoding regulatory DNA in the human genome.

    Science.gov (United States)

    Elkon, Ran; Agami, Reuven

    2017-08-08

    Genetic variants associated with common diseases are usually located in noncoding parts of the human genome. Delineation of the full repertoire of functional noncoding elements, together with efficient methods for probing their biological roles, is therefore of crucial importance. Over the past decade, DNA accessibility and various epigenetic modifications have been associated with regulatory functions. Mapping these features across the genome has enabled researchers to begin to document the full complement of putative regulatory elements. High-throughput reporter assays to probe the functions of regulatory regions have also been developed but these methods separate putative regulatory elements from the chromosome so that any effects of chromatin context and long-range regulatory interactions are lost. Definitive assignment of function(s) to putative cis-regulatory elements requires perturbation of these elements. Genome-editing technologies are now transforming our ability to perturb regulatory elements across entire genomes. Interpretation of high-throughput genetic screens that incorporate genome editors might enable the construction of an unbiased map of functional noncoding elements in the human genome.

  5. 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.

  6. Mapping sequences in loops of nuclear DNA by their progressive detachment from the nuclear cage.

    OpenAIRE

    Cook, P R; Brazell, I A

    1980-01-01

    Nuclear DNA is organised into loops, probably by attachment to a supramolecular structure. We describe a method which enables us to map the position of sequences within a loop relative to the point of attachment. Nuclear DNA is isolated unbroken by lysing HeLa cells in 2M NaCl to release structures which retain many of the morphological features of nuclei. Their DNA is supercoiled and so must remain unbroken and looped during lysis. Nucleoids are digested to various degrees with a restriction...

  7. 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.

  8. Human NEIL3 is mainly a monofunctional DNA glycosylase removing spiroiminohydantoin and guanidinohydantoin

    OpenAIRE

    Krokeide, Silje Z.; Laerdahl, Jon K.; Salah, Medya; Luna, Luisa; Cederkvist, F. Henning; Fleming, Aaron M.; Burrows, Cynthia J.; Dalhus, Bjørn; Bjørås, Magnar

    2013-01-01

    Base excision repair is the major pathway for removal of oxidative DNA base damage. This pathway is initiated by DNA glycosylases, which recognize and excise damaged bases from DNA. In this work, we have purified the glycosylase domain (GD) of human DNA glycosylase NEIL3. The substrate specificity has been characterized and we have elucidated the catalytic mechanisms. GD NEIL3 excised the hydantoin lesions spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh) in single-stranded (ss) and doub...

  9. Biobanks and human health research: Balancing progress and ...

    African Journals Online (AJOL)

    Biobanks are repositories that store human biological materials and their associated data. They are rapidly becoming part of national and international networks and give rise to unique ethico-regulatory issues. Whether consent is informed and whether this term should be used when specimens are collected for biobank ...

  10. 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. ...

  11. Molecular cloning and characterization of human papilloma virus DNA derived from a laryngeal papilloma.

    Science.gov (United States)

    Gissmann, L; Diehl, V; Schultz-Coulon, H J; zur Hausen, H

    1982-01-01

    Papilloma virus DNA from a laryngeal papilloma was cloned in phage lambda L 47 and characterized after cleavage with different restriction enzymes. Hybridization with the DNAs of human papilloma virus types 1, 2, 3, 4, 5, and 8 showed no homology under stringent hybridization conditions. Human papilloma virus type 6 DNA, however, was partially identical to laryngeal papilloma virus DNA; different restriction enzyme fragments hybridizing with the other DNA were identified on each genome. The degree of homology was determined by reassociation kinetics to be 25%. According to the present nomenclature, laryngeal papilloma virus therefore represents a different type of human papilloma virus and is tentatively designated as human papilloma virus type 11. Sequences homologous to laryngeal papilloma virus DNA were also found in four of nine additional laryngeal papillomas. Attempt to detect homologous DNA in 12 carcinomas of the larynx were negative. Images PMID:6292500

  12. 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.

  13. The identification of gene expression profiles associated with progression of human diabetic neuropathy

    Science.gov (United States)

    Hur, Junguk; Sullivan, Kelli A.; Pande, Manjusha; Hong, Yu; Sima, Anders A. F.; Jagadish, Hosagrahar V.; Kretzler, Matthias

    2011-01-01

    Diabetic neuropathy is a common complication of diabetes. While multiple pathways are implicated in the pathophysiology of diabetic neuropathy, there are no specific treatments and no means to predict diabetic neuropathy onset or progression. Here, we identify gene expression signatures related to diabetic neuropathy and develop computational classification models of diabetic neuropathy progression. Microarray experiments were performed on 50 samples of human sural nerves collected during a 52-week clinical trial. A series of bioinformatics analyses identified differentially expressed genes and their networks and biological pathways potentially responsible for the progression of diabetic neuropathy. We identified 532 differentially expressed genes between patient samples with progressing or non-progressing diabetic neuropathy, and found these were functionally enriched in pathways involving inflammatory responses and lipid metabolism. A literature-derived co-citation network of the differentially expressed genes revealed gene subnetworks centred on apolipoprotein E, jun, leptin, serpin peptidase inhibitor E type 1 and peroxisome proliferator-activated receptor gamma. The differentially expressed genes were used to classify a test set of patients with regard to diabetic neuropathy progression. Ridge regression models containing 14 differentially expressed genes correctly classified the progression status of 92% of patients (P diabetic neuropathy progression in human sural nerve biopsies and describe their potential utility in classifying diabetic neuropathy. Our results identifying the unique gene signature of patients with progressive diabetic neuropathy will facilitate the development of new mechanism-based diagnostics and therapies. PMID:21926103

  14. Chromatin inactivation precedes de novo dna methylation during the progressive epigenetic silencing of the rassf1a promoter

    Energy Technology Data Exchange (ETDEWEB)

    Strunnikova Maria; Schagdarsurengin, Undraga; Kehlen, Astrid; Garbe, James C.; Stampfer, Martha R.; Dammann, Reinhard

    2005-02-23

    Epigenetic inactivation of the RASSF1A tumor suppressor by CpG island methylation was frequently detected in cancer. However, the mechanisms of this aberrant DNA methylation are unknown. In the RASSF1A promoter, we characterized four Sp1 sites, which are frequently methylated in cancer. We examined the functional relationship between DNA methylation, histone modification, Sp1 binding, and RASSF1A expression in proliferating human mammary epithelial cells. With increasing passages, the transcription of RASSF1A was dramatically silenced. This inactivation was associated with deacetylation and lysine 9 trimethylation of histone H3 and an impaired binding of Sp1 at the RASSF1A promoter. In mammary epithelial cells that had overcome a stress-associated senescence barrier, a spreading of DNA methylation in the CpG island promoter was observed. When the RASSF1A-silenced cells were treated with inhibitors of DNA methyltransferase and histone deacetylase, binding of Sp1 and expression of RASSF1 A reoccurred. In summary, we observed that histone H3 deacetylation and H3 lysine 9 trimethylation occur in the same time window as gene inactivation and precede DNA methylation. Our data suggest that in epithelial cells, histone inactivation may trigger de novo DNA methylation of the RASSF1A promoter and this system may serve as a model for CpG island inactivation of tumor suppressor genes.

  15. 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.

  16. Long-Term Imaging of DNA Damage and Cell Cycle Progression in Budding Yeast Using Spinning Disk Confocal Microscopy.

    Science.gov (United States)

    Montecchi, Riccardo; Schwob, Etienne

    2018-01-01

    Live cell imaging can monitor biological processes in time and space by providing quantitative measurements of cell behavior on a single-cell basis and in live conditions. However the illumination required to visualize fluorescently tagged endogenous proteins often perturbs cellular physiology, a problem particularly acute for yeast cells that are small, highly photosensitive and with scarce protein content. Analyzing the activation of the DNA damage response (DDR) in various yeast mutants or growth conditions, as well as its consequences for cell cycle progression and cell viability over extended periods of time therefore requires a special microscopy setup that does not by itself create DNA damage or perturb cell growth. Here, we provide a quick guide, strains and advice for imaging the DDR in S. cerevisiae for extended time (3-12 h) using spinning-disk confocal microscopy in conditions of limited photobleaching and photodamage. DDR is a conserved mechanism that allows the cell to respond to various stresses, especially those altering DNA integrity or topology. Acquiring time-lapse images of the DDR at high temporal and spatial resolution is of great interest, in particular when studying the effects of mutations or drugs which compromise genomic stability and cell cycle progression.

  17. Plans and progress for building a Great Lakes fauna DNA barcode reference library

    Science.gov (United States)

    DNA reference libraries provide researchers with an important tool for assessing regional biodiversity by allowing unknown genetic sequences to be assigned identities, while also providing a means for taxonomists to validate identifications. Expanding the representation of Great...

  18. Identification of some human pathogenic fungi using four DNA ...

    African Journals Online (AJOL)

    Stocks from pathogenic fungi isolated from infected areas on different patients, around Lagos-Nigeria were analysed using molecular methods (DNA extraction, PCR-RFLP and DNA sequencing). Four DNA extraction protocols were employed in the identification of the fungal isolates. Sixteen different fungal isolates were ...

  19. 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

    Analyses of degraded DNA are typically hampered by contamination, especially when employing universal primers such as commonly used in environmental DNA studies. In addition to false-positive results, the amplification of contaminant DNA may cause false-negative results because of competition, or...

  20. 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.

  1. 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.

  2. 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.

  3. Human adipogenesis is associated with genome-wide DNA methylation and gene-expression changes

    DEFF Research Database (Denmark)

    Broholm, Christa; Olsson, Anders Henrik; Perfilyev, Alexander

    2016-01-01

    AIM: To define the genomic distribution and function of DNA methylation changes during human adipogenesis. METHODS: We isolated adipocyte-derived stem cells from 13 individuals and analyzed genome-wide DNA methylation and gene expression in cultured adipocyte-derived stem cells and mature adipocy...... for cell cycle and adipokine signaling. CONCLUSION: Human adipogenesis is associated with significant DNA methylation changes across the entire genome and may impact regulation of cell cycle and adipokine signaling....

  4. Resveratrol and ascorbic acid prevent DNA damage induced by cryopreservation in human semen.

    Science.gov (United States)

    Branco, Cátia S; Garcez, Márcia E; Pasqualotto, Fábio F; Erdtman, Bernardo; Salvador, Mirian

    2010-04-01

    Cryopreservation of human semen can cause DNA damages, which compromise the fertilization and normal embryo development. The present study showed that the antioxidant resveratrol prevents these damages both in fertile and infertile men. The addition of ascorbic acid before cryopreservation can reduce DNA damages only in infertile men. Although further studies are needed, the present work showed that resveratrol could be considered in human cryopreservation procedures to avoid/minimize DNA damages and preserve sperm integrity. Copyright 2009 Elsevier Inc. All rights reserved.

  5. 1999 Gordon Research Conference on Mammalian DNA Repair. Final Progress Report

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1999-02-12

    This Conference will examine DNA repair as the key component in genomic surveillance that is so crucial to the overall integrity and function of mammalian cells. Recent discoveries have catapulted the field of DNA repair into a pivotal position for fundamental investigations into oncology, aging, environmental health, and developmental biology. We hope to highlight the most promising and exciting avenues of research in robust discussions at this conference. This Mammalian DNA Repair Gordon Conference differs from the past conferences in this series, in which the programs were broader in scope, with respect to topics and biological systems covered. A conference sponsored by the Genetics Society in April 1998 emphasized recombinational mechanisms for double-strand break repair and the role of mismatch repair deficiency in colorectal cancer. These topics will therefore receive somewhat less emphasis in the upcoming Conference. In view of the recent mechanistic advances in mammalian DNA repair, an upcoming comprehensive DNA repair meeting next autumn at Hilton Head; and the limited enrollment for Gordon Conferences we have decided to focus session-by-session on particular areas of controversy and/or new developments specifically in mammalian systems. Thus, the principal presentations will draw upon results from other cellular systems only to the extent that they impact our understanding of mammalian DNA repair.

  6. Progress in Tetanus Prophylaxis: The Advent of Human Antitoxin

    Science.gov (United States)

    Perey, Bernard J. F.

    1966-01-01

    Injections of tetanus antitoxin of animal origin frequently cause serious disability and sometimes death. Despite world-wide knowledge of these effects, millions of prophylactic injections of equine tetanus antitoxin are given annually, and it is continually proposed that the dosage be increased in order to obtain higher “protective” levels in the serum, a procedure which would increase the incidence and severity of reactions. Furthermore, equine antitoxin frequently fails to prevent tetanus. Tetanus antitoxin of human origin is available which carries no risk of complications and confers a higher degree of immunity more quickly than equine antitoxin. The cost of treating reactions to horse serum, together with the financial loss incurred by work-absence, far outweighs the cost of human antitoxin. In the author's opinion, the use, in this country, of antitoxin of animal origin is no longer medically acceptable and may well prove legally indefensible. PMID:5902706

  7. 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.

  8. Alternative DNA structure formation in the mutagenic human c-MYC promoter.

    Science.gov (United States)

    Del Mundo, Imee Marie A; Zewail-Foote, Maha; Kerwin, Sean M; Vasquez, Karen M

    2017-05-05

    Mutation 'hotspot' regions in the genome are susceptible to genetic instability, implicating them in diseases. These hotspots are not random and often co-localize with DNA sequences potentially capable of adopting alternative DNA structures (non-B DNA, e.g. H-DNA and G4-DNA), which have been identified as endogenous sources of genomic instability. There are regions that contain overlapping sequences that may form more than one non-B DNA structure. The extent to which one structure impacts the formation/stability of another, within the sequence, is not fully understood. To address this issue, we investigated the folding preferences of oligonucleotides from a chromosomal breakpoint hotspot in the human c-MYC oncogene containing both potential G4-forming and H-DNA-forming elements. We characterized the structures formed in the presence of G4-DNA-stabilizing K+ ions or H-DNA-stabilizing Mg2+ ions using multiple techniques. We found that under conditions favorable for H-DNA formation, a stable intramolecular triplex DNA structure predominated; whereas, under K+-rich, G4-DNA-forming conditions, a plurality of unfolded and folded species were present. Thus, within a limited region containing sequences with the potential to adopt multiple structures, only one structure predominates under a given condition. The predominance of H-DNA implicates this structure in the instability associated with the human c-MYC oncogene. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. A micro-RNA expression signature for human NAFLD progression.

    Science.gov (United States)

    Guo, Yan; Xiong, Yanhua; Sheng, Quanghu; Zhao, Shilin; Wattacheril, Julia; Flynn, Charles Robb

    2016-10-01

    The spectrum of nonalcoholic fatty liver disease (NAFLD) describes disease conditions deteriorating from nonalcoholic fatty liver (NAFL) to nonalcoholic steatohepatitis (NASH) to cirrhosis (CIR) to hepatocellular carcinoma (HCC). From a molecular and biochemical perspective, our understanding of the etiology of this disease is limited by the broad spectrum of disease presentations, the lack of a thorough understanding of the factors contributing to disease susceptibility, and ethical concerns related to repeat sampling of the liver. To better understand the factors associated with disease progression, we investigated by next-generation RNA sequencing the altered expression of microRNAs (miRNAs) in liver biopsies of class III obese subjects (body mass index ≥40 kg/m(2)) biopsied at the time of elective bariatric surgery. Clinical characteristics and unbiased RNA expression profiles for 233 miRs, 313 transfer RNAs (tRNAs), and 392 miscellaneous small RNAs (snoRNAs, snRNAs, rRNAs) were compared among 36 liver biopsy specimens stratified by disease severity. The abundances of 3 miRNAs that were found to be differentially regulated (miR-301a-3p and miR-34a-5p increased and miR-375 decreased) with disease progression were validated by RT-PCR. No tRNAs or miscellaneous RNAs were found to be associated with disease severity. Similar patterns of increased miR-301a and decreased miR-375 expression were observed in 134 hepatocellular carcinoma (HCC) samples deposited in The Cancer Genome Atlas (TCGA). Our analytical results suggest that NAFLD severity is associated with a specific pattern of altered hepatic microRNA expression that may drive the hallmark of this disorder: altered lipid and carbohydrate metabolism. The three identified miRNAs can potentially be used as biomarkers to access the severity of NAFLD. The persistence of this miRNA expression pattern in an external validation cohort of HCC samples suggests that specific microRNA expression patterns may permit and

  10. Application of Circulating Tumor DNA as a Non-Invasive Tool for Monitoring the Progression of Colorectal Cancer.

    Directory of Open Access Journals (Sweden)

    Jiaolin Zhou

    Full Text Available Liquid biopsy has been proposed to be a promising noninvasive tool to obtain information on tumor progression. Through a clinical observation of a case series of 6 consecutive patients, we aim to determine the value of circulating tumor DNA (ctDNA for monitoring the tumor burden during the treatment of colorectal cancer (CRC.We used capture sequencing of 545 genes to identify somatic alternations in primary tumor tissues of the six CRC patients who underwent radical surgery and in 23 plasma samples collected at serial time points. We compared the mutation patterns and variant allele frequencies (VAFs between the matched tissue and the plasma samples and evaluated the potential advantage of using ctDNA as a better tumor load indicator to detect disease relapse over carcinoembryonic antigen (CEA, cancer antigen (CA 19-9 and imaging studies.We identified low-frequency mutations with a mean VAF of 0.88% (corresponding to a mean tumor burden of 0.20ng/mL in the preoperative plasmas of four patients with locally advanced CRC and a subset of mutations shared by their primary tumors. The tumor loads appeared a sudden decrease upon surgery or other adjuvant treatments and then generally maintained at low levels (0.092ng/mL until disease recurred. ctDNA increased by 13-fold when disease relapsed in one patient while the CEA and CA 19-9 levels remained normal. In this patient, all six somatic mutations identified in the preoperative plasma were detected in the recrudescent plasma again, with five mutations showing allele fraction increase.We described a multi-time-point profile of ctDNA of CRC patients during the course of comprehensive treatment and observed a correlation of ctDNA level with the clinically evaluated tumor progression. This demonstrated a new strategy by analyzing the heterogeneous ctDNA to evaluate and monitor the tumor burden in the treatment and follow-up of CRC patients, with potentially better potency than conventional biomarkers.

  11. The ability of human nuclear DNA to cause false positive low-abundance heteroplasmy calls varies across the mitochondrial genome.

    Science.gov (United States)

    Albayrak, Levent; Khanipov, Kamil; Pimenova, Maria; Golovko, George; Rojas, Mark; Pavlidis, Ioannis; Chumakov, Sergei; Aguilar, Gerardo; Chávez, Arturo; Widger, William R; Fofanov, Yuriy

    2016-12-12

    Low-abundance mutations in mitochondrial populations (mutations with minor allele frequency ≤ 1%), are associated with cancer, aging, and neurodegenerative disorders. While recent progress in high-throughput sequencing technology has significantly improved the heteroplasmy identification process, the ability of this technology to detect low-abundance mutations can be affected by the presence of similar sequences originating from nuclear DNA (nDNA). To determine to what extent nDNA can cause false positive low-abundance heteroplasmy calls, we have identified mitochondrial locations of all subsequences that are common or similar (one mismatch allowed) between nDNA and mitochondrial DNA (mtDNA). Performed analysis revealed up to a 25-fold variation in the lengths of longest common and longest similar (one mismatch allowed) subsequences across the mitochondrial genome. The size of the longest subsequences shared between nDNA and mtDNA in several regions of the mitochondrial genome were found to be as low as 11 bases, which not only allows using these regions to design new, very specific PCR primers, but also supports the hypothesis of the non-random introduction of mtDNA into the human nuclear DNA. Analysis of the mitochondrial locations of the subsequences shared between nDNA and mtDNA suggested that even very short (36 bases) single-end sequencing reads can be used to identify low-abundance variation in 20.4% of the mitochondrial genome. For longer (76 and 150 bases) reads, the proportion of the mitochondrial genome where nDNA presence will not interfere found to be 44.5 and 67.9%, when low-abundance mutations at 100% of locations can be identified using 417 bases long single reads. This observation suggests that the analysis of low-abundance variations in mitochondria population can be extended to a variety of large data collections such as NCBI Sequence Read Archive, European Nucleotide Archive, The Cancer Genome Atlas, and International Cancer Genome

  12. Human Exploration Systems and Mobility Capability Roadmap Progress Review

    Science.gov (United States)

    Culbert, Chris; Taylor, Jeff

    2005-01-01

    Contents include the following: Capability Roadmap Team. Capability Description and Capability Breakdown Structure. Benefits of the Human Systems and Mobility Capability. Roadmap Process and Approach. Drivers and Assumptions for the whole team. Current State-of-the-Art, Assumptions and Requirements will be covered in the appropriate sections. Capability Presentations by Leads under Roadmap (Repeated for each capability under roadmap). Capability Description, Benefits, Current State-of-the-Art. Capability Requirements and Assumptions. Roadmap for Capability. Capability Readiness Level. Technology Readiness Level. Figures of Merit. Summary of Top Level Capability. Significant Technical Challenges. Summary and Forward Work.

  13. Human imprinting disorders: Principles, practice, problems and progress.

    Science.gov (United States)

    Mackay, Deborah J G; Temple, I Karen

    2017-11-01

    Epigenetic regulation orchestrates gene expression with exquisite precision, over a huge dynamic range and across developmental space and time, permitting genomically-homogeneous humans to develop and adapt to their surroundings. Every generation, these epigenetic marks are re-set twice: in the germline, to enable differentiation of sperm and eggs, and at fertilisation, to create the totipotent zygote that then begins growth and differentiation into a new human. A small group of genes evades the second, zygotic wave of epigenetic reprogramming, and these genes retain an epigenetic 'imprint' of the parent from whom they were inherited. Imprinted genes are (as a general rule) expressed from one parental allele only. Some imprinted genes are critical regulators of growth and development, and thus disruption of their normal monoallelic expression causes congenital imprinting disorders, with clinical features impacting growth, development, behaviour and metabolism. Imprinting disorders as a group have characteristics that challenge diagnosis and management, including clinical and molecular heterogeneity, overlapping clinical features, somatic mosaicism, and multi-locus involvement. New insights into the biology and epigenomics of the early embryo offers new clues about the origin and importance of imprinting disorders. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  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. Prevalence of human papillomavirus DNA in female cervical lesions from Rio de Janeiro, Brazil

    Directory of Open Access Journals (Sweden)

    S. M. B. Cavalcanti

    1994-12-01

    Full Text Available A hundred-sixty paraffin-embedded specimens from female cervical lesions were examined for human papillomavirus (HPV types 6, 11, 16 and 18 infections by non-isotopic in situ hybridization. The data were compared with histologic diagnosis. Eighty-eight (55 biopsies contained HPV DNA sequences. In low grade cervical intraepithelial neoplasias (CIN I, HPV infection was detected in 78.7 of the cases, the benign HPV 6 was the most prevalent type. HPV DNA was detected in 58 of CIN II and CIN III cases and in 41.8 of squamous cell carcinomas (SCC. Histologically normal women presented 20 of HPV infection. Oncogenic HPV was found in 10 of these cases, what may indicate a higher risk of developing CINs and cancer. Twenty-five percent of the infected tissues contained mixed infections. HPV 16 was the most common type infecting the cervix and its prevalence raised significantly with the severity of the lesions, pointing its role in cancer pathogenesis. White women presented twice the cervical lesions of mulatto and African origin women, although HPV infection rates were nearly the same for the three groups (approximately 50. Our results showed that HPV typing by in situ hybridization is a useful tool for distinguishing between low and high risk cervical lesions. Further studies are required to elucidate risk factors associated with HPV infection and progression to malignancy in Brazilian population.

  16. Chimeric DNA Vaccines against ErbB2{sup +} Carcinomas: From Mice to Humans

    Energy Technology Data Exchange (ETDEWEB)

    Quaglino, Elena; Riccardo, Federica; Macagno, Marco; Bandini, Silvio; Cojoca, Rodica; Ercole, Elisabetta [Molecular Biotechnology Center, Department of Clinical and Biological Sciences, University of Turin, 10126 Turin (Italy); Amici, Augusto [Department of Molecular Cellular and Animal Biology, University of Camerino, 62032 Camerino (Italy); Cavallo, Federica, E-mail: federica.cavallo@unito.it [2 Department of Molecular Cellular and Animal Biology, University of Camerino, 62032 Camerino (Italy)

    2011-08-10

    DNA vaccination exploits a relatively simple and flexible technique to generate an immune response against microbial and tumor-associated antigens (TAAs). Its effectiveness is enhanced by the application of an electrical shock in the area of plasmid injection (electroporation). In our studies we exploited a sophisticated electroporation device approved for clinical use (Cliniporator, IGEA, Carpi, Italy). As the target antigen is an additional factor that dramatically modulates the efficacy of a vaccine, we selected ErbB2 receptor as a target since it is an ideal oncoantigen. It is overexpressed on the cell membrane by several carcinomas for which it plays an essential role in driving their progression. Most oncoantigens are self-tolerated molecules. To circumvent immune tolerance we generated two plasmids (RHuT and HuRT) coding for chimeric rat/human ErbB2 proteins. Their immunogenicity was compared in wild type mice naturally tolerant for mouse ErbB2, and in transgenic mice that are also tolerant for rat or human ErbB2. In several of these mice, RHuT and HuRT elicited a stronger anti-tumor response than plasmids coding for fully human or fully rat ErbB2. The ability of heterologous moiety to blunt immune tolerance could be exploited to elicit a significant immune response in patients. A clinical trial to delay the recurrence of ErbB2{sup +} carcinomas of the oral cavity, oropharynx and hypopharynx is awaiting the approval of the Italian authorities.

  17. Single-stranded DNA catenation mediated by human EVL and a type I topoisomerase

    Science.gov (United States)

    Takaku, Motoki; Takahashi, Daisuke; Machida, Shinichi; Ueno, Hiroyuki; Hosoya, Noriko; Ikawa, Shukuko; Miyagawa, Kiyoshi; Shibata, Takehiko; Kurumizaka, Hitoshi

    2010-01-01

    The human Ena/Vasp-like (EVL) protein is considered to be a bifunctional protein, involved in both actin remodeling and homologous recombination. In the present study, we found that human EVL forms heat-stable multimers of circular single-stranded DNA (ssDNA) molecules in the presence of a type I topoisomerase in vitro. An electron microscopic analysis revealed that the heat-stable ssDNA multimers formed by EVL and topoisomerase were ssDNA catemers. The ssDNA catenation did not occur when either EVL or topoisomerase was omitted from the reaction mixture. A deletion analysis revealed that the ssDNA catenation completely depended on the annealing activity of EVL. Human EVL was captured from a human cell extract by TOPO IIIα-conjugated beads, and the interaction between EVL and TOPO IIIα was confirmed by a surface plasmon resonance analysis. Purified TOPO IIIα catalyzed the ssDNA catenation with EVL as efficiently as the Escherichia coli topoisomerase I. Since the ssDNA cutting and rejoining reactions, which are the sub-steps of ssDNA catenation, may be an essential process in homologous recombination, EVL and TOPO IIIα may function in the processing of DNA intermediates formed during homologous recombination. PMID:20639531

  18. Structural and dynamical effects induced by the anticancer drug topotecan on the human topoisomerase I - DNA complex.

    Directory of Open Access Journals (Sweden)

    Giordano Mancini

    Full Text Available BACKGROUND: Human topoisomerase I catalyzes the relaxation of DNA supercoils in fundamental cell processes like transcription, replication and chromosomal segregation. It is the only target of the camptothecin family of anticancer drugs. Among these, topotecan has been used to treat lung and ovarian carcinoma for several years. Camptothecins reversibly binds to the covalent intermediate DNA-enzyme, stabilizing the cleavable complex and reducing the religation rate. The stalled complex then collides with the progression of the replication fork, producing lethal double strand DNA breaks and eventually cell death. METHODOLOGY/PRINCIPAL FINDINGS: Long lasting molecular dynamics simulations of the DNA-topoisomerase I binary complex and of the DNA-topoisomerase-topotecan ternary complex have been performed and compared. The conformational space sampled by the binary complex is reduced by the presence of the drug, as observed by principal component and cluster analyses. This conformational restraint is mainly due to the reduced flexibility of residues 633-643 (the region connecting the linker to the core domain that causes an overall mobility loss in the ternary complex linker domain. During the simulation, DNA/drug stacking interactions are fully maintained, and hydrogen bonds are maintained with the enzyme. Topotecan keeps the catalytic residue Lys532 far from the DNA, making it unable to participate to the religation reaction. Arg364 is observed to interact with both the B and E rings of topotecan with two stable direct hydrogen bonds. An interesting constrain exerted by the protein on the geometrical arrangement of topotecan is also observed. CONCLUSIONS/SIGNIFICANCE: Atomistic-scale understanding of topotecan interactions with the DNA-enzyme complex is fundamental to the explaining of its poisonous effect and of the drug resistance observed in several single residue topoisomerase mutants. We observed significant alterations due to topotecan in

  19. The human papillomavirus DNA helicase E1 binds, stimulates, and confers processivity to cellular DNA polymerase epsilon

    Science.gov (United States)

    Chojnacki, Michaelle

    2018-01-01

    Abstract The papillomavirus (PV) helicase protein E1 recruits components of the cellular DNA replication machinery to the PV replication fork, such as Replication Protein A (RPA), DNA polymerase α-primase (pol α) and topoisomerase I (topo I). Here we show that E1 binds to DNA polymerase ϵ (pol ϵ) and dramatically stimulates the DNA synthesis activity of pol ϵ. This stimulation of pol ϵ by E1 is highly specific and occurs even in the absence of the known pol ϵ cofactors Replication Factor C (RFC), Proliferating Cell Nuclear Antigen (PCNA) and RPA. This stimulation is due to an increase in the processivity of pol ϵ and occurs independently of pol ϵ’s replication cofactors. This increase in processivity is dependent on the ability of the E1 helicase to hydrolyze ATP, suggesting it is dependent on E1’s helicase action. In addition, RPA, thought to be vital for processive DNA synthesis by both pol ϵ and pol δ, was found to be dispensable for processive synthesis by pol ϵ in the presence of E1. Overall, E1 appears to be conferring processivity to pol ϵ by directly tethering pol ϵ to the DNA parental strand and towing ϵ behind the E1 helicase as the replication fork progresses; and thereby apparently obviating the need for RPA for leading strand synthesis. Thus far only pol α and pol δ have been implicated in the DNA replication of mammalian viruses; this is the first reported example of a virus recruiting pol ϵ. Furthermore, this demonstrates a unique capacity of a viral helicase having evolved to stimulate a cellular replicative DNA polymerase. PMID:29155954

  20. Primer in Genetics and Genomics, Article 3-Explaining Human Diversity: The Role of DNA.

    Science.gov (United States)

    Read, Catherine Y

    2017-05-01

    Genetic variation lays the foundation for diversity and enables humans to adapt to changing environments. The order of the nucleotides adenine, guanine, cytosine, and thymine on the deoxyribonucleic acid (DNA) molecules of the nuclear chromosomes and mitochondrial DNA (mtDNA) plays an important role in normal cell division, tissue development, and reproduction but is susceptible to alteration from a large number of random, inherited, or environmental events. Variations can range from a change in a single nucleotide to duplication of entire chromosomes. Single nucleotide polymorphisms are the major source of human heterogeneity. Other variations that can alter phenotypes and adversely impact growth, development, and health include copy number variations, aneuploidies, and structural alterations such as deletions, translocations, inversions, duplications, insertions, or mutations in mtDNA. In addition, DNA rearrangements in somatic cells underlie the uncontrolled cell growth found in cancer. This article explores the mechanisms by which variations in DNA arise and the impact those changes can have on human health.

  1. 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.

  2. Genotyping human ancient mtDNA control and coding region polymorphisms with a multiplexed Single-Base-Extension assay: the singular maternal history of the Tyrolean Iceman

    Directory of Open Access Journals (Sweden)

    Egarter-Vigl Eduard

    2009-06-01

    Full Text Available Abstract Background Progress in the field of human ancient DNA studies has been severely restricted due to the myriad sources of potential contamination, and because of the pronounced difficulty in identifying authentic results. Improving the robustness of human aDNA results is a necessary pre-requisite to vigorously testing hypotheses about human evolution in Europe, including possible admixture with Neanderthals. This study approaches the problem of distinguishing between authentic and contaminating sequences from common European mtDNA haplogroups by applying a multiplexed Single-Base-Extension assay, containing both control and coding region sites, to DNA extracted from the Tyrolean Iceman. Results The multiplex assay developed for this study was able to confirm that the Iceman's mtDNA belongs to a new European mtDNA clade with a very limited distribution amongst modern data sets. Controlled contamination experiments show that the correct results are returned by the multiplex assay even in the presence of substantial amounts of exogenous DNA. The overall level of discrimination achieved by targeting both control and coding region polymorphisms in a single reaction provides a methodology capable of dealing with most cases of homoplasy prevalent in European haplogroups. Conclusion The new genotyping results for the Iceman confirm the extreme fallibility of human aDNA studies in general, even when authenticated by independent replication. The sensitivity and accuracy of the multiplex Single-Base-Extension methodology forms part of an emerging suite of alternative techniques for the accurate retrieval of ancient DNA sequences from both anatomically modern humans and Neanderthals. The contamination of laboratories remains a pressing concern in aDNA studies, both in the pre and post-PCR environments, and the adoption of a forensic style assessment of a priori risks would significantly improve the credibility of results.

  3. Genotyping human ancient mtDNA control and coding region polymorphisms with a multiplexed Single-Base-Extension assay: the singular maternal history of the Tyrolean Iceman.

    Science.gov (United States)

    Endicott, Phillip; Sanchez, Juan J; Pichler, Irene; Brotherton, Paul; Brooks, Jerome; Egarter-Vigl, Eduard; Cooper, Alan; Pramstaller, Peter

    2009-06-19

    Progress in the field of human ancient DNA studies has been severely restricted due to the myriad sources of potential contamination, and because of the pronounced difficulty in identifying authentic results. Improving the robustness of human aDNA results is a necessary pre-requisite to vigorously testing hypotheses about human evolution in Europe, including possible admixture with Neanderthals. This study approaches the problem of distinguishing between authentic and contaminating sequences from common European mtDNA haplogroups by applying a multiplexed Single-Base-Extension assay, containing both control and coding region sites, to DNA extracted from the Tyrolean Iceman. The multiplex assay developed for this study was able to confirm that the Iceman's mtDNA belongs to a new European mtDNA clade with a very limited distribution amongst modern data sets. Controlled contamination experiments show that the correct results are returned by the multiplex assay even in the presence of substantial amounts of exogenous DNA. The overall level of discrimination achieved by targeting both control and coding region polymorphisms in a single reaction provides a methodology capable of dealing with most cases of homoplasy prevalent in European haplogroups. The new genotyping results for the Iceman confirm the extreme fallibility of human aDNA studies in general, even when authenticated by independent replication. The sensitivity and accuracy of the multiplex Single-Base-Extension methodology forms part of an emerging suite of alternative techniques for the accurate retrieval of ancient DNA sequences from both anatomically modern humans and Neanderthals. The contamination of laboratories remains a pressing concern in aDNA studies, both in the pre and post-PCR environments, and the adoption of a forensic style assessment of a priori risks would significantly improve the credibility of results.

  4. 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.

  5. 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.

  6. Perspectives on the Holocene in Britain: human DNA

    Science.gov (United States)

    Evison, Martin P.

    1999-10-01

    Analysis of genetically-based variation in human populations has been possible for nearly a century. Recently, DNA sequence-based methods have begun to replace protein-based methods leading to a higher resolution, but more complex results. Patterns of gene distribution may be established with statistical reliability, but understanding them in terms of history is a process of interpretation; multi-disciplinary research is essential. A brief synopsis of the Holocene in Britain is presented, based on the genetic data , but with reference to archaeology, palaeoecology, ethnology and linguistics. The genetic history of Britain is a Holocene history. Patterns of gene distributions can be seen as the consequence of social processes and of selection. The major events are: the reoccupation of Britain in the Late Upper Palaeolithic/Early Mesolithic (c. 10,000-7,500 bp), social and demographic development during the Mesolithic (7,500-6,000 bp), and the Mesolithic-Neolithic transition (6,000-4,000 bp). Interpretations of the transition based on social interaction, intermarriage and minor genetic input are most consistent with the broad evidence. An extreme indigenist position is not supported.

  7. Reprogramming of human cancer cells to pluripotency for models of cancer progression

    Science.gov (United States)

    Kim, Jungsun; Zaret, Kenneth S

    2015-01-01

    The ability to study live cells as they progress through the stages of cancer provides the opportunity to discover dynamic networks underlying pathology, markers of early stages, and ways to assess therapeutics. Genetically engineered animal models of cancer, where it is possible to study the consequences of temporal-specific induction of oncogenes or deletion of tumor suppressors, have yielded major insights into cancer progression. Yet differences exist between animal and human cancers, such as in markers of progression and response to therapeutics. Thus, there is a need for human cell models of cancer progression. Most human cell models of cancer are based on tumor cell lines and xenografts of primary tumor cells that resemble the advanced tumor state, from which the cells were derived, and thus do not recapitulate disease progression. Yet a subset of cancer types have been reprogrammed to pluripotency or near-pluripotency by blastocyst injection, by somatic cell nuclear transfer and by induced pluripotent stem cell (iPS) technology. The reprogrammed cancer cells show that pluripotency can transiently dominate over the cancer phenotype. Diverse studies show that reprogrammed cancer cells can, in some cases, exhibit early-stage phenotypes reflective of only partial expression of the cancer genome. In one case, reprogrammed human pancreatic cancer cells have been shown to recapitulate stages of cancer progression, from early to late stages, thus providing a model for studying pancreatic cancer development in human cells where previously such could only be discerned from mouse models. We discuss these findings, the challenges in developing such models and their current limitations, and ways that iPS reprogramming may be enhanced to develop human cell models of cancer progression. PMID:25712212

  8. Licochalcone B Arrests Cell Cycle Progression and Induces Apoptosis in Human Breast Cancer MCF-7 Cells.

    Science.gov (United States)

    Yu, Lina; Ma, Jun; Han, Jichun; Wang, Bo; Chen, Xiaoyu; Gao, Caixia; Li, Defang; Zheng, Qiusheng

    2016-01-01

    Recent patent of licochalcone B (LCB) as an antiinflammatory agent has been developed. Emerging evidence shows that LCB may be a promising alternative compound with anti-cancer activities. However, the anticancer mechanism of LCB in MCF-7 cells has not been fully investigated. We aimed to unearth the anti-cancer effect and mechanism of LCB in MCF-7 cells. Cell proliferation activity and cell-cycle progression were determined by sulforhodamine B assay and flow cytometry, respectively. The mRNA and protein levels of cell cycle-related proteins and apoptosis-associated proteins were examined by RT-qPCR and western blot, respectively. Mitochondrial membrane potential (MMP) was measured by flow cytometry after JC-1 staining. We found that LCB inhibited MCF-7 cells proliferation in a concentration- and time-dependent manner. Moreover, LCB-treatment led to S phase arrest in MCF-7 cells, which could be elucidated by the decreased mRNA and protein levels of Cyclin A, Cdk2 and Cdc25 A, and the increased protein level of p21. LCB also induced such apoptosis morphology as phosphatidylserine externalization, chromatin condensation and DNA fragmentation. Moreover, LCB led to the loss of MMP, resulting in the release of cytochrome C. The above apoptotic events were supported by the fact that LCB upregulated the mRNA and protein levels of Caspase 3, Caspase 9 and Bax, and downregulated the mRNA and protein level of Bcl-2, which was triggered by the increased p53 protein level in LCB-treated MCF-7 cells. These findings suggested that LCB could be a promising agent for treatment of human breast cancer.

  9. Impact of HIV Type 1 DNA Levels on Spontaneous Disease Progression: A Meta-Analysis

    DEFF Research Database (Denmark)

    Tsiara, Chrissa G; Nikolopoulos, Georgios K; Bagos, Pantelis G

    2012-01-01

    , and to compare the prognostic information obtained by HIV-1 DNA with that derived from plasma HIV-1 RNA. Eligible articles were identified through a comprehensive search of Medline, ISI Web of Science, Scopus, and Google Scholar. The analysis included univariate and bivariate random-effects models...

  10. Sequencing of megabase plus DNA by hybridization: Method development ENT. Final technical progress report

    Energy Technology Data Exchange (ETDEWEB)

    Crkvenjakov, R.; Drmanac, R.

    1991-01-31

    Sequencing by hybridization (SBH) is the only sequencing method based on the experimental determination of the content of oligonucleotide sequences. The data acquisition relies on the natural process of base pairing. It is possible to determine the content of complementary oligosequences in the target DNA by the process of hybridization with oligonucleotide probes of known sequences.

  11. Roles of POLD4, smallest subunit of DNA polymerase {delta}, in nuclear structures and genomic stability of human cells

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Qin Miao [Division of Molecular Carcinogenesis, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya (Japan); Akashi, Tomohiro [Division of Molecular Mycology and Medicine, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya (Japan); Masuda, Yuji; Kamiya, Kenji [Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553 (Japan); Takahashi, Takashi [Division of Molecular Carcinogenesis, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya (Japan); Suzuki, Motoshi, E-mail: msuzuki@med.nagoya-u.ac.jp [Division of Molecular Carcinogenesis, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya (Japan)

    2010-01-01

    Mammalian DNA polymerase {delta} (pol {delta}) is essential for DNA replication, though the functions of this smallest subunit of POLD4 have been elusive. We investigated pol {delta} activities in vitro and found that it was less active in the absence of POLD4, irrespective of the presence of the accessory protein PCNA. shRNA-mediated reduction of POLD4 resulted in a marked decrease in colony formation activity by Calu6, ACC-LC-319, and PC-10 cells. We also found that POLD4 reduction was associated with an increased population of karyomere-like cells, which may be an indication of DNA replication stress and/or DNA damage. The karyomere-like cells retained an ability to progress through the cell cycle, suggesting that POLD4 reduction induces modest genomic instability, while allowing cells to grow until DNA damage reaches an intolerant level. Our results indicate that POLD4 is required for the in vitro pol {delta} activity, and that it functions in cell proliferation and maintenance of genomic stability of human cells.

  12. DNA alterations in Cd133+ and Cd133- tumour cells enriched from intra-operative human colon tumour biopsies.

    Science.gov (United States)

    Cervantes-Madrid, Diana; Wettergren, Yvonne; Falk, Peter; Lundholm, Kent; Asting, Annika G

    2017-03-27

    Tumour stem cells are considered important to promote disease progression, recurrence and treatment resistance following chemotherapy in colon cancer. However, genomic analyses of colorectal cancer have mainly been performed on integrated tumour tissue consisting of several different cell types in addition to differentiated tumour cells. The purpose of the present study was to compare genomic alterations in two cell fractions enriched of CD133+ and CD133-/EpCAM+ cells, respectively, obtained from fresh intraoperative human tumour biopsies. The tumour biopsies were fractionated into CD133+ and CD133-/EpCAM+ cells by immunomagnetic separation, confirmed by immunocytochemistry and Q-PCR. DNA were extracted and used for array comparative genome hybridization (aCGH) after whole genome amplification. Frozen tumour tissue biopsies were used for DNA/RNA extraction and Q-PCR analyses to check for DNA alterations detected in the cell fractions. The number and size of DNA alterations were equally distributed across the cell fractions; however, large deletions were detected on chromosome 1, 7 and 19 in CD133-/EpCAM+ cells. Deletions were frequent in both cell fractions and a deletion on chromosome 19p was confirmed in 90% of the patients. Isolation of enriched cells derived from tumour tissue revealed mainly genomic deletions, which were not observed in tumour tissue DNA analyses. CD133+ cells were genetically heterogeneous among patients without any defined profile compared to CD133-/EpCAM+ cells.

  13. Complex forms of mitochondrial DNA in human B cells transformed by Epstein-Barr virus (EBV)

    DEFF Research Database (Denmark)

    Christiansen, Gunna; Christiansen, C; Zeuthen, J

    1983-01-01

    Human lymphocytes and lymphoid cell lines were analyzed for the presence of complex forms of mitochondrial DNA (mtDNA) by electron microscopy. A high frequency (9%-14.5%) of catenated dimers, circular dimers, or oligomers were found in samples from Epstein-Barr-virus-(EBV) transformed lymphoblast......Human lymphocytes and lymphoid cell lines were analyzed for the presence of complex forms of mitochondrial DNA (mtDNA) by electron microscopy. A high frequency (9%-14.5%) of catenated dimers, circular dimers, or oligomers were found in samples from Epstein-Barr-virus-(EBV) transformed...

  14. 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

    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......-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....

  15. Prevalence of human papillomavirus and Epstein-Barr virus DNA in penile cancer cases from Brazil

    Directory of Open Access Journals (Sweden)

    Larissa Alves Afonso

    2012-02-01

    Full Text Available Penile cancer is a potentially mutilating disease. Although its occurrence is relatively rare worldwide, penile cancer rates can be high in developing countries. A few studies have been conducted on the involvement of human papillomavirus (HPV in penile carcinoma, which have found HPV present in 30-70% of penile malignant lesions, with a higher prevalence of HPV 16 and 18. It has been assumed that cofactors, such as Epstein-Barr virus (EBV infections, may play a role in the progression of penile neoplasia. The aim of this study was to determine HPV and EBV prevalence in 135 penile malignant lesions from Brazilian men through the use of MY09/11 polymerase chain reaction (PCR, type-specific PCR and restriction fragment length polymorphism analysis. HPV prevalence among the men tested was 60.7%. Of the men who tested positive, 27 presented with HPV 16 (29.7%, five with HPV 18 (5.5%, 21 with HPV 45 (23.1% and nine with HPV 6 (9.9%. Seven mixed infections were detected (9.2%, while 11 cases remained untyped (13.4%. Regarding EBV positivity, 46.7% of the samples contained EBV DNA with EBV-1 as the most prevalent type (74.6%. More than 23% of the men were co-infected with both HPV and EBV, while 35% presented exclusively with HPV DNA and 20% presented only with EBV DNA. Penile carcinoma aetiology has not been fully elucidated and the role of HPV and EBV infections individually or synergistically is still controversial. Hence, more studies are needed to determine their possible role in carcinogenesis.

  16. DNA excision repair in cell extracts from human cell lines exhibiting hypersensitivity to DNA-damaging agents

    Energy Technology Data Exchange (ETDEWEB)

    Hansson, J.; Keyse, S.M.; Lindahl, T.; Wood, R.D. (Imperial Cancer Research Fund, South Mimms, (United Kingdom))

    1991-07-01

    Whole cell extracts from human lymphoid cell lines can perform in vitro DNA repair synthesis in plasmids damaged by agents including UV or cis-diamminedichloroplatinum(II) (cis-DDP). Extracts from xeroderma pigmentosum (XP) cells are defective in repair synthesis. We have now studied in vitro DNA repair synthesis using extracts from lymphoblastoid cell lines representing four human hereditary syndromes with increased sensitivity to DNA-damaging agents. Extracts of cell lines from individuals with the sunlight-sensitive disorders dysplastic nevus syndrome or Cockayne's syndrome (complementation groups A and B) showed normal DNA repair synthesis in plasmids with UV photoproducts. This is consistent with in vivo measurements of the overall DNA repair capacity in such cell lines. A number of extracts were prepared from two cell lines representing the variant form of XP (XP-V). Half of the extracts prepared showed normal levels of in vitro DNA repair synthesis in plasmids containing UV lesions, but the remainder of the extracts from the same cell lines showed deficient repair synthesis, suggesting the possibility of an unusually labile excision repair protein in XP-V. Fanconi's anemia (FA) cells show cellular hypersensitivity to cross-linking agents including cis-DDP. Extracts from cell lines belonging to two different complementation groups of FA showed normal DNA repair synthesis in plasmids containing cis-DDP or UV adducts. Thus, there does not appear to be an overall excision repair defect in FA, but the data do not exclude a defect in the repair of interstrand DNA cross-links.

  17. Comparison of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens

    Directory of Open Access Journals (Sweden)

    Nakatsu Cindy H

    2010-05-01

    Full Text Available Abstract Background The influence of diet on intestinal microflora has been investigated mainly using conventional microbiological approaches. Although these studies have advanced knowledge on human intestinal microflora, it is imperative that new methods are applied to facilitate scientific progress. Culture-independent molecular fingerprinting method of Polymerase Chain Reaction and Denaturing Gradient Gel Electrophoresis (PCR-DGGE has been used to study microbial communities in a variety of environmental samples. However, these protocols must be optimized prior to their application in order to enhance the quality and accuracy of downstream analyses. In this study, the relative efficacy of four commercial DNA extraction kits (Mobio Ultra Clean® Fecal DNA Isolation Kit, M; QIAamp® DNA Stool Mini Kit, Q; FastDNA® SPIN Kit, FSp; FastDNA® SPIN Kit for Soil, FSo were evaluated. Further, PCR-DGGE technique was also assessed for its feasibility in detecting differences in human intestinal bacterial fingerprint profiles. Method Total DNA was extracted from varying weights of human fecal specimens using four different kits, followed by PCR amplification of bacterial 16S rRNA genes, and DGGE separation of the amplicons. Results Regardless of kit, maximum DNA yield was obtained using 10 to 50 mg (wet wt of fecal specimens and similar DGGE profiles were obtained. However, kits FSp and FSo extracted significantly larger amounts of DNA per g dry fecal specimens and produced more bands on their DGGE profiles than kits M and Q due to their use of bead-containing lysing matrix and vigorous shaking step. DGGE of 16S rRNA gene PCR products was suitable for capturing the profiles of human intestinal microbial community and enabled rapid comparative assessment of inter- and intra-subject differences. Conclusion We conclude that extraction kits that incorporated bead-containing lysing matrix and vigorous shaking produced high quality DNA from human fecal

  18. 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

  19. Mitochondrial DNA copy number variation across human cancers.

    Science.gov (United States)

    Reznik, Ed; Miller, Martin L; Şenbabaoğlu, Yasin; Riaz, Nadeem; Sarungbam, Judy; Tickoo, Satish K; Al-Ahmadie, Hikmat A; Lee, William; Seshan, Venkatraman E; Hakimi, A Ari; Sander, Chris

    2016-02-22

    Mutations, deletions, and changes in copy number of mitochondrial DNA (mtDNA), are observed throughout cancers. Here, we survey mtDNA copy number variation across 22 tumor types profiled by The Cancer Genome Atlas project. We observe a tendency for some cancers, especially of the bladder, breast, and kidney, to be depleted of mtDNA, relative to matched normal tissue. Analysis of genetic context reveals an association between incidence of several somatic alterations, including IDH1 mutations in gliomas, and mtDNA content. In some but not all cancer types, mtDNA content is correlated with the expression of respiratory genes, and anti-correlated to the expression of immune response and cell-cycle genes. In tandem with immunohistochemical evidence, we find that some tumors may compensate for mtDNA depletion to sustain levels of respiratory proteins. Our results highlight the extent of mtDNA copy number variation in tumors and point to related therapeutic opportunities.

  20. Polymorphisms in human DNA repair genes and head and neck ...

    Indian Academy of Sciences (India)

    Genetic polymorphisms in some DNA repair proteins are associated with a number of malignant transformations like head and neck squamous cell carcinoma (HNSCC). Xeroderma pigmentosum group D (XPD) and X-ray repair cross-complementing proteins 1 (XRCC1) and 3 (XRCC3) genes are involved in DNA repair ...

  1. Quantification and presence of human ancient DNA in burial place ...

    African Journals Online (AJOL)

    ... was shown to consists of intact DNA that is virtually free of RNA, resulting in a more accurate representation of gene expression using RTPCR and PCR amplification methods. In this study, the results demonstrate that RT-PCR method can be useful for the improved ancient DNA extraction in anthropology and archeology.

  2. DNA molecules and human therapeutics | Danquah | African Journal ...

    African Journals Online (AJOL)

    Nucleic acid molecules are championing a new generation of reverse engineered biopharmaceuticals. In terms of potential application in gene medicine, plasmid DNA (pDNA) vectors have exceptional therapeutic and immunological profiles as they are free from safety concerns associated with viral vectors, display ...

  3. 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

    The present study investigated the effect of a single bout of exhaustive exercise on the generation of DNA strand breaks and oxidative DNA damage under normal conditions and at high-altitude hypoxia (4559 meters for 3 days). Twelve healthy subjects performed a maximal bicycle exercise test; lymph...

  4. Sunlight-induced DNA damage in human mononuclear cells

    DEFF Research Database (Denmark)

    Møller, Peter; Wallin, Hakan; Holst, Erik

    2002-01-01

    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...... 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...

  5. Ki-67 Contributes to Normal Cell Cycle Progression and Inactive X Heterochromatin in p21 Checkpoint-Proficient Human Cells.

    Science.gov (United States)

    Sun, Xiaoming; Bizhanova, Aizhan; Matheson, Timothy D; Yu, Jun; Zhu, Lihua Julie; Kaufman, Paul D

    2017-09-01

    The Ki-67 protein is widely used as a tumor proliferation marker. However, whether Ki-67 affects cell cycle progression has been controversial. Here we demonstrate that depletion of Ki-67 in human hTERT-RPE1, WI-38, IMR90, and hTERT-BJ cell lines and primary fibroblast cells slowed entry into S phase and coordinately downregulated genes related to DNA replication. Some gene expression changes were partially relieved in Ki-67-depleted hTERT-RPE1 cells by codepletion of the Rb checkpoint protein, but more thorough suppression of the transcriptional and cell cycle defects was observed upon depletion of the cell cycle inhibitor p21. Notably, induction of p21 upon depletion of Ki-67 was a consistent hallmark of cell types in which transcription and cell cycle distribution were sensitive to Ki-67; these responses were absent in cells that did not induce p21. Furthermore, upon Ki-67 depletion, a subset of inactive X (Xi) chromosomes in female hTERT-RPE1 cells displayed several features of compromised heterochromatin maintenance, including decreased H3K27me3 and H4K20me1 labeling. These chromatin alterations were limited to Xi chromosomes localized away from the nuclear lamina and were not observed in checkpoint-deficient 293T cells. Altogether, our results indicate that Ki-67 integrates normal S-phase progression and Xi heterochromatin maintenance in p21 checkpoint-proficient human cells. Copyright © 2017 American Society for Microbiology.

  6. Lead Exposure Disrupts Global DNA Methylation in Human Embryonic Stem Cells and Alters Their Neuronal Differentiation

    Science.gov (United States)

    Senut, Marie-Claude; Sen, Arko; Cingolani, Pablo; Shaik, Asra; Land, Susan J.; Ruden, Douglas M.

    2014-01-01

    Exposure to lead (Pb) during childhood can result in learning disabilities and behavioral problems. Although described in animal models, whether Pb exposure also alters neuronal differentiation in the developing brains of exposed children is unknown. Here, we investigated the effects of physiologically relevant concentrations of Pb (from 0.4 to 1.9μM) on the capacity of human embryonic stem cells (hESCs) to progress to a neuronal fate. We found that neither acute nor chronic exposure to Pb prevented hESCs from generating neural progenitor cells (NPCs). NPCs derived from hESCs chronically exposed to 1.9μM Pb throughout the neural differentiation process generated 2.5 times more TUJ1-positive neurons than those derived from control hESCs. Pb exposure of hESCs during the stage of neural rosette formation resulted in a significant decrease in the expression levels of the neural marker genes PAX6 and MSI1. Furthermore, the resulting NPCs differentiated into neurons with shorter neurites and less branching than control neurons, as assessed by Sholl analysis. DNA methylation studies of control, acutely treated hESCs and NPCs derived from chronically exposed hESCs using the Illumina HumanMethylation450 BeadChip demonstrated that Pb exposure induced changes in the methylation status of genes involved in neurogenetic signaling pathways. In summary, our study shows that exposure to Pb subtly alters the neuronal differentiation of exposed hESCs and that these changes could be partly mediated by modifications in the DNA methylation status of genes crucial to brain development. PMID:24519525

  7. DNA damage and gene therapy of xeroderma pigmentosum, a human DNA repair-deficient disease

    Energy Technology Data Exchange (ETDEWEB)

    Dupuy, Aurélie [Laboratory of Genetic Instability and Oncogenesis UMR8200CNRS, Institut Gustave Roussy and University Paris-Sud, Villejuif (France); Sarasin, Alain, E-mail: alain.sarasin@gustaveroussy.fr [Laboratory of Genetic Instability and Oncogenesis UMR8200CNRS, Institut Gustave Roussy and University Paris-Sud, Villejuif (France); Service de Génétique, Institut Gustave Roussy (France)

    2015-06-15

    Graphical abstract: - Highlights: • Full correction of mutation in the XPC gene by engineered nucleases. • Meganucleases and TALENs are inhibited by 5-MeC for inducing double strand breaks. • Gene therapy of XP cells is possible using homologous recombination for DSB repair. - Abstract: Xeroderma pigmentosum (XP) is a genetic disease characterized by hypersensitivity to ultra-violet and a very high risk of skin cancer induction on exposed body sites. This syndrome is caused by germinal mutations on nucleotide excision repair genes. No cure is available for these patients except a complete protection from all types of UV radiations. We reviewed the various techniques to complement or to correct the genetic defect in XP cells. We, particularly, developed the correction of XP-C skin cells using the fidelity of the homologous recombination pathway during repair of double-strand break (DSB) in the presence of XPC wild type sequences. We used engineered nucleases (meganuclease or TALE nuclease) to induce a DSB located at 90 bp of the mutation to be corrected. Expression of specific TALE nuclease in the presence of a repair matrix containing a long stretch of homologous wild type XPC sequences allowed us a successful gene correction of the original TG deletion found in numerous North African XP patients. Some engineered nucleases are sensitive to epigenetic modifications, such as cytosine methylation. In case of methylated sequences to be corrected, modified nucleases or demethylation of the whole genome should be envisaged. Overall, we showed that specifically-designed TALE-nuclease allowed us to correct a 2 bp deletion in the XPC gene leading to patient's cells proficient for DNA repair and showing normal UV-sensitivity. The corrected gene is still in the same position in the human genome and under the regulation of its physiological promoter. This result is a first step toward gene therapy in XP patients.

  8. A checklist of the bats of Peninsular Malaysia and progress towards a DNA barcode reference library.

    Directory of Open Access Journals (Sweden)

    Voon-Ching Lim

    Full Text Available Several published checklists of bat species have covered Peninsular Malaysia as part of a broader region and/or in combination with other mammal groups. Other researchers have produced comprehensive checklists for specific localities within the peninsula. To our knowledge, a comprehensive checklist of bats specifically for the entire geopolitical region of Peninsular Malaysia has never been published, yet knowing which species are present in Peninsular Malaysia and their distributions across the region are crucial in developing suitable conservation plans. Our literature search revealed that 110 bat species have been documented in Peninsular Malaysia; 105 species have precise locality records while five species lack recent and/or precise locality records. We retrieved 18 species from records dated before the year 2000 and seven species have only ever been recorded once. Our search of Barcode of Life Datasystems (BOLD found that 86 (of the 110 species have public records of which 48 species have public DNA barcodes available from bats sampled in Peninsular Malaysia. Based on Neighbour-Joining tree analyses and the allocation of DNA barcodes to Barcode Index Number system (BINs by BOLD, several DNA barcodes recorded under the same species name are likely to represent distinct taxa. We discuss these cases in detail and highlight the importance of further surveys to determine the occurences and resolve the taxonomy of particular bat species in Peninsular Malaysia, with implications for conservation priorities.

  9. Human gamma-satellite DNA maintains open chromatin structure and protects a transgene from epigenetic silencing.

    Science.gov (United States)

    Kim, Jung-Hyun; Ebersole, Thomas; Kouprina, Natalay; Noskov, Vladimir N; Ohzeki, Jun-Ichirou; Masumoto, Hiroshi; Mravinac, Brankica; Sullivan, Beth A; Pavlicek, Adam; Dovat, Sinisa; Pack, Svetlana D; Kwon, Yoo-Wook; Flanagan, Patrick T; Loukinov, Dmitri; Lobanenkov, Victor; Larionov, Vladimir

    2009-04-01

    The role of repetitive DNA sequences in pericentromeric regions with respect to kinetochore/heterochromatin structure and function is poorly understood. Here, we use a mouse erythroleukemia cell (MEL) system for studying how repetitive DNA assumes or is assembled into different chromatin structures. We show that human gamma-satellite DNA arrays allow a transcriptionally permissive chromatin conformation in an adjacent transgene and efficiently protect it from epigenetic silencing. These arrays contain CTCF and Ikaros binding sites. In MEL cells, this gamma-satellite DNA activity depends on binding of Ikaros proteins involved in differentiation along the hematopoietic pathway. Given our discovery of gamma-satellite DNA in pericentromeric regions of most human chromosomes and a dynamic chromatin state of gamma-satellite arrays in their natural location, we suggest that gamma-satellite DNA represents a unique region of the functional centromere with a possible role in preventing heterochromatin spreading beyond the pericentromeric region.

  10. Use of laptop computers connected to internet through Wi-Fi decreases human sperm motility and increases sperm DNA fragmentation.

    Science.gov (United States)

    Avendaño, Conrado; Mata, Ariela; Sanchez Sarmiento, César A; Doncel, Gustavo F

    2012-01-01

    To evaluate the effects of laptop computers connected to local area networks wirelessly (Wi-Fi) on human spermatozoa. Prospective in vitro study. Center for reproductive medicine. Semen samples from 29 healthy donors. Motile sperm were selected by swim up. Each sperm suspension was divided into two aliquots. One sperm aliquot (experimental) from each patient was exposed to an internet-connected laptop by Wi-Fi for 4 hours, whereas the second aliquot (unexposed) was used as control, incubated under identical conditions without being exposed to the laptop. Evaluation of sperm motility, viability, and DNA fragmentation. Donor sperm samples, mostly normozoospermic, exposed ex vivo during 4 hours to a wireless internet-connected laptop showed a significant decrease in progressive sperm motility and an increase in sperm DNA fragmentation. Levels of dead sperm showed no significant differences between the two groups. To our knowledge, this is the first study to evaluate the direct impact of laptop use on human spermatozoa. Ex vivo exposure of human spermatozoa to a wireless internet-connected laptop decreased motility and induced DNA fragmentation by a nonthermal effect. We speculate that keeping a laptop connected wirelessly to the internet on the lap near the testes may result in decreased male fertility. Further in vitro and in vivo studies are needed to prove this contention. Copyright © 2012 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  11. DNA Catenation Maintains Structure of Human Metaphase Chromosomes

    DEFF Research Database (Denmark)

    L. V. Bauer, David; Marie, Rodolphe; Rasmussen, Kristian Hagsted

    2012-01-01

    Mitotic chromosome structure is pivotal to cell division but difficult to observe in fine detail using conventional methods. DNA catenation has been implicated in both sister chromatid cohesion and chromosome condensation, but has never been observed directly. We have used a lab-on-a-chip microfl......Mitotic chromosome structure is pivotal to cell division but difficult to observe in fine detail using conventional methods. DNA catenation has been implicated in both sister chromatid cohesion and chromosome condensation, but has never been observed directly. We have used a lab......-on-a-chip microfluidic device and fluorescence microscopy, coupled with a simple image analysis pipeline, to digest chromosomal proteins and examine the structure of the remaining DNA, which maintains the canonical ‘X’ shape. By directly staining DNA, we observe that DNA catenation between sister chromatids (separated...... by fluid flow) is composed of distinct fibres of DNA concentrated at the centromeres. Disrupting the catenation of the chromosomes with Topoisomerase IIa significantly alters overall chromosome shape, suggesting that DNA catenation must be simultaneously maintained for correct chromosome condensation...

  12. Forensic DNA Phenotyping: Predicting human appearance from crime scene material for investigative purposes.

    Science.gov (United States)

    Kayser, Manfred

    2015-09-01

    Forensic DNA Phenotyping refers to the prediction of appearance traits of unknown sample donors, or unknown deceased (missing) persons, directly from biological materials found at the scene. "Biological witness" outcomes of Forensic DNA Phenotyping can provide investigative leads to trace unknown persons, who are unidentifiable with current comparative DNA profiling. This intelligence application of DNA marks a substantially different forensic use of genetic material rather than that of current DNA profiling presented in the courtroom. Currently, group-specific pigmentation traits are already predictable from DNA with reasonably high accuracies, while several other externally visible characteristics are under genetic investigation. Until individual-specific appearance becomes accurately predictable from DNA, conventional DNA profiling needs to be performed subsequent to appearance DNA prediction. Notably, and where Forensic DNA Phenotyping shows great promise, this is on a (much) smaller group of potential suspects, who match the appearance characteristics DNA-predicted from the crime scene stain or from the deceased person's remains. Provided sufficient funding being made available, future research to better understand the genetic basis of human appearance will expectedly lead to a substantially more detailed description of an unknown person's appearance from DNA, delivering increased value for police investigations in criminal and missing person cases involving unknowns. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  13. [DNA identification of human papillomavirus in anogenital condyloma of a male population].

    Science.gov (United States)

    Turazza, E I; Ojeda, R D; Ratto, P; Barrera, A; Ríos, A; Kahn, T; Bercovich, J; Jaimovich, L; Grinstein, S

    1993-01-01

    Human Papillomaviruses (HPV) are the cause of benign human anogenital lesions where HPV 6 and HPV 11 are most commonly found. Conversely, HPV 16, 18, 31 and 33 are frequently detected in genital carcinomas and are thus considered as oncogenic types. In order to analyze the prevalence of specific HPV types in an Argentine male population, 43 anogenital lesions from different patients with diagnosis of condyloma acuminata were analyzed. These lesions were localized in different regions of the male genitalia comprising the corona glandis, urethral meatus, skin of the penis, scrotum and anus. The biopsies were screened for the presence of HPV 6, 11, 16, 18, 30, 31 and 33 by Southern blot at different stringent conditions of hybridization (Tm -48 degrees C and Tm -20 degrees C). HPV DNA was found in 41 examined cases (95.3%) with a clear prevalence of HPV 6 and HPV 11 types (51.2% and 23.3% respectively). Six samples (14.0%) were positive only under nonstringent conditions of hybridization. Mixed infections between HPV 16, 18, 30, 31, 33 or a HPV 30 related type with HPV 6 or HPV 11 were detected in 8 specimens (18.6%). Only one case was between HPV 16 and HPV 30. Two additional samples were only positive for HPV 30. Experiments in progress about the prevalence of HPV types in female lesions as well as in normal subjects will contribute to complete the description of the epidemiology of these infections in Argentina.

  14. Molecular cloning and mammalian expression of human beta 2-glycoprotein I cDNA

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Schousboe, Inger; Boel, Espen

    1991-01-01

    Human β2-glycoprotein (β2gpI) cDNA was isolated from a liver cDNA library and sequenced. The cDNA encoded a 19-residue hydrophobic signal peptide followed by the mature β2gpI of 326 amino acid residues. In liver and in the hepatoma cell line HepG2 there are two mRNA species of about 1.4 and 4.3 kb...

  15. Human circulating plasma DNA significantly decreases while lymphocyte DNA damage increases under chronic occupational exposure to low-dose gamma-neutron and tritium β-radiation

    Energy Technology Data Exchange (ETDEWEB)

    Korzeneva, Inna B., E-mail: inna.korzeneva@molgen.vniief.ru [Russian Federal Nuclear Center – All-Russian Research Institute of Experimental Physics (RFNC-VNIIEF) 607190, Sarov, 37 Mira ave., Nizhniy Novgorod Region (Russian Federation); Kostuyk, Svetlana V.; Ershova, Liza S. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, 115478 Moscow, 1 Moskvorechye str. (Russian Federation); Osipov, Andrian N. [Federal Medial and Biological Center named after Burnazyan of the Federal Medical and Biological Agency (FMBTz named after Burnazyan of FMBA), Moscow (Russian Federation); State Research Center - Burnasyan Federal Medical Biophysical Center of Federal Medical Biological Agency, Zhivopisnaya, 46, Moscow, 123098 (Russian Federation); Zhuravleva, Veronika F.; Pankratova, Galina V. [Russian Federal Nuclear Center – All-Russian Research Institute of Experimental Physics (RFNC-VNIIEF) 607190, Sarov, 37 Mira ave., Nizhniy Novgorod Region (Russian Federation); Porokhovnik, Lev N.; Veiko, Natalia N. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, 115478 Moscow, 1 Moskvorechye str. (Russian Federation)

    2015-09-15

    Highlights: • The chronic exposure to low-dose IR induces DSBs in human lymphocytes (TM index). • Exposure to IR decreases the level of human circulating DNA (cfDNA index). • IR induces an increase of DNase1 activity (DNase1 index) in plasma. • IR induces an increase of the level of antibodies to DNA (Ab DNA index) in plasma. • The ratio cfDNA/(DNase 1 × Ab DNA × TM) is a potential marker of human exposure to IR. - Abstract: The blood plasma of healthy people contains cell-fee (circulating) DNA (cfDNA). Apoptotic cells are the main source of the cfDNA. The cfDNA concentration increases in case of the organism’s cell death rate increase, for example in case of exposure to high-dose ionizing radiation (IR). The objects of the present research are the blood plasma and blood lymphocytes of people, who contacted occupationally with the sources of external gamma/neutron radiation or internal β-radiation of tritium N = 176). As the controls (references), blood samples of people, who had never been occupationally subjected to the IR sources, were used (N = 109). With respect to the plasma samples of each donor there were defined: the cfDNA concentration (the cfDNA index), DNase1 activity (the DNase1 index) and titre of antibodies to DNA (the Ab DNA index). The general DNA damage in the cells was defined (using the Comet assay, the tail moment (TM) index). A chronic effect of the low-dose ionizing radiation on a human being is accompanied by the enhancement of the DNA damage in lymphocytes along with a considerable cfDNA content reduction, while the DNase1 content and concentration of antibodies to DNA (Ab DNA) increase. All the aforementioned changes were also observed in people, who had not worked with the IR sources for more than a year. The ratio cfDNA/(DNase1 × Ab DNA × TM) is proposed to be used as a marker of the chronic exposure of a person to the external low-dose IR. It was formulated the assumption that the joint analysis of the cfDNA, DNase1, Ab

  16. Comparison of three different techniques of human sperm DNA isolation for methylation assay.

    Science.gov (United States)

    Yuan, Hong-fang; Kuete, Martin; Su, Li; Yang, Fan; Hu, Zhi-yong; Tian, Bo-zhen; Zhang, Hui-ping; Zhao, Kai

    2015-12-01

    Human sperm DNA is an important genetic and epigenetic material, whose chromatin structure differs from that of somatic cells. As such, conventional methods for DNA extraction of somatic cells may not be suitable for obtaining sperm DNA. In this study, we evaluated and compared three sperm DNA extraction techniques, namely, modified guanidinium thiocyanate method (method A), traditional phenol-chloroform method (method B), and TianGen kit method (method C). Spectrophotometry and agarose gel electrophoresis analyses showed that method A produced DNA with higher quantity and purity than those of methods B and C (Psperm DNA methylation assay further indicated that methods A and B were effective, and the former yielded higher quantitative accuracy. In conclusion, the modified guanidinium thiocyanate method provided high quality and reliable results and could be an optimal technique for extracting sperm DNA for methylation assay.

  17. Nuclear DNA Content Varies with Cell Size across Human Cell Types

    Science.gov (United States)

    Gillooly, James F.; Hein, Andrew; Damiani, Rachel

    2015-01-01

    Variation in the size of cells, and the DNA they contain, is a basic feature of multicellular organisms that affects countless aspects of their structure and function. Within humans, cell size is known to vary by several orders of magnitude, and differences in nuclear DNA content among cells have been frequently observed. Using published data, here we describe how the quantity of nuclear DNA across 19 different human cell types increases with cell volume. This observed increase is similar to intraspecific relationships between DNA content and cell volume in other species, and interspecific relationships between diploid genome size and cell volume. Thus, we speculate that the quantity of nuclear DNA content in somatic cells of humans is perhaps best viewed as a distribution of values that reflects cell size distributions, rather than as a single, immutable quantity. PMID:26134319

  18. Nuclear DNA Content Varies with Cell Size across Human Cell Types.

    Science.gov (United States)

    Gillooly, James F; Hein, Andrew; Damiani, Rachel

    2015-07-01

    Variation in the size of cells, and the DNA they contain, is a basic feature of multicellular organisms that affects countless aspects of their structure and function. Within humans, cell size is known to vary by several orders of magnitude, and differences in nuclear DNA content among cells have been frequently observed. Using published data, here we describe how the quantity of nuclear DNA across 19 different human cell types increases with cell volume. This observed increase is similar to intraspecific relationships between DNA content and cell volume in other species, and interspecific relationships between diploid genome size and cell volume. Thus, we speculate that the quantity of nuclear DNA content in somatic cells of humans is perhaps best viewed as a distribution of values that reflects cell size distributions, rather than as a single, immutable quantity. Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.

  19. Repair of DNA damage in light sensitive human skin diseases

    Energy Technology Data Exchange (ETDEWEB)

    Horkay, I.; Varga, L.; Tam' asi P., Gundy, S.

    1978-12-01

    Repair of uv-light induced DNA damage and changes in the semiconservative DNA synthesis were studied by in vitro autoradiography in the skin of patients with lightdermatoses (polymorphous light eruption, porphyria cutanea tarda, erythropoietic protoporphyria) and xeroderma pigmentosum as well as in that of healthy controls. In polymorphous light eruption the semiconservative DNA replication rate was more intensive in the area of the skin lesions and in the repeated phototest site, the excision repair synthesis appeared to be unaltered. In cutaneous prophyrias a decreased rate of the repair incorporation could be detected. Xeroderma pigmentosum was characterized by a strongly reduced repair synthesis.

  20. Assessing DNA methylation in the developing human intestinal epithelium: potential link to inflammatory bowel disease.

    Science.gov (United States)

    Kraiczy, J; Nayak, K; Ross, A; Raine, T; Mak, T N; Gasparetto, M; Cario, E; Rakyan, V; Heuschkel, R; Zilbauer, M

    2016-05-01

    DNA methylation is one of the major epigenetic mechanisms implicated in regulating cellular development and cell-type-specific gene expression. Here we performed simultaneous genome-wide DNA methylation and gene expression analysis on purified intestinal epithelial cells derived from human fetal gut, healthy pediatric biopsies, and children newly diagnosed with inflammatory bowel disease (IBD). Results were validated using pyrosequencing, real-time PCR, and immunostaining. The functional impact of DNA methylation changes on gene expression was assessed by employing in-vitro assays in intestinal cell lines. DNA methylation analyses allowed identification of 214 genes for which expression is regulated via DNA methylation, i.e. regulatory differentially methylated regions (rDMRs). Pathway and functional analysis of rDMRs suggested a critical role for DNA methylation in regulating gene expression and functional development of the human intestinal epithelium. Moreover, analysis performed on intestinal epithelium of children newly diagnosed with IBD revealed alterations in DNA methylation within genomic loci, which were found to overlap significantly with those undergoing methylation changes during intestinal development. Our study provides novel insights into the physiological role of DNA methylation in regulating functional maturation of the human intestinal epithelium. Moreover, we provide data linking developmentally acquired alterations in the DNA methylation profile to changes seen in pediatric IBD.

  1. Phosphorylation of human INO80 is involved in DNA damage tolerance

    Energy Technology Data Exchange (ETDEWEB)

    Kato, Dai; Waki, Mayumi; Umezawa, Masaki; Aoki, Yuka [Department of Biological Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510 (Japan); Utsugi, Takahiko [Bio Matrix Research Inc., 105 Higashifukai, Nagareyama, Chiba 275-0101 (Japan); Ohtsu, Masaya [Department of Biological Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510 (Japan); Murakami, Yasufumi, E-mail: yasufumi@rs.noda.tus.ac.jp [Department of Biological Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510 (Japan); Bio Matrix Research Inc., 105 Higashifukai, Nagareyama, Chiba 275-0101 (Japan)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Depletion of hINO80 significantly reduced PCNA ubiquitination. Black-Right-Pointing-Pointer Depletion of hINO80 significantly reduced nuclear dots intensity of RAD18 after UV irradiation. Black-Right-Pointing-Pointer Western blot analyses showed phosphorylated hINO80 C-terminus. Black-Right-Pointing-Pointer Overexpression of phosphorylation mutant hINO80 reduced PCNA ubiquitination. -- Abstract: Double strand breaks (DSBs) are the most serious type of DNA damage. DSBs can be generated directly by exposure to ionizing radiation or indirectly by replication fork collapse. The DNA damage tolerance pathway, which is conserved from bacteria to humans, prevents this collapse by overcoming replication blockages. The INO80 chromatin remodeling complex plays an important role in the DNA damage response. The yeast INO80 complex participates in the DNA damage tolerance pathway. The mechanisms regulating yINO80 complex are not fully understood, but yeast INO80 complex are necessary for efficient proliferating cell nuclear antigen (PCNA) ubiquitination and for recruitment of Rad18 to replication forks. In contrast, the function of the mammalian INO80 complex in DNA damage tolerance is less clear. Here, we show that human INO80 was necessary for PCNA ubiquitination and recruitment of Rad18 to DNA damage sites. Moreover, the C-terminal region of human INO80 was phosphorylated, and overexpression of a phosphorylation-deficient mutant of human INO80 resulted in decreased ubiquitination of PCNA during DNA replication. These results suggest that the human INO80 complex, like the yeast complex, was involved in the DNA damage tolerance pathway and that phosphorylation of human INO80 was involved in the DNA damage tolerance pathway. These findings provide new insights into the DNA damage tolerance pathway in mammalian cells.

  2. Concerted action of two novel tRNA mtDNA point mutations in chronic progressive external ophthalmoplegia.

    Science.gov (United States)

    Kornblum, Cornelia; Zsurka, Gábor; Wiesner, Rudolf J; Schröder, Rolf; Kunz, Wolfram S

    2008-04-01

    CPEO (chronic progressive external ophthalmoplegia) is a common mitochondrial disease phenotype in adults which is due to mtDNA (mitochondrial DNA) point mutations in a subset of patients. Attributing pathogenicity to novel tRNA mtDNA mutations still poses a challenge, particularly when several mtDNA sequence variants are present. In the present study we report a CPEO patient for whom sequencing of the mitochondrial genome revealed three novel tRNA mtDNA mutations: G5835A, del4315A, T1658C in tRNATyr, tRNAIle and tRNAVal genes. In skeletal muscle, the tRNAVal and tRNAIle mutations were homoplasmic, whereas the tRNATyr mutation was heteroplasmic. To address the pathogenic relevance, we performed two types of functional tests: (i) single skeletal muscle fibre analysis comparing G5835A mutation loads and biochemical phenotypes of corresponding fibres, and (ii) Northern-blot analyses of mitochondrial tRNATyr, tRNAIle and tRNAVal. We demonstrated that both the G5835A tRNATyr and del4315A tRNAIle mutation have serious functional consequences. Single-fibre analyses displayed a high threshold of the tRNATyr mutation load for biochemical phenotypic expression at the single-cell level, indicating a rather mild pathogenic effect. In contrast, skeletal muscle tissue showed a severe decrease in respiratory-chain activities, a reduced overall COX (cytochrome c oxidase) staining intensity and abundant COX-negative fibres. Northern-blot analyses showed a dramatic reduction of tRNATyr and tRNAIle levels in muscle, with impaired charging of tRNAIle, whereas tRNAVal levels were only slightly decreased, with amino-acylation unaffected. Our findings suggest that the heteroplasmic tRNATyr and homoplasmic tRNAIle mutation act together, resulting in a concerted effect on the biochemical and histological phenotype. Thus homoplasmic mutations may influence the functional consequences of pathogenic heteroplasmic mtDNA mutations.

  3. The progressive realization of human rights to water: the legal basis, policy implications, and monitoring challenge

    Directory of Open Access Journals (Sweden)

    Nastiti Anindrya

    2018-01-01

    Full Text Available Since 2010, the United Nations General Assembly had explicitly recognized the human right to water and sanitation and obliged States to provide for its progressive realization and entitles everyone to sufficient, safe, acceptable, physically accessible and affordable water for essential personal and domestic uses. This paper scrutinizes the legal basis and the policy implication for human right to water in Indonesia, before and following the annulment of the Water Resource Law 7/2004. This paper considers that one of the greatest challenges is to find an appropriate and internationally-comparable methodology in measuring the progressive realization of human rights to water and sanitation. We also highlight the importance of composite indicators and concludes that single variable indicators are insufficient to capture the range of issues involved in the realization of the human rights to water.

  4. A Mini-Library of Sequenced Human DNA Fragments: Linking Bench Experiments with Informatics

    Science.gov (United States)

    Dalgleish, Raymond; Shanks, Morag E.; Monger, Karen; Butler, Nicola J.

    2012-01-01

    We describe the development of a mini-library of human DNA fragments for use in an enquiry-based learning (EBL) undergraduate practical incorporating "wet-lab" and bioinformatics tasks. In spite of the widespread emergence of the polymerase chain reaction (PCR), the cloning and analysis of DNA fragments in "Escherichia coli"…

  5. 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...

  6. DNA from Pre-Clovis Human Coprolites in Oregon, North America

    DEFF Research Database (Denmark)

    Gilbert, M Thomas P; Jenkins, Dennis L; Götherstrom, Anders

    2008-01-01

    Caves, south-central Oregon, by 12,300 (14)C yr. B.P., through recovery of human mtDNA from coprolites, directly dated by accelerator mass spectrometry. The mtDNA corresponds to Native American founding haplogroups A2 and B2. The dates of the coprolites are >1000 (14)C years earlier than currently...

  7. Role of extracellular DNA oxidative modification in radiation induced bystander effects in human endotheliocytes

    Energy Technology Data Exchange (ETDEWEB)

    Kostyuk, Svetlana V. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation); Ermakov, Aleksei V., E-mail: avePlato@mail.ru [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation); Alekseeva, Anna Yu.; Smirnova, Tatiana D.; Glebova, Kristina V.; Efremova, Liudmila V. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation); Baranova, Ancha [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation); School of System Biology, George Mason University, Fairfax, VA 22030 (United States); Veiko, Natalya N. [Research Centre for Medical Genetics, Russian Academy of Medical Sciences, Moscow (Russian Federation)

    2012-01-03

    The development of the bystander effect induced by low doses of irradiation in human umbilical vein endothelial cells (HUVECs) depends on extracellular DNA (ecDNA) signaling pathway. We found that the changes in the levels of ROS and NO production by human endothelial cells are components of the radiation induced bystander effect that can be registered at a low dose. We exposed HUVECs to X-ray radiation and studied effects of ecDNA{sup R} isolated from the culture media conditioned by the short-term incubation of irradiated cells on intact HUVECs. Effects of ecDNA{sup R} produced by irradiated cells on ROS and NO production in non-irradiated HUVECs are similar to bystander effect. These effects at least partially depend on TLR9 signaling. We compared the production of the nitric oxide and the ROS in human endothelial cells that were (1) irradiated at a low dose; (2) exposed to the ecDNA{sup R} extracted from the media conditioned by irradiated cells; and (3) exposed to human DNA oxidized in vitro. We found that the cellular responses to all three stimuli described above are essentially similar. We conclude that irradiation-related oxidation of the ecDNA is an important component of the ecDNA-mediated bystander effect.

  8. Genome-wide DNA methylation levels and altered cortisol stress reactivity following childhood trauma in humans

    NARCIS (Netherlands)

    Houtepen, Lotte C|info:eu-repo/dai/nl/413662802; Vinkers, Christiaan H|info:eu-repo/dai/nl/304824755; Carrillo-Roa, Tania; Hiemstra, Marieke; van Lier, Pol A; Meeus, Wim; Branje, Susan; Heim, Christine M; Nemeroff, Charles B; Mill, Jonathan; Schalkwyk, Leonard C; Creyghton, Menno P|info:eu-repo/dai/nl/336269471; Kahn, René S|info:eu-repo/dai/nl/073778532; Joëls, Marian|info:eu-repo/dai/nl/070219249; Binder, Elisabeth B; Boks, Marco P M|info:eu-repo/dai/nl/286852071

    2016-01-01

    DNA methylation likely plays a role in the regulation of human stress reactivity. Here we show that in a genome-wide analysis of blood DNA methylation in 85 healthy individuals, a locus in the Kit ligand gene (KITLG; cg27512205) showed the strongest association with cortisol stress reactivity (P=5.8

  9. Human SIRT6 promotes DNA end resection through CtIP deacetylation

    DEFF Research Database (Denmark)

    Kaidi, Abderrahmane; Weinert, Brian T; Choudhary, Chunaram

    2010-01-01

    SIRT6 belongs to the sirtuin family of protein lysine deacetylases, which regulate aging and genome stability. We found that human SIRT6 has a role in promoting DNA end resection, a crucial step in DNA double-strand break (DSB) repair by homologous recombination. SIRT6 depletion impaired the accu...

  10. Problem-Solving Test: Analysis of DNA Damage Recognizing Proteins in Yeast and Human Cells

    Science.gov (United States)

    Szeberenyi, Jozsef

    2013-01-01

    The experiment described in this test was aimed at identifying DNA repair proteins in human and yeast cells. Terms to be familiar with before you start to solve the test: DNA repair, germline mutation, somatic mutation, inherited disease, cancer, restriction endonuclease, radioactive labeling, [alpha-[superscript 32]P]ATP, [gamma-[superscript…

  11. Quantum dot based DNA nanosensors for amplification-free detection of human topoisomerase I

    DEFF Research Database (Denmark)

    Jepsen, Morten Leth; Ottaviani, Alessio; Knudsen, Birgitta R.

    2014-01-01

    We develop a quantum dot based DNA nanosensor specifically targeting the cleavage–religation activity of an essential DNA-modifying enzyme, human topoisomerase I. The assay has shown great promise in biological crude samples and thus is expected to contribute to clinical diagnostics and anti...

  12. The DNA sequence, annotation and analysis of human chromosome 3

    DEFF Research Database (Denmark)

    Muzny, Donna M; Scherer, Steven E; Kaul, Rajinder

    2006-01-01

    After the completion of a draft human genome sequence, the International Human Genome Sequencing Consortium has proceeded to finish and annotate each of the 24 chromosomes comprising the human genome. Here we describe the sequencing and analysis of human chromosome 3, one of the largest human chr...

  13. Mechanisms of interstrand DNA crosslink repair and human disorders.

    Science.gov (United States)

    Hashimoto, Satoru; Anai, Hirofumi; Hanada, Katsuhiro

    2016-01-01

    Interstrand DNA crosslinks (ICLs) are the link between Watson-Crick strands of DNAs with the covalent bond and prevent separation of DNA strands. Since the ICL lesion affects both strands of the DNA, the ICL repair is not simple. So far, nucleotide excision repair (NER), structure-specific endonucleases, translesion DNA synthesis (TLS), homologous recombination (HR), and factors responsible for Fanconi anemia (FA) are identified to be involved in ICL repair. Since the presence of ICL lesions causes severe defects in transcription and DNA replication, mutations in these DNA repair pathways give rise to a various hereditary disorders. NER plays an important role for the ICL recognition and removal in quiescent cells, and defects of NER causes congential progeria syndrome, such as xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. On the other hand, the ICL repair in S phase requires more complicated orchestration of multiple factors, including structure-specific endonucleases, and TLS, and HR. Disturbed this ICL repair orchestration in S phase causes genome instability resulting a cancer prone disease, Fanconi anemia. So far more than 30 factors in ICL repair have already identified. Recently, a new factor, UHRF1, was discovered as a sensor of ICLs. In addition to this, numbers of nucleases that are involved in the first incision, also called unhooking, of ICL lesions have also been identified. Here we summarize the recent studies of ICL associated disorders and repair mechanism, with emphasis in the first incision of ICLs.

  14. An Improved Methodology to Overcome Key Issues in Human Fecal Metagenomic DNA Extraction

    Directory of Open Access Journals (Sweden)

    Jitendra Kumar

    2016-12-01

    Full Text Available Microbes are ubiquitously distributed in nature, and recent culture-independent studies have highlighted the significance of gut microbiota in human health and disease. Fecal DNA is the primary source for the majority of human gut microbiome studies. However, further improvement is needed to obtain fecal metagenomic DNA with sufficient amount and good quality but low host genomic DNA contamination. In the current study, we demonstrate a quick, robust, unbiased, and cost-effective method for the isolation of high molecular weight (>23 kb metagenomic DNA (260/280 ratio >1.8 with a good yield (55.8 ± 3.8 ng/mg of feces. We also confirm that there is very low human genomic DNA contamination (eubacterial: human genomic DNA marker genes = 227.9:1 in the human feces. The newly-developed method robustly performs for fresh as well as stored fecal samples as demonstrated by 16S rRNA gene sequencing using 454 FLX+. Moreover, 16S rRNA gene analysis indicated that compared to other DNA extraction methods tested, the fecal metagenomic DNA isolated with current methodology retains species richness and does not show microbial diversity biases, which is further confirmed by qPCR with a known quantity of spike-in genomes. Overall, our data highlight a protocol with a balance between quality, amount, user-friendliness, and cost effectiveness for its suitability toward usage for culture-independent analysis of the human gut microbiome, which provides a robust solution to overcome key issues associated with fecal metagenomic DNA isolation in human gut microbiome studies.

  15. An Improved Methodology to Overcome Key Issues in Human Fecal Metagenomic DNA Extraction.

    Science.gov (United States)

    Kumar, Jitendra; Kumar, Manoj; Gupta, Shashank; Ahmed, Vasim; Bhambi, Manu; Pandey, Rajesh; Chauhan, Nar Singh

    2016-12-01

    Microbes are ubiquitously distributed in nature, and recent culture-independent studies have highlighted the significance of gut microbiota in human health and disease. Fecal DNA is the primary source for the majority of human gut microbiome studies. However, further improvement is needed to obtain fecal metagenomic DNA with sufficient amount and good quality but low host genomic DNA contamination. In the current study, we demonstrate a quick, robust, unbiased, and cost-effective method for the isolation of high molecular weight (>23kb) metagenomic DNA (260/280 ratio >1.8) with a good yield (55.8±3.8ng/mg of feces). We also confirm that there is very low human genomic DNA contamination (eubacterial: human genomic DNA marker genes=227.9:1) in the human feces. The newly-developed method robustly performs for fresh as well as stored fecal samples as demonstrated by 16S rRNA gene sequencing using 454 FLX+. Moreover, 16S rRNA gene analysis indicated that compared to other DNA extraction methods tested, the fecal metagenomic DNA isolated with current methodology retains species richness and does not show microbial diversity biases, which is further confirmed by qPCR with a known quantity of spike-in genomes. Overall, our data highlight a protocol with a balance between quality, amount, user-friendliness, and cost effectiveness for its suitability toward usage for culture-independent analysis of the human gut microbiome, which provides a robust solution to overcome key issues associated with fecal metagenomic DNA isolation in human gut microbiome studies. Copyright © 2016 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.

  16. Protection by quercetin and quercetin-rich fruit juice against induction of oxidative DNA damage and formation of BPDE-DNA adducts in human lymphocytes

    NARCIS (Netherlands)

    Wilms, L.C.; Hollman, P.C.H.; Boots, A.W.; Kleinjans, J.C.S.

    2005-01-01

    Flavonoids are claimed to protect against cardiovascular disease, certain forms of cancer and ageing, possibly by preventing initial DNA damage. Therefore, we investigated the protective effects of the flavonoid quercetin against the formation of oxidative DNA damage and bulky DNA adducts in human

  17. Human DNA polymerase delta double-mutant D316A;E318A interferes with DNA mismatch repair in vitro

    DEFF Research Database (Denmark)

    Liu, Dekang; Frederiksen, Jane H; Liberti, Sascha E

    2017-01-01

    DNA mismatch repair (MMR) is a highly-conserved DNA repair mechanism, whose primary role is to remove DNA replication errors preventing them from manifesting as mutations, thereby increasing the overall genome stability. Defects in MMR are associated with increased cancer risk in humans and other...

  18. Cadmium Chloride Induces DNA Damage and Apoptosis of Human Liver Carcinoma Cells via Oxidative Stress

    National Research Council Canada - National Science Library

    Skipper, Anthony; Sims, Jennifer N; Yedjou, Clement G; Tchounwou, Paul B

    2016-01-01

    ... mechanisms of action are not fully elucidated. In this research, we hypothesized that oxidative stress plays a key role in cadmium chloride-induced toxicity, DNA damage, and apoptosis of human liver carcinoma (HepG₂) cells...

  19. DNA methylation and transcriptional trajectories during human development and reprogramming of isogenic pluripotent stem cells

    NARCIS (Netherlands)

    Roost, Matthias S; Slieker, Roderick C; Bialecka, Monika; van Iperen, Liesbeth; Gomes Fernandes, Maria M; He, Nannan; Suchiman, H Eka D; Szuhai, Karoly; Carlotti, Françoise; de Koning, Eelco J P; Mummery, Christine L; Heijmans, Bastiaan T; Chuva de Sousa Lopes, Susana M

    2017-01-01

    Determining cell identity and maturation status of differentiated pluripotent stem cells (PSCs) requires knowledge of the transcriptional and epigenetic trajectory of organs during development. Here, we generate a transcriptional and DNA methylation atlas covering 21 organs during human fetal

  20. The molecular and cellular response of normal and progressed human bronchial epithelial cells to HZE particles

    Science.gov (United States)

    Story, Michael; Ding, Liang-Hao; Minna, John; Park, Seong-mi; Larsen, Jill

    We have used a model of non-oncogenically immortalized normal human bronchial epithelial cells to determine the response of such cells to particles found outside the protection of the earth’s electromagnetic field. We have identified an enhanced frequency of cellular transformation, as measured by growth in soft agar, for both 56Fe and 28Si (1 GeV/n) that is maximal (4-6 fold) at 0.25 Gy and 0.40 Gy, respectively. At 4 months post-irradiation 38 individual soft agar clones were isolated. These clones were characterized extensively for cellular and molecular changes. Gene expression analysis suggested that these clones had down-regulated several genes associated with anti-oxidant pathways including GLS2, GPX1 and 4, SOD2, PIG3, and NQO1 amongst others. As a result, many of these transformed clones were exposed to high levels of intracellular radical oxygen species (ROS), although there appeared not to be any enhanced mitochondrial ROS. DNA repair pathways associated with ATM/ATR signaling were also upregulated. However, these transformants do not develop into tumors when injected into immune-compromised mice, suggesting that they have not progressed sufficiently to become oncogenic. Therefore we chose 6 soft agar clones for continuous culture for an additional 14 months. Amongst the 6 clones, only one clone showed any significant change in phenotype. Clone 3kt-ff.2a, propagated for 18 months, were 2-fold more radioresistant, had a shortened doubling time and the background rate of transformation more than doubled. Furthermore, the morphology of transformed clones changed. Clones from this culture are being compared to the original clone as well as the parental HBEC3KT and will be injected into immune-compromised mice for oncogenic potential. Oncogenically progressed HBECs, HBEC3KT cells that overexpress a mutant RAS gene and where p53 has been knocked down, designated HBEC3KTR53, responded quite differently to HZE particle exposure. First, these cells are more

  1. The Conjugative Relaxase TrwC Promotes Integration of Foreign DNA in the Human Genome.

    Science.gov (United States)

    González-Prieto, Coral; Gabriel, Richard; Dehio, Christoph; Schmidt, Manfred; Llosa, Matxalen

    2017-06-15

    Bacterial conjugation is a mechanism of horizontal DNA transfer. The relaxase TrwC of the conjugative plasmid R388 cleaves one strand of the transferred DNA at the oriT gene, covalently attaches to it, and leads the single-stranded DNA (ssDNA) into the recipient cell. In addition, TrwC catalyzes site-specific integration of the transferred DNA into its target sequence present in the genome of the recipient bacterium. Here, we report the analysis of the efficiency and specificity of the integrase activity of TrwC in human cells, using the type IV secretion system of the human pathogen Bartonella henselae to introduce relaxase-DNA complexes. Compared to Mob relaxase from plasmid pBGR1, we found that TrwC mediated a 10-fold increase in the rate of plasmid DNA transfer to human cells and a 100-fold increase in the rate of chromosomal integration of the transferred DNA. We used linear amplification-mediated PCR and plasmid rescue to characterize the integration pattern in the human genome. DNA sequence analysis revealed mostly reconstituted oriT sequences, indicating that TrwC is active and recircularizes transferred DNA in human cells. One TrwC-mediated site-specific integration event was detected, proving that TrwC is capable of mediating site-specific integration in the human genome, albeit with very low efficiency compared to the rate of random integration. Our results suggest that TrwC may stabilize the plasmid DNA molecules in the nucleus of the human cell, probably by recircularization of the transferred DNA strand. This stabilization would increase the opportunities for integration of the DNA by the host machinery.IMPORTANCE Different biotechnological applications, including gene therapy strategies, require permanent modification of target cells. Long-term expression is achieved either by extrachromosomal persistence or by integration of the introduced DNA. Here, we studied the utility of conjugative relaxase TrwC, a bacterial protein with site

  2. Roles of the human Rad51 L1 and L2 loops in DNA binding.

    Science.gov (United States)

    Matsuo, Yusuke; Sakane, Isao; Takizawa, Yoshimasa; Takahashi, Masayuki; Kurumizaka, Hitoshi

    2006-07-01

    The human Rad51 protein, a eukaryotic ortholog of the bacterial RecA protein, is a key enzyme that functions in homologous recombination and recombinational repair of double strand breaks. The Rad51 protein contains two flexible loops, L1 and L2, which are proposed to be sites for DNA binding, based on a structural comparison with RecA. In the present study, we performed mutational and fluorescent spectroscopic analyses on the L1 and L2 loops to examine their role in DNA binding. Gel retardation and DNA-dependent ATP hydrolysis measurements revealed that the substitution of the tyrosine residue at position 232 (Tyr232) within the L1 loop with alanine, a short side chain amino acid, significantly decreased the DNA-binding ability of human Rad51, without affecting the protein folding or the salt-induced, DNA-independent ATP hydrolysis. Even the conservative replacement with tryptophan affected the DNA binding, indicating that Tyr232 is involved in DNA binding. The importance of the L1 loop was confirmed by the fluorescence change of a tryptophan residue, replacing the Asp231, Ser233, or Gly236 residue, upon DNA binding. The alanine replacement of phenylalanine at position 279 (Phe279) within the L2 loop did not affect the DNA-binding ability of human Rad51, unlike the Phe203 mutation of the RecA L2 loop. The Phe279 side chain may not be directly involved in the interaction with DNA. However, the fluorescence intensity of the tryptophan replacing the Rad51-Phe279 residue was strongly reduced upon DNA binding, indicating that the L2 loop is also close to the DNA-binding site.

  3. "Human Potential" and Progressive Pedagogy: A Long Cultural History of the Ambiguity of "Race" and "Intelligence"

    Science.gov (United States)

    Oland, Trine

    2012-01-01

    This article examines the cultural constructs of progressive pedagogy in Danish school pedagogy and its emerging focus on the child's human potential from the 1920s to the 1950s. It draws on Foucault's notion of "dispositifs" and the "elements of history," encircling a complex transformation of continuity and discontinuity of…

  4. Cytogenetic responses to ionizing radiation exposure of human fibroblasts with knocked-down expressions of various DNA damage signaling genes

    Science.gov (United States)

    Zhang, Ye; Rohde, Larry; Wu, Honglu

    Changes of gene expression profile are one of the most important biological responses in living cells after ionizing radiation (IR) exposure. Although some studies have demonstrated that genes with up-regulated expression induced by IR may play important roles in DNA damage sensing, cell cycle checkpoint and chromosomal repair, the relationship between the regulation of gene expression by IR and its impact on cytogenetic responses to ionizing radiation has not been systematically studied. Here, the expression of 25 genes selected based on their transcriptional changes in response to IR or from their known DNA repair roles were individually knocked down by siRNA transfection in human fibroblast cells. Chromosome aberrations (CA) and micronuclei (MN) formation were measured as the cytogenetic endpoints. Our results showed that the yields of MN and/or CA formation were significantly increased by suppressed expression of some of the selected genes in DSB and other DNA repair pathways. Knocked-down expression of other genes showed significant impact on cell cycle progression, possibly because of severe impairment of DNA damage repair. Of these 11 genes that affected the cytogenetic response, 9 were up-regulated in the cells exposed to gamma radiation, suggesting that genes transcriptionally modulated by IR were critical to regulating the biological consequences after IR. Failure to express these IR-responsive genes, such as by gene mutation, could seriously change the outcome of the post IR scenario and lead to carcinogenesis.

  5. Development of a traceable molecular hygiene control method (TMHCM) for human DNA content in foods.

    Science.gov (United States)

    Şakalar, Ergün; Ergün, Şeyma Özçirak; Pala, Çiğdem; Akar, Emine; Ataşoğlu, Cengiz

    2017-06-15

    The aim of this study was to develop a molecular technique to determine the level of human originated DNA contamination in unhygienic food products. In the study, four model foods were prepared under both hygienic (H) and non-hygienic (NH) conditions and the human originated microbial loads of these products were determined. DNA was extracted from the model foods and human buccal samples by GIDAGEN Multi-fast DNA isolation kit. A primer specific region of human mitochondrial D-Loop was designed. The level of human DNA contamination in the model foods was determined by real-time PCR. The sensitivity of the technique developed here was 0.00001ng DNA/PCR. In addition, the applicability of the traceable molecular hygiene control method (TMHCM) was tested in 60 food samples from the market. The results of this study demonstrate that DNA based TMHCM can be used to predict to what extent foods meet the human oriented hygienic conditions. Copyright © 2016. Published by Elsevier Ltd.

  6. 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. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  7. The m.3244G>A mutation in mtDNA is another cause of progressive external ophthalmoplegia

    Science.gov (United States)

    Sotiriou, Evangelia; Çoku, Jorida; Tanji, Kurenai; Huang, Hua-bin; Hirano, Michio; DiMauro, Salvatore

    2009-01-01

    We sequenced all mitochondrial tRNA genes in a 61-year-old man with chronic progressive external ophthalmoplegia and mitochondrial myopathy but without mtDNA rearrangements, and identified a heteroplasmic m.3244G>A mutation in the tRNALeu (UUR) gene. This mutation had been previously associated with the MELAS phenotype, but not described in any detail. The mutation load in muscle was 84% and COX-negative fibers harbored greater levels of mutant genomes than COX-positive fibers. The m.G3244G>A mutation affects a highly conserved nucleotide in the dihydrouridine loop and has been associated with a wobble modification deficiency of the mutant tRNA. PMID:19285865

  8. DNA Damage Reduces the Quality, but Not the Quantity of Human Papillomavirus 16 E1 and E2 DNA Replication

    Directory of Open Access Journals (Sweden)

    Molly L. Bristol

    2016-06-01

    Full Text Available Human papillomaviruses (HPVs are causative agents in almost all cervical carcinomas. HPVs are also causative agents in head and neck cancer, the cases of which are increasing rapidly. Viral replication activates the DNA damage response (DDR pathway; associated proteins are recruited to replication foci, and this pathway may serve to allow for viral genome amplification. Likewise, HPV genome double-strand breaks (DSBs could be produced during replication and could lead to linearization and viral integration. Many studies have shown that viral integration into the host genome results in unregulated expression of the viral oncogenes, E6 and E7, promoting HPV-induced carcinogenesis. Previously, we have demonstrated that DNA-damaging agents, such as etoposide, or knocking down viral replication partner proteins, such as topoisomerase II β binding protein I (TopBP1, does not reduce the level of DNA replication. Here, we investigated whether these treatments alter the quality of DNA replication by HPV16 E1 and E2. We confirm that knockdown of TopBP1 or treatment with etoposide does not reduce total levels of E1/E2-mediated DNA replication; however, the quality of replication is significantly reduced. The results demonstrate that E1 and E2 continue to replicate under genomically-stressed conditions and that this replication is mutagenic. This mutagenesis would promote the formation of substrates for integration of the viral genome into that of the host, a hallmark of cervical cancer.

  9. DNA Damage Reduces the Quality, but Not the Quantity of Human Papillomavirus 16 E1 and E2 DNA Replication.

    Science.gov (United States)

    Bristol, Molly L; Wang, Xu; Smith, Nathan W; Son, Minkyeong P; Evans, Michael R; Morgan, Iain M

    2016-06-22

    Human papillomaviruses (HPVs) are causative agents in almost all cervical carcinomas. HPVs are also causative agents in head and neck cancer, the cases of which are increasing rapidly. Viral replication activates the DNA damage response (DDR) pathway; associated proteins are recruited to replication foci, and this pathway may serve to allow for viral genome amplification. Likewise, HPV genome double-strand breaks (DSBs) could be produced during replication and could lead to linearization and viral integration. Many studies have shown that viral integration into the host genome results in unregulated expression of the viral oncogenes, E6 and E7, promoting HPV-induced carcinogenesis. Previously, we have demonstrated that DNA-damaging agents, such as etoposide, or knocking down viral replication partner proteins, such as topoisomerase II β binding protein I (TopBP1), does not reduce the level of DNA replication. Here, we investigated whether these treatments alter the quality of DNA replication by HPV16 E1 and E2. We confirm that knockdown of TopBP1 or treatment with etoposide does not reduce total levels of E1/E2-mediated DNA replication; however, the quality of replication is significantly reduced. The results demonstrate that E1 and E2 continue to replicate under genomically-stressed conditions and that this replication is mutagenic. This mutagenesis would promote the formation of substrates for integration of the viral genome into that of the host, a hallmark of cervical cancer.

  10. Fecal collection, ambient preservation, and DNA extraction for PCR amplification of bacterial and human markers from human feces.

    Science.gov (United States)

    Nechvatal, Jordan M; Ram, Jeffrey L; Basson, Marc D; Namprachan, Phanramphoei; Niec, Stephanie R; Badsha, Kawsar Z; Matherly, Larry H; Majumdar, Adhip P N; Kato, Ikuko

    2008-02-01

    Feces contain intestinal bacteria and exfoliated epithelial cells that may provide useful information concerning gastrointestinal tract health. Intestinal bacteria that synthesize or metabolize potential carcinogens and produce anti-tumorigenic products may have relevance to colorectal cancer, the second most common cause of cancer deaths in the USA. To facilitate epidemiological studies relating bacterial and epithelial cell DNA and RNA markers, preservative/extraction methods suitable for self-collection and shipping of fecal samples at room temperature were tested. Purification and PCR amplification of fecal DNA were compared after preservation of stool samples in RNAlater (R) or Paxgene (P), or after drying over silica gel (S) or on Whatman FTA cards (W). Comparisons were made to samples frozen in liquid nitrogen (N2). DNA purification methods included Whatman (accompanying FTA cards), Mo-Bio Fecal (MB), Qiagen Stool (QS), and others. Extraction methods were compared for amount of DNA extracted, DNA amplifiable in a real-time SYBR-Green quantitative PCR format, and the presence of PCR inhibitors. DNA can be extracted after room temperature storage for five days from W, R, S and P, and from N2 frozen samples. High amounts of total DNA and PCR-amplifiable Bacteroides spp. DNA (34%+/-9% of total DNA) with relatively little PCR inhibition were especially obtained with QS extraction applied to R preserved samples (method QS-R). DNA for human reduced folate carrier (SLC19A1) genomic sequence was also detected in 90% of the QS-R extracts. Thus, fecal DNA is well preserved by methods suitable for self-collection that may be useful in future molecular epidemiological studies of intestinal bacteria and human cancer markers.

  11. Comparison of DNA extraction methods for human gut microbial community profiling.

    Science.gov (United States)

    Lim, Mi Young; Song, Eun-Ji; Kim, Sang Ho; Lee, Jangwon; Nam, Young-Do

    2017-12-21

    The human gut harbors a vast range of microbes that have significant impact on health and disease. Therefore, gut microbiome profiling holds promise for use in early diagnosis and precision medicine development. Accurate profiling of the highly complex gut microbiome requires DNA extraction methods that provide sufficient coverage of the original community as well as adequate quality and quantity. We tested nine different DNA extraction methods using three commercial kits (TianLong Stool DNA/RNA Extraction Kit (TS), QIAamp DNA Stool Mini Kit (QS), and QIAamp PowerFecal DNA Kit (QP)) with or without additional bead-beating step using manual or automated methods and compared them in terms of DNA extraction ability from human fecal sample. All methods produced DNA in sufficient concentration and quality for use in sequencing, and the samples were clustered according to the DNA extraction method. Inclusion of bead-beating step especially resulted in higher degrees of microbial diversity and had the greatest effect on gut microbiome composition. Among the samples subjected to bead-beating method, TS kit samples were more similar to QP kit samples than QS kit samples. Our results emphasize the importance of mechanical disruption step for a more comprehensive profiling of the human gut microbiome. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

  12. Vitreous humour as a potential DNA source for postmortem human identification.

    Directory of Open Access Journals (Sweden)

    Renata Zbiec

    2007-06-01

    Full Text Available PURPOSE
    The aim of this study was the assessment of vitreous humor as a potential DNA for forensic human postmortem identification.

    MATERIAL AND METHODS
    Vitreous humor samples were collected using two alternative approaches from 25 corpses of either sex during autopsies. DNA was extracted by standard organic method. Recovered DNA was quantitiated fluorometrically. AmpFlSTR SGM Plus kit and ABI 310 Genetic Analyzer (Applera were used to obtain genetic profiles.

    RESULTS
    Different DNA yields were quantitated in vitreous body depending on cause of death and sampling approach.

    CONCLUSION
    Vitreous humor is a potential DNA for forensic human postmortem identification depending on a sampling method used.

  13. The RNA splicing factor ASF/SF2 inhibits human topoisomerase I mediated DNA relaxation

    DEFF Research Database (Denmark)

    Andersen, Félicie Faucon; Tange, Thomas Ø.; Sinnathamby, Thayaline

    2002-01-01

    Human topoisomerase I interacts with and phosphorylates the SR-family of RNA splicing factors, including ASF/SF2, and has been suggested to play an important role in the regulation of RNA splicing. Here we present evidence to support the theory that the regulation can go the other way around...... with the SR-proteins controlling topoisomerase I DNA activity. We demonstrate that the splicing factor ASF/SF2 inhibits relaxation by interfering with the DNA cleavage and/or DNA binding steps of human topoisomerase I catalysis. The inhibition of relaxation correlated with the ability of various deletion...

  14. Frequent mutations in EGFR, KRAS and TP53 genes in human lung cancer tumors detected by ion torrent DNA sequencing.

    Directory of Open Access Journals (Sweden)

    Xin Cai

    Full Text Available Lung cancer is the most common malignancy and the leading cause of cancer deaths worldwide. While smoking is by far the leading cause of lung cancer, other environmental and genetic factors influence the development and progression of the cancer. Since unique mutations patterns have been observed in individual cancer samples, identification and characterization of the distinctive lung cancer molecular profile is essential for developing more effective, tailored therapies. Until recently, personalized DNA sequencing to identify genetic mutations in cancer was impractical and expensive. The recent technological advancements in next-generation DNA sequencing, such as the semiconductor-based Ion Torrent sequencing platform, has made DNA sequencing cost and time effective with more reliable results. Using the Ion Torrent Ampliseq Cancer Panel, we sequenced 737 loci from 45 cancer-related genes to identify genetic mutations in 76 human lung cancer samples. The sequencing analysis revealed missense mutations in KRAS, EGFR, and TP53 genes in the breast cancer samples of various histologic types. Thus, this study demonstrates the necessity of sequencing individual human cancers in order to develop personalized drugs or combination therapies to effectively target individual, breast cancer-specific mutations.

  15. PIK3CA and TP53 gene mutations in human breast cancer tumors frequently detected by ion torrent DNA sequencing.

    Science.gov (United States)

    Bai, Xusheng; Zhang, Enke; Ye, Hua; Nandakumar, Vijayalakshmi; Wang, Zhuo; Chen, Lihong; Tang, Chuanning; Li, Jianhui; Li, Huijin; Zhang, Wei; Han, Wei; Lou, Feng; Zhang, Dandan; Sun, Hong; Dong, Haichao; Zhang, Guangchun; Liu, Zhiyuan; Dong, Zhishou; Guo, Baishuai; Yan, He; Yan, Chaowei; Wang, Lu; Su, Ziyi; Li, Yangyang; Jones, Lindsey; Huang, Xue F; Chen, Si-Yi; Gao, Jinglong

    2014-01-01

    Breast cancer is the most common malignancy and the leading cause of cancer deaths in women worldwide. While specific genetic mutations have been linked to 5-10% of breast cancer cases, other environmental and epigenetic factors influence the development and progression of the cancer. Since unique mutations patterns have been observed in individual cancer samples, identification and characterization of the distinctive breast cancer molecular profile is needed to develop more effective target therapies. Until recently, identifying genetic cancer mutations via personalized DNA sequencing was impractical and expensive. The recent technological advancements in next-generation DNA sequencing, such as the semiconductor-based Ion Torrent sequencing platform, has made DNA sequencing cost and time effective with more reliable results. Using the Ion Torrent Ampliseq Cancer Panel, we sequenced 737 loci from 45 cancer-related genes to identify genetic mutations in 105 human breast cancer samples. The sequencing analysis revealed missense mutations in PIK3CA, and TP53 genes in the breast cancer samples of various histologic types. Thus, this study demonstrates the necessity of sequencing individual human cancers in order to develop personalized drugs or combination therapies to effectively target individual, breast cancer-specific mutations.

  16. Frequent mutations in EGFR, KRAS and TP53 genes in human lung cancer tumors detected by ion torrent DNA sequencing.

    Science.gov (United States)

    Cai, Xin; Sheng, Jianhui; Tang, Chuanning; Nandakumar, Vijayalakshmi; Ye, Hua; Ji, Hong; Tang, Haiying; Qin, Yu; Guan, Hongwei; Lou, Feng; Zhang, Dandan; Sun, Hong; Dong, Haichao; Zhang, Guangchun; Liu, Zhiyuan; Dong, Zhishou; Guo, Baishuai; Yan, He; Yan, Chaowei; Wang, Lu; Su, Ziyi; Li, Yangyang; Jones, Lindsey; Huang, Xue F; Chen, Si-Yi; Wu, Taihua; Lin, Hongli

    2014-01-01

    Lung cancer is the most common malignancy and the leading cause of cancer deaths worldwide. While smoking is by far the leading cause of lung cancer, other environmental and genetic factors influence the development and progression of the cancer. Since unique mutations patterns have been observed in individual cancer samples, identification and characterization of the distinctive lung cancer molecular profile is essential for developing more effective, tailored therapies. Until recently, personalized DNA sequencing to identify genetic mutations in cancer was impractical and expensive. The recent technological advancements in next-generation DNA sequencing, such as the semiconductor-based Ion Torrent sequencing platform, has made DNA sequencing cost and time effective with more reliable results. Using the Ion Torrent Ampliseq Cancer Panel, we sequenced 737 loci from 45 cancer-related genes to identify genetic mutations in 76 human lung cancer samples. The sequencing analysis revealed missense mutations in KRAS, EGFR, and TP53 genes in the breast cancer samples of various histologic types. Thus, this study demonstrates the necessity of sequencing individual human cancers in order to develop personalized drugs or combination therapies to effectively target individual, breast cancer-specific mutations.

  17. DNA topoisomerases and the DNA lesion in human genetic instability syndromes.

    Science.gov (United States)

    Poot, M; Hoehn, H

    1993-04-01

    Genetically determined chromosomal instability entails, among other sequelae, a condition of elevated cancer risk. Patients with the autosomal recessive disorder Fanconi's anemia (FA) often develop leukemias of the monocytic lineage together with pancytopenia, whereas the Bloom's syndrome (BS) mutation confers an early and elevated incidence of neoplasia of no particular type. Cultured cells from FA patients show spontaneously elevated rates of chromosome aberrations and a hypersensitivity to DNA cross-linking agents. Cytogenetic evaluation of cells from BS patients revealed elevated rates of sister chromatid exchanges, which were sensitive to the bromodeoxyuridine (BrdU) concentration used in the assay. Such a BrdU sensitivity was also found in cultured cells from healthy subjects exposed to the intracellular superoxide generator paraquat or to bleomycin. Skin fibroblasts from FA and BS patients show poor growth, which in the case of FA could be mitigated by lowering the oxygen concentration to 5%. Lymphoblastoid B-cell lines derived from peripheral blood samples from FA and BS patients show elevated numbers of cells arrested in the G2 phase of the cell cycle. This phenomenon could also be provoked by exposing cell lines from healthy subjects to compounds interfering with the function of DNA topoisomerase I (camptothecin) or II (m-AMSA). To test for a putative deficiency of either DNA topoisomerase, B-cell cultures from FA and BS patients were compared with cell cultures from healthy subjects regarding their sensitivity towards camptothecin and m-AMSA. No difference in sensitivity to these agents was found in patient vs. control cell lines, thus ruling out a deficiency in DNA topoisomerase I or II as the prime defect in these conditions of elevated cancer risk. The similarity between the cell cycle kinetic patterns found in untreated FA cell lines and in normal cell lines exposed to camptothecin or m-AMSA suggest that the DNA lesion in FA, presumably being caused

  18. Linkage of DNA Methylation Quantitative Trait Loci to Human Cancer Risk

    Directory of Open Access Journals (Sweden)

    Holger Heyn

    2014-04-01

    Full Text Available Epigenetic regulation and, in particular, DNA methylation have been linked to the underlying genetic sequence. DNA methylation quantitative trait loci (meQTL have been identified through significant associations between the genetic and epigenetic codes in physiological and pathological contexts. We propose that interrogating the interplay between polymorphic alleles and DNA methylation is a powerful method for improving our interpretation of risk alleles identified in genome-wide association studies that otherwise lack mechanistic explanation. We integrated patient cancer risk genotype data and genome-scale DNA methylation profiles of 3,649 primary human tumors, representing 13 solid cancer types. We provide a comprehensive meQTL catalog containing DNA methylation associations for 21% of interrogated cancer risk polymorphisms. Differentially methylated loci harbor previously reported and as-yet-unidentified cancer genes. We suggest that such regulation at the DNA level can provide a considerable amount of new information about the biology of cancer-risk alleles.

  19. Age and metabolic risk factors associated with oxidatively damaged DNA in human peripheral blood mononuclear cells

    DEFF Research Database (Denmark)

    Løhr, Mille; Jensen, Annie; Eriksen, Louise

    2015-01-01

    Aging is associated with oxidative stress-generated damage to DNA and this could be related to metabolic disturbances. This study investigated the association between levels of oxidatively damaged DNA in peripheral blood mononuclear cells (PBMCs) and metabolic risk factors in 1,019 subjects, aged...... 18-93 years. DNA damage was analyzed as strand breaks by the comet assay and levels of formamidopyrimidine (FPG-) and human 8-oxoguanine DNA glycosylase 1 (hOGG1)-sensitive sites There was an association between age and levels of FPG-sensitive sites for women, but not for men. The same tendency...... was observed for the level of hOGG1-sensitive sites, whereas there was no association with the level of strand breaks. The effect of age on oxidatively damaged DNA in women disappeared in multivariate models, which showed robust positive associations between DNA damage and plasma levels of triglycerides...

  20. Seasonal variations of DNA damage in human lymphocytes: Correlation with different environmental variables

    Energy Technology Data Exchange (ETDEWEB)

    Giovannelli, Lisa [Dipartimento di Farmacologia Preclinica e Clinica, Universita di Firenze, Viale Pieraccini 6, 50139 Florence (Italy)]. E-mail: lisa.giovannelli@unifi.it; Pitozzi, Vanessa [Dipartimento di Farmacologia Preclinica e Clinica, Universita di Firenze, Viale Pieraccini 6, 50139 Florence (Italy); Moretti, Silvia [Department of Dermatological Sciences, University of Florence, Florence (Italy); Boddi, Vieri [Department of Public Health, University of Florence, Florence (Italy); Dolara, Piero [Dipartimento di Farmacologia Preclinica e Clinica, Universita di Firenze, Viale Pieraccini 6, 50139 Florence (Italy)

    2006-01-29

    Several types of DNA damage, including DNA breaks and DNA base oxidation, display a seasonal trend. In the present work, a sample of 79 healthy subjects living in the city of Florence, Italy, was used to analyse this effect. Three possible causative agents were taken into consideration: solar radiation, air temperature and air ozone level. DNA damage was measured in isolated human lymphocytes at different times during the year and the observed damage was correlated with the levels of these three agents in the days preceding blood sampling. Three time windows were chosen: 3, 7 and 30 days before blood sampling. DNA strand breaks and the oxidized purinic bases cleaved by the formamidopyrimidine glycosylase (FPG sites) were measured by means of the comet assay. The results of multivariate regression analysis showed a positive correlation between lymphocyte DNA damage and air temperature, and a less strong correlation with global solar radiation and air ozone levels.

  1. An exploration of sequence specific DNA-duplex/pyrene interactions for intercalated and surface-associated pyrene species. Technical progress report

    Energy Technology Data Exchange (ETDEWEB)

    Netzel, T.L.

    1994-01-07

    The use of both short (5-atom) and long (12-atom) covalent linking chains to attach, respectively, a pyrenesulfonate or a pyrenebutyrate moiety to a central region of a DNA duplex allows construction of DNA-duplex/pyrene assemblies of two types. Long linking chains permit pyrene to intercalate within the DNA duplex, while the short chains constrain pyrene to remain in the outer-surface region of the major-groove of the duplex. Electrochemical data suggest that reductive electron-transfer (ET) quenching of photoexcited pyrene (pyrene*) labels will be most exothermic for guanosine than for the other three DNA nucleosides and that oxidative ET quenching of pyrene* will be most exothermic for thymidine than for the other three DNA nucleosides. The study combines two effects, (1) differential DNA/pyrene geometries in covalent assemblies with different length linking chains and (2) differential ET quenching reactivities among the DNA nucleotides to explore sequence specific and duplex/pyrene association specific effects on DNA-base ionization reactions. This report describes progress in synthesizing target pyrene-labeled nucleosides and oligonucleotides, in commissioning our fluorescence lifetime measurement system, and in the photochemical behavior of pyrene-labeled nucleosides, single strands of DNA, and duplexes of DNA.

  2. Preliminary perspectives on DNA collection in anti-human trafficking efforts.

    Science.gov (United States)

    Katsanis, Sara H; Kim, Joyce; Minear, Mollie A; Chandrasekharan, Subhashini; Wagner, Jennifer K

    2014-01-01

    Forensic DNA methodologies have potential applications in the investigation of human trafficking cases. DNA and relationship testing may be useful for confirmation of biological relationship claims in immigration, identification of trafficked individuals who are missing persons, and family reunification of displaced individuals after mass disasters and conflicts. As these applications rely on the collection of DNA from non-criminals and potentially vulnerable individuals, questions arise as to how to address the ethical challenges of collection, security, and privacy of collected samples and DNA profiles. We administered a survey targeted to victims' advocates to gain preliminary understanding of perspectives regarding human trafficking definitions, DNA and sex workers, and perceived trust of authorities potentially involved in DNA collection. We asked respondents to consider the use of DNA for investigating adoption fraud, sex trafficking, and post-conflict child soldier cases. We found some key differences in perspectives on defining what qualifies as "trafficking." When we varied terminology between "sex worker" and "sex trafficking victim" we detected differences in perception on which authorities can be trusted. Respondents were supportive of the hypothetical models proposed to collect DNA. Most were favorable of DNA specimens being controlled by an authority outside of law enforcement. Participants voiced concerns focused on privacy, misuse of DNA samples and data, unintentional harms, data security, and infrastructure. These preliminary data indicate that while there is perceived value in programs to use DNA for investigating cases of human trafficking, these programs may need to consider levels of trust in authorities as their logistics are developed and implemented.

  3. A novel method to capture methylated human DNA from stool: implications for colorectal cancer screening.

    Science.gov (United States)

    Zou, Hongzhi; Harrington, Jonathan; Rego, Rafaela L; Ahlquist, David A

    2007-09-01

    Assay of methylated DNA markers in stool is a promising approach for colorectal cancer (CRC) screening. A method to capture hypermethylated CpG islands from stool would enrich target analyte and allow optimal assay sensitivity. Methyl-binding domain (MBD) protein was produced using a pET6HMBD plasmid with MBD DNA sequence cloned from rat MeCP2 gene and bound to a column of nickel-agarose resin. We first established the feasibility of using the MBD column to extract methylated human DNA in a high background of fecal bacterial DNA. To explore the impact of MBD enrichment on detection sensitivity, the tumor-associated methylated vimentin gene was assayed with methylation-specific PCR from stools to which low amounts of cancer cell DNA (0-50 ng) were added and from stools from CRC patients and healthy individuals. Stools from cancer patients were selected with low amounts of human DNA (median 7 ng, range 0.5-832 ng). With MBD enrichment, methylated vimentin was detected in stools enriched with >/=10 ng of cancer cell DNA and in CRC stool with a range of native human DNA amounts from 4 to 832 ng. Without MBD enrichment, methylated vimentin was not detected in the enriched stools and was detected in only 1 cancer stool with high human DNA (832 ng). In stools from healthy individuals methylated vimentin was not detected, with or without MBD enrichment. MBD capture increases assay sensitivity for detecting methylated DNA markers in stool. Applied clinical studies for stool cancer screening are indicated.

  4. [Progresses on Neandertal genomics].

    Science.gov (United States)

    Bi, Cai-Li; Guo, Guang-Yan; Zhang, Xiao; Tian, Yan-Hui; Shen, Yin-Zhu

    2012-06-01

    Neandertal is our closest known relative and also an archaic hominid reserving the richest fossils. Whether the Neandertals exchanged their DNA with modern human or not is a matter of debate on the modern human origin. The progresses on the mitochondrial and nuclear genomes of Neandertals in recent years were reviewed in this paper. Recent study has revealed possible genetic contribution of Neandertals to the modern human to some extent, which arose the rethinking of modern human origin. The experiences gained in the research on Neandertals will benefit the study on archaic hominids, unravel the mystery of modern human origin, and enrich the relative theoretical systems in evolutionary biological field.

  5. Chromosome region-specific libraries for human genome analysis. Progress report, September 1, 1991--August 31, 1992

    Energy Technology Data Exchange (ETDEWEB)

    Kao, Fa-Ten

    1992-08-01

    During the grant period progress has been made in the successful demonstration of regional mapping of microclones derived from microdissection libraries; successful demonstration of the feasibility of converting microclones with short inserts into yeast artificial chromosome clones with very large inserts for high resolution physical mapping of the dissected region; Successful demonstration of the usefulness of region-specific microclones to isolate region-specific cDNA clones as candidate genes to facilitate search for the crucial genes underlying genetic diseases assigned to the dissected region; and the successful construction of four region-specific microdissection libraries for human chromosome 2, including 2q35-q37, 2q33-q35, 2p23-p25 and 2p2l-p23. The 2q35-q37 library has been characterized in detail. The characterization of the other three libraries is in progress. These region-specific microdissection libraries and the unique sequence microclones derived from the libraries will be valuable resources for investigators engaged in high resolution physical mapping and isolation of disease-related genes residing in these chromosomal regions.

  6. Aberrant DNA methylation of WNT pathway genes in the development and progression of CIMP-negative colorectal cancer.

    Science.gov (United States)

    Galamb, Orsolya; Kalmár, Alexandra; Péterfia, Bálint; Csabai, István; Bodor, András; Ribli, Dezső; Krenács, Tibor; Patai, Árpád V; Wichmann, Barnabás; Barták, Barbara Kinga; Tóth, Kinga; Valcz, Gábor; Spisák, Sándor; Tulassay, Zsolt; Molnár, Béla

    2016-08-02

    The WNT signaling pathway has an essential role in colorectal carcinogenesis and progression, which involves a cascade of genetic and epigenetic changes. We aimed to analyze DNA methylation affecting the WNT pathway genes in colorectal carcinogenesis in promoter and gene body regions using whole methylome analysis in 9 colorectal cancer, 15 adenoma, and 6 normal tumor adjacent tissue (NAT) samples by methyl capture sequencing. Functional methylation was confirmed on 5-aza-2'-deoxycytidine-treated colorectal cancer cell line datasets. In parallel with the DNA methylation analysis, mutations of WNT pathway genes (APC, β-catenin/CTNNB1) were analyzed by 454 sequencing on GS Junior platform. Most differentially methylated CpG sites were localized in gene body regions (95% of WNT pathway genes). In the promoter regions, 33 of the 160 analyzed WNT pathway genes were differentially methylated in colorectal cancer vs. normal, including hypermethylated AXIN2, CHP1, PRICKLE1, SFRP1, SFRP2, SOX17, and hypomethylated CACYBP, CTNNB1, MYC; 44 genes in adenoma vs. NAT; and 41 genes in colorectal cancer vs. adenoma comparisons. Hypermethylation of AXIN2, DKK1, VANGL1, and WNT5A gene promoters was higher, while those of SOX17, PRICKLE1, DAAM2, and MYC was lower in colon carcinoma compared to adenoma. Inverse correlation between expression and methylation was confirmed in 23 genes, including APC, CHP1, PRICKLE1, PSEN1, and SFRP1. Differential methylation affected both canonical and noncanonical WNT pathway genes in colorectal normal-adenoma-carcinoma sequence. Aberrant DNA methylation appears already in adenomas as an early event of colorectal carcinogenesis.

  7. Comparing different post-mortem human samples as DNA sources for downstream genotyping and identification.

    Science.gov (United States)

    Calacal, Gayvelline C; Apaga, Dame Loveliness T; Salvador, Jazelyn M; Jimenez, Joseph Andrew D; Lagat, Ludivino J; Villacorta, Renato Pio F; Lim, Maria Cecilia F; Fortun, Raquel D R; Datar, Francisco A; De Ungria, Maria Corazon A

    2015-11-01

    The capability of DNA laboratories to perform genotyping procedures from post-mortem remains, including those that had undergone putrefaction, continues to be a challenge in the Philippines, a country characterized by very humid and warm conditions all year round. These environmental conditions accelerate the decomposition of human remains that were recovered after a disaster and those that were left abandoned after a crime. When considerable tissue decomposition of human remains has taken place, there is no other option but to extract DNA from bone and/or teeth samples. Routinely, femur shafts are obtained from recovered bodies for human identification because the calcium matrix protects the DNA contained in the osteocytes. In the Philippines, there is difficulty in collecting femur samples after natural disasters or even human-made disasters, because these events are usually characterized by a large number of fatalities. Identification of casualties is further delayed by limitation in human and material resources. Hence, it is imperative to test other types of biological samples that are easier to collect, transport, process and store. We analyzed DNA that were obtained from body fluid, bone marrow, muscle tissue, clavicle, femur, metatarsal, patella, rib and vertebral samples from five recently deceased untreated male cadavers and seven male human remains that were embalmed, buried for ∼ 1 month and then exhumed. The bodies had undergone different environmental conditions and were in various stages of putrefaction. A DNA extraction method utilizing a detergent-washing step followed by an organic procedure was used. The utility of bone marrow and vitreous fluid including bone marrow and vitreous fluid that was transferred on FTA(®) cards and subjected to autosomal STR and Y-STR DNA typing were also evaluated. DNA yield was measured and the presence or absence of PCR inhibitors in DNA extracts was assessed using Plexor(®)HY. All samples were amplified using

  8. Forensic aspects of mass disasters: strategic considerations for DNA-based human identification.

    Science.gov (United States)

    Budowle, Bruce; Bieber, Frederick R; Eisenberg, Arthur J

    2005-07-01

    Many mass disasters result in loss of lives. Law enforcement and/or public safety and health officials often have the responsibility for identifying the human remains found at the scene, so they can be returned to their families. The recovered human remains range from being relatively intact to highly degraded. DNA-based identity testing is a powerful tool for victim identification in that the data are not restricted to any particular one to one body landmark comparison and DNA profile comparisons can be used to associate separated remains or body parts. Even though DNA typing is straightforward, a disaster is a chaotic environment that can complicate effective identification of the remains. With some planning, or at least identification of the salient features to consider, stress can be reduced for those involved in the identification process. General guidelines are provided for developing an action plan for identification of human remains from a mass disaster by DNA analysis. These include: (1) sample collection, preservation, shipping and storage; (2) tracking and chain of custody issues; (3) laboratory facilities; (4) quality assurance and quality control practices; (5) parsing out work; (6) extraction and typing; (7) interpretation of results; (8) automation; (9) software for tracking and managing data; (10) the use of an advisory panel; (11) education and communication; and (12) privacy issues. In addition, key technologies that may facilitate the identification process are discussed, such as resin based DNA extraction, real-time PCR for quantitation of DNA, use of mini-STRs, SNP detection procedures, and software. Many of the features necessary for DNA typing of human remains from a mass disaster are the same as those for missing persons' cases. Therefore, developing a missing persons DNA identification program would also provide the basis for a mass disaster human remains DNA identification program.

  9. Human embryonic stem cells have enhanced repair of multiple forms of DNA damage

    DEFF Research Database (Denmark)

    Maynard, Scott; Swistowska, Anna Maria; Lee, Jae Wan

    2008-01-01

    Embryonic stem cells need to maintain genomic integrity so that they can retain the ability to differentiate into multiple cell types without propagating DNA errors. Previous studies have suggested that mechanisms of genome surveillance, including DNA repair, are superior in mouse embryonic stem...... cells compared with various differentiated murine cells. Using single-cell gel electrophoresis (comet assay) we found that human embryonic stem cells (BG01, I6) have more efficient repair of different types of DNA damage (generated from H2O2, UV-C, ionizing radiation, or psoralen) than human primary...... fibroblasts (WI-38, hs27) and, with the exception of UV-C damage, HeLa cells. Microarray gene expression analysis showed that mRNA levels of several DNA repair genes are elevated in human embryonic stem cells compared with their differentiated forms (embryoid bodies). These data suggest that genomic...

  10. Polymorphisms in human DNA repair genes and head and neck ...

    Indian Academy of Sciences (India)

    Sci. USA 97, 9886–9891. Viswanathan H. and Wilson J. A. 2004 Alcohol—the neglected factor in head and neck cancer. Clin. Otolaryngol. 29, 295–300. Vogel U., Hedayati M., Dybdahl M., Grossman L. and Nexo B. A.. 2001 Polymorphisms of the DNA repair gene XPD, correlations with risk of basal cell carcinoma revisited.

  11. DNA Genotype of Human Papilloma Virus Infection among ...

    African Journals Online (AJOL)

    Objective: To determine DNA genotype of HPV infection among adolescent girls at KNH - Youth Clinic and use it as an advocacy tool for the introduction of the HPV vaccine provision in the clinic. Design: Cross-sectional study. Setting: Kenyatta National Hospital Youth clinic. Subjects: Adolescent (and youths) girls aged ...

  12. Detection of human papillomavirus DNA with in situ hybridisation in ...

    African Journals Online (AJOL)

    studied and different methods used in HPV DNA detection. According to the Federation Dentaire Internationa~ intra- oral cancer, of which squamous cell carcinoma accounts for the majority of cases, is the third most common malj§nant disease among men in developing countries.'2 The g'iortality rate remains unacceptably ...

  13. Stability of human telomere quadruplexes at high DNA concentrations.

    Science.gov (United States)

    Kejnovská, Iva; Vorlíčková, Michaela; Brázdová, Marie; Sagi, Janos

    2014-04-01

    For mimicking macromolecular crowding of DNA quadruplexes, various crowding agents have been used, typically PEG, with quadruplexes of micromolar strand concentrations. Thermal and thermodynamic stabilities of these quadruplexes increased with the concentration of the agents, the rise depended on the crowder used. A different phenomenon was observed, and is presented in this article, when the crowder was the quadruplex itself. With DNA strand concentrations ranging from 3 µM to 9 mM, the thermostability did not change up to ∼2 mM, above which it increased, indicating that the unfolding quadruplex units were not monomolecular above ∼2 mM. The results are explained by self-association of the G-quadruplexes above this concentration. The ΔG(°) 37 values, evaluated only below 2 mM, did not become more negative, as with the non-DNA crowders, instead, slightly increased. Folding topology changed from antiparallel to hybrid above 2 mM, and then to parallel quadruplexes at high, 6-9 mM strand concentrations. In this range, the concentration of the DNA phosphate anions approached the concentration of the K(+) counterions used. Volume exclusion is assumed to promote the topological changes of quadruplexes toward the parallel, and the decreased screening of anions could affect their stability. Copyright © 2013 Wiley Periodicals, Inc.

  14. Evaluation of the DNA damaging effects of amitraz on human ...

    Indian Academy of Sciences (India)

    Amitraz is formamidine pesticide widely used as insecticide and acaricide. In veterinary medicine, amitraz has important uses against ticks, mites and lice on animals. Also, amitraz is used in apiculture to control Varroa destructor. It this study, the alkaline Comet assay was used to evaluate DNA damaging effects of amitraz ...

  15. Circulating mitochondrial DNA as biomarker linking environmental chemical exposure to early preclinical lesions elevation of mtDNA in human serum after exposure to carcinogenic halo-alkane-based pesticides.

    Directory of Open Access Journals (Sweden)

    Lygia T Budnik

    Full Text Available There is a need for a panel of suitable biomarkers for detection of environmental chemical exposure leading to the initiation or progression of degenerative diseases or potentially, to cancer. As the peripheral blood may contain increased levels of circulating cell-free DNA in diseased individuals, we aimed to evaluate this DNA as effect biomarker recognizing vulnerability after exposure to environmental chemicals. We recruited 164 individuals presumably exposed to halo-alkane-based pesticides. Exposure evaluation was based on human biomonitoring analysis; as biomarker of exposure parent halo-methanes, -ethanes and their metabolites, as well as the hemoglobin-adducts methyl valine and hydroxyl ethyl valine in blood were used, complemented by expert evaluation of exposure and clinical intoxication symptoms as well as a questionnaire. Assessment showed exposures to halo alkanes in the concentration range being higher than non-cancer reference doses (RfD but (mostly lower than the occupational exposure limits. We quantified circulating DNA in serum from 86 individuals with confirmed exposure to off-gassing halo-alkane pesticides (in storage facilities or in home environment and 30 non-exposed controls, and found that exposure was significantly associated with elevated serum levels of circulating mitochondrial DNA (in size of 79 bp, mtDNA-79, p = 0.0001. The decreased integrity of mtDNA (mtDNA-230/mtDNA-79 in exposed individuals implicates apoptotic processes (p = 0.015. The relative amounts of mtDNA-79 in serum were positively associated with the lag-time after intoxication to these chemicals (r = 0.99, p<0.0001. Several months of post-exposure the specificity of this biomarker increased from 30% to 97% in patients with intoxication symptoms. Our findings indicate that mitochondrial DNA has a potential to serve as a biomarker recognizing vulnerable risk groups after exposure to toxic/carcinogenic chemicals.

  16. DNA Repair in Human Pluripotent Stem Cells Is Distinct from That in Non-Pluripotent Human Cells

    Science.gov (United States)

    Luo, Li Z.; Park, Sang-Won; Bates, Steven E.; Zeng, Xianmin; Iverson, Linda E.; O'Connor, Timothy R.

    2012-01-01

    The potential for human disease treatment using human pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells (iPSCs), also carries the risk of added genomic instability. Genomic instability is most often linked to DNA repair deficiencies, which indicates that screening/characterization of possible repair deficiencies in pluripotent human stem cells should be a necessary step prior to their clinical and research use. In this study, a comparison of DNA repair pathways in pluripotent cells, as compared to those in non-pluripotent cells, demonstrated that DNA repair capacities of pluripotent cell lines were more heterogeneous than those of differentiated lines examined and were generally greater. Although pluripotent cells had high DNA repair capacities for nucleotide excision repair, we show that ultraviolet radiation at low fluxes induced an apoptotic response in these cells, while differentiated cells lacked response to this stimulus, and note that pluripotent cells had a similar apoptotic response to alkylating agent damage. This sensitivity of pluripotent cells to damage is notable since viable pluripotent cells exhibit less ultraviolet light-induced DNA damage than do differentiated cells that receive the same flux. In addition, the importance of screening pluripotent cells for DNA repair defects was highlighted by an iPSC line that demonstrated a normal spectral karyotype, but showed both microsatellite instability and reduced DNA repair capacities in three out of four DNA repair pathways examined. Together, these results demonstrate a need to evaluate DNA repair capacities in pluripotent cell lines, in order to characterize their genomic stability, prior to their pre-clinical and clinical use. PMID:22412831

  17. Emphysema Distribution and Diffusion Capacity Predict Emphysema Progression in Human Immunodeficiency Virus Infection

    Science.gov (United States)

    Leung, Janice M; Malagoli, Andrea; Santoro, Antonella; Besutti, Giulia; Ligabue, Guido; Scaglioni, Riccardo; Dai, Darlene; Hague, Cameron; Leipsic, Jonathon; Sin, Don D.; Man, SF Paul; Guaraldi, Giovanni

    2016-01-01

    Background Chronic obstructive pulmonary disease (COPD) and emphysema are common amongst patients with human immunodeficiency virus (HIV). We sought to determine the clinical factors that are associated with emphysema progression in HIV. Methods 345 HIV-infected patients enrolled in an outpatient HIV metabolic clinic with ≥2 chest computed tomography scans made up the study cohort. Images were qualitatively scored for emphysema based on percentage involvement of the lung. Emphysema progression was defined as any increase in emphysema score over the study period. Univariate analyses of clinical, respiratory, and laboratory data, as well as multivariable logistic regression models, were performed to determine clinical features significantly associated with emphysema progression. Results 17.4% of the cohort were emphysema progressors. Emphysema progression was most strongly associated with having a low baseline diffusion capacity of carbon monoxide (DLCO) and having combination centrilobular and paraseptal emphysema distribution. In adjusted models, the odds ratio (OR) for emphysema progression for every 10% increase in DLCO percent predicted was 0.58 (95% confidence interval [CI] 0.41–0.81). The equivalent OR (95% CI) for centrilobular and paraseptal emphysema distribution was 10.60 (2.93–48.98). Together, these variables had an area under the curve (AUC) statistic of 0.85 for predicting emphysema progression. This was an improvement over the performance of spirometry (forced expiratory volume in 1 second to forced vital capacity ratio), which predicted emphysema progression with an AUC of only 0.65. Conclusion Combined paraseptal and centrilobular emphysema distribution and low DLCO could identify HIV patients who may experience emphysema progression. PMID:27902753

  18. Disease progression from chronic hepatitis C to cirrhosis and hepatocellular carcinoma is associated with increasing DNA promoter methylation.

    Science.gov (United States)

    Zekri, Abd El-Rahman Nabawy; Nassar, Auhood Abdel-Monem; El-Din El-Rouby, Mahmoud Nour; Shousha, Hend Ibrahim; Barakat, Ahmed Barakat; El-Desouky, Eman Desouky; Zayed, Naglaa Ali; Ahmed, Ola Sayed; El-Din Youssef, Amira Salah; Kaseb, Ahmed Omar; Abd El-Aziz, Ashraf Omar; Bahnassy, Abeer Ahmed

    2014-01-01

    Changes in DNA methylation patterns are believed to be early events in hepatocarcinogenesis. A better understanding of methylation states and how they correlate with disease progression will aid in finding potential strategies for early detection of HCC. The aim of our study was to analyze the methylation frequency of tumor suppressor genes, P14, P15, and P73, and a mismatch repair gene (O6MGMT) in HCV related chronic liver disease and HCC to identify candidate epigenetic biomarkers for HCC prediction. 516 Egyptian patients with HCV-related liver disease were recruited from Kasr Alaini multidisciplinary HCC clinic from April 2010 to January 2012. Subjects were divided into 4 different clinically defined groups - HCC group (n=208), liver cirrhosis group (n=108), chronic hepatitis C group (n=100), and control group (n=100) - to analyze the methylation status of the target genes in patient plasma using EpiTect Methyl qPCR Array technology. Methylation was considered to be hypermethylated if >10% and/or intermediately methylated if >60%. In our series, a significant difference in the hypermethylation status of all studied genes was noted within the different stages of chronic liver disease and ultimately HCC. Hypermethylation of the P14 gene was detected in 100/208 (48.1%), 52/108 (48.1%), 16/100 (16%) and 8/100 (8%) among HCC, liver cirrhosis, chronic hepatitis and control groups, respectively, with a statistically significant difference between the studied groups (p-value 0.008). We also detected P15 hypermethylation in 92/208 (44.2%), 36/108 (33.3%), 20/100 (20%) and 4/100 (4%) , respectively (p-value 0.006). In addition, hypermethylation of P73 was detected in 136/208 (65.4%), 72/108 (66.7%), 32/100 (32%) and 4/100 (4%) (p-value <0.001). Also, we detected O6MGMT hypermethylation in 84/208 (40.4%), 60/108 (55.3%), 20/100 (20%) and 4/100 (4%), respectively (p value <0.001. The epigenetic changes observed in this study indicate that HCC tumors exhibit specific DNA

  19. DHX9 helicase is involved in preventing genomic instability induced by alternatively structured DNA in human cells

    Science.gov (United States)

    Jain, Aklank; Bacolla, Albino; del Mundo, Imee M.; Zhao, Junhua; Wang, Guliang; Vasquez, Karen M.

    2013-01-01

    Sequences that have the capacity to adopt alternative (i.e. non-B) DNA structures in the human genome have been implicated in stimulating genomic instability. Previously, we found that a naturally occurring intra-molecular triplex (H-DNA) caused genetic instability in mammals largely in the form of DNA double-strand breaks. Thus, it is of interest to determine the mechanism(s) involved in processing H-DNA. Recently, we demonstrated that human DHX9 helicase preferentially unwinds inter-molecular triplex DNA in vitro. Herein, we used a mutation-reporter system containing H-DNA to examine the relevance of DHX9 activity on naturally occurring H-DNA structures in human cells. We found that H-DNA significantly increased mutagenesis in small-interfering siRNA-treated, DHX9-depleted cells, affecting mostly deletions. Moreover, DHX9 associated with H-DNA in the context of supercoiled plasmids. To further investigate the role of DHX9 in the recognition/processing of H-DNA, we performed binding assays in vitro and chromatin immunoprecipitation assays in U2OS cells. DHX9 recognized H-DNA, as evidenced by its binding to the H-DNA structure and enrichment at the H-DNA region compared with a control region in human cells. These composite data implicate DHX9 in processing H-DNA structures in vivo and support its role in the overall maintenance of genomic stability at sites of alternatively structured DNA. PMID:24049074

  20. Teaching Progress: A Critique of the Grand Narrative of Human Rights as Pedagogy for Marginalized Students

    Directory of Open Access Journals (Sweden)

    Robyn Linde

    2015-10-01

    Full Text Available With the adoption of the Universal Declaration of Human Rights in 1948, education about human rights became an important focus of the new human rights regime and a core method of spreading its values throughout the world. This story of human rights is consistently presented as a progressive teleology that contextualizes the expansion of rights within a larger grand narrative of liberalization, emancipation, and social justice. This paper examines the disjuncture between the grand narrative on international movements for human rights and social justice and the lived experiences of marginalized students in urban environments in the United States. Drawing on our experience as professors who teach human rights, social justice, and social movements courses at an urban, four-year college in Providence, R.I., with a student body which includes large populations of students who are of color, first-generation, economically disadvantaged, and nontraditional in other ways, we explore the relevance and impact of these grand narratives for the lives of our students and their sense of agency. In particular, we advocate for a critical and transformational approach to human rights pedagogy to counter and overcome the pervasive individualization that undergirds the grand narrative of human rights. We argue that a critical (and radical human rights pedagogy must evaluate the position of the individual in modern life if liberation through human rights law and activism is to be possible.

  1. Replication of alpha-satellite DNA arrays in endogenous human centromeric regions and in human artificial chromosome.

    Science.gov (United States)

    Erliandri, Indri; Fu, Haiqing; Nakano, Megumi; Kim, Jung-Hyun; Miga, Karen H; Liskovykh, Mikhail; Earnshaw, William C; Masumoto, Hiroshi; Kouprina, Natalay; Aladjem, Mirit I; Larionov, Vladimir

    2014-10-01

    In human chromosomes, centromeric regions comprise megabase-size arrays of 171 bp alpha-satellite DNA monomers. The large distances spanned by these arrays preclude their replication from external sites and imply that the repetitive monomers contain replication origins. However, replication within these arrays has not previously been profiled and the role of alpha-satellite DNA in initiation of DNA replication has not yet been demonstrated. Here, replication of alpha-satellite DNA in endogenous human centromeric regions and in de novo formed Human Artificial Chromosome (HAC) was analyzed. We showed that alpha-satellite monomers could function as origins of DNA replication and that replication of alphoid arrays organized into centrochromatin occurred earlier than those organized into heterochromatin. The distribution of inter-origin distances within centromeric alphoid arrays was comparable to the distribution of inter-origin distances on randomly selected non-centromeric chromosomal regions. Depletion of CENP-B, a kinetochore protein that binds directly to a 17 bp CENP-B box motif common to alpha-satellite DNA, resulted in enrichment of alpha-satellite sequences for proteins of the ORC complex, suggesting that CENP-B may have a role in regulating the replication of centromeric regions. Mapping of replication initiation sites in the HAC revealed that replication preferentially initiated in transcriptionally active regions. Published by Oxford University Press on behalf of Nucleic Acids Research 2014. This work is written by (a) US Government employee(s) and is in the public domain in the US.

  2. Validation of human papillomavirus genotyping by signature DNA sequence analysis.

    Science.gov (United States)

    Lee, Sin Hang; Vigliotti, Veronica S; Vigliotti, Jessica S; Pappu, Suri

    2009-05-22

    Screening with combined cytologic and HPV testing has led to the highest number of excessive colposcopic referrals due to high false positive rates of the current HPV testing in the USA. How best to capitalize on the enhanced sensitivity of HPV DNA testing while minimizing false-positive results from its lower specificity is an important task for the clinical pathologists. The HPV L1 gene DNA in liquid-based Pap cytology specimens was initially amplified by the degenerate MY09/MY11 PCR primers and then re-amplified by the nested GP5+/GP6+ primers, or the heminested GP6/MY11, heminested GP5/MY09 primers or their modified equivalent without sample purification or DNA extraction. The nested PCR products were used for direct automated DNA sequencing. A 34- to 50-base sequence including the GP5+ priming site was selected as the signature sequence for routine genotyping by online BLAST sequence alignment algorithms. Of 3,222 specimens, 352 were found to contain HPV DNA, with 92% of the positive samples infected by only 1 of the 35 HPV genotypes detected and 8% by more than 1 HPV genotype. The most common genotype was HPV-16 (68 isolates), followed by HPV-52 (25 isolates). More than half (53.7%) of the total number of HPV isolates relied on a nested PCR for detection although the majority of HPV-16, -18, -31, -33 -35 and -58 isolates were detected by a single MY09/MY11 PCR. Alignment of a 34-base sequence downstream of the GP5+ site failed to distinguish some isolates of HPV-16, -31 and -33. Novel variants of HPV with less than "100% identities" signature sequence match with those stored in the Genbank database were also detected by signature DNA sequencing in this rural and suburban population of the United States. Laboratory staff must be familiar with the limitations of the consensus PCR primers, the locations of the signature sequence in the L1 gene for some HPV genotypes, and HPV genotype sequence variants in order to perform accurate HPV genotyping.

  3. Validation of human papillomavirus genotyping by signature DNA sequence analysis

    Directory of Open Access Journals (Sweden)

    Vigliotti Jessica S

    2009-05-01

    Full Text Available Abstract Background Screening with combined cytologic and HPV testing has led to the highest number of excessive colposcopic referrals due to high false positive rates of the current HPV testing in the USA. How best to capitalize on the enhanced sensitivity of HPV DNA testing while minimizing false-positive results from its lower specificity is an important task for the clinical pathologists. Methods The HPV L1 gene DNA in liquid-based Pap cytology specimens was initially amplified by the degenerate MY09/MY11 PCR primers and then re-amplified by the nested GP5+/GP6+ primers, or the heminested GP6/MY11, heminested GP5/MY09 primers or their modified equivalent without sample purification or DNA extraction. The nested PCR products were used for direct automated DNA sequencing. A 34- to 50-base sequence including the GP5+ priming site was selected as the signature sequence for routine genotyping by online BLAST sequence alignment algorithms. Results Of 3,222 specimens, 352 were found to contain HPV DNA, with 92% of the positive samples infected by only 1 of the 35 HPV genotypes detected and 8% by more than 1 HPV genotype. The most common genotype was HPV-16 (68 isolates, followed by HPV-52 (25 isolates. More than half (53.7% of the total number of HPV isolates relied on a nested PCR for detection although the majority of HPV-16, -18, -31, -33 -35 and -58 isolates were detected by a single MY09/MY11 PCR. Alignment of a 34-base sequence downstream of the GP5+ site failed to distinguish some isolates of HPV-16, -31 and -33. Novel variants of HPV with less than "100% identities" signature sequence match with those stored in the Genbank database were also detected by signature DNA sequencing in this rural and suburban population of the United States. Conclusion Laboratory staff must be familiar with the limitations of the consensus PCR primers, the locations of the signature sequence in the L1 gene for some HPV genotypes, and HPV genotype sequence

  4. Evaluation of methods for the extraction and purification of DNA from the human microbiome.

    Directory of Open Access Journals (Sweden)

    Sanqing Yuan

    Full Text Available DNA extraction is an essential step in all cultivation-independent approaches to characterize microbial diversity, including that associated with the human body. A fundamental challenge in using these approaches has been to isolate DNA that is representative of the microbial community sampled.In this study, we statistically evaluated six commonly used DNA extraction procedures using eleven human-associated bacterial species and a mock community that contained equal numbers of those eleven species. These methods were compared on the basis of DNA yield, DNA shearing, reproducibility, and most importantly representation of microbial diversity. The analysis of 16S rRNA gene sequences from a mock community showed that the observed species abundances were significantly different from the expected species abundances for all six DNA extraction methods used.Protocols that included bead beating and/or mutanolysin produced significantly better bacterial community structure representation than methods without both of them. The reproducibility of all six methods was similar, and results from different experimenters and different times were in good agreement. Based on the evaluations done it appears that DNA extraction procedures for bacterial community analysis of human associated samples should include bead beating and/or mutanolysin to effectively lyse cells.

  5. Islet expression of the DNA repair enzyme 8-oxoguanosine DNA glycosylase (Ogg1 in human type 2 diabetes

    Directory of Open Access Journals (Sweden)

    Yoon Kun-Ho

    2002-04-01

    Full Text Available Abstract Background It has become increasingly clear that β-cell failure plays a critical role in the pathogenesis of type 2 diabetes. Free-radical mediated β-cell damage has been intensively studied in type 1 diabetes, but not in human type 2 diabetes. Therefore, we studied the protein expression of the DNA repair enzyme Ogg1 in pancreases from type 2 diabetics. Ogg1 was studied because it is the major enzyme involved in repairing 7,8-dihydro-8-oxoguanosine DNA adducts, a lesion previously observed in a rat model of type 2 diabetes. Moreover, in a gene expression screen, Ogg1 was over-expressed in islets from a human type 2 diabetic. Methods Immunofluorescent staining of Ogg1 was performed on pancreatic specimens from healthy controls and patients with diabetes for 2–23 years. The intensity and islet area stained for Ogg1 was evaluated by semi-quantitative scoring. Results Both the intensity and the area of islet Ogg1 staining were significantly increased in islets from the type 2 diabetic subjects compared to the healthy controls. A correlation between increased Ogg1 fluorescent staining intensity and duration of diabetes was also found. Most of the staining observed was cytoplasmic, suggesting that mitochondrial Ogg1 accounts primarily for the increased Ogg1 expression. Conclusion We conclude that oxidative stress related DNA damage may be a novel important factor in the pathogenesis of human type 2 diabetes. An increase of Ogg1 in islet cell mitochondria is consistent with a model in which hyperglycemia and consequent increased β-cell oxidative metabolism lead to DNA damage and the induction of Ogg1 expression.

  6. Human in vitro skin organ culture as a model system for evaluating DNA repair.

    Science.gov (United States)

    Liu, Hannah; Tuchinda, Papapit; Fishelevich, Rita; Harberts, Erin; Gaspari, Anthony A

    2014-06-01

    UV-exposures result in accumulation of genetic lesions that facilitate the development of skin cancer. Numerous pharmacologic agents are currently under development to both inhibit formation of DNA lesions and enhance repair. Drugs must be evaluated in vitro, currently performed in cell culture systems, before being tested on humans. Current systems do not account for the architecture and diverse cellularity of intact human skin. To establish a novel, functionally viable, and reproducible in vitro skin organ culture system for studying the effects of various pharmacologic agents on DNA repair. Human skin was obtained from neonatal foreskins. Intact skin punches derived from foreskins were cultured in vitro prior to exposure to UV-irradiation, and evaluated for DNA-damage using a DNA dot blot. Serial skin biopsies were obtained from patients with actinic keratoses treated with topical imiquimod. Expression of immune-stimulating and DNA repair genes was evaluated in ex vivo and in vitro samples. DNA dot blots revealed active repair of UV induced lesions in our in vitro skin organ culture. The photo-protective effect of sunscreen was detected, while imiquimod treatment did not enhance DNA repair in vitro. The DNA repair molecules XPA and XPF were up-regulated in the skin of imiquimod treated patients with actinic keratoses and imiquimod treated bone marrow-derived cell lines, but not keratinocytes. Our in vitro human skin organ culture model detected repair of UV-induced DNA lesions, and may be easily adapted to investigate various photo-protective drugs intended to prevent or treat skin cancer. Copyright © 2014 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  7. Construction and analysis of SSH cDNA library of human vascular endothelial cells related to gastrocarcinoma

    OpenAIRE

    Liu, Yong-Bo; Wei, Zhao-Xia; Li, Li; Li, Hang-Sheng; Chen, Hui; Li, Xiao-Wen

    2003-01-01

    AIM: To construct subtracted cDNA libraries of human vascular endothelial cells (VECs) related to gastrocarcinoma using suppression substractive hybridization (SSH) and to analyze cDNA libraries of gastrocarcinoma and VECs in Cancer Gene Anatomy Project (CGAP) database.

  8. PLASMID DNA DAMAGE CAUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITIN

    Science.gov (United States)

    Plasmid DNA damage caused by methylated arsenicals, ascorbic acid and human liver ferritin. Arsenic causes cancer in human skin, urinary bladder, lung, liver and kidney and is a significant world-wide public health problem. Although the metabolism of inorganic arsenic is ...

  9. Human CD4+ T cells require exogenous cystine for glutathione and DNA synthesis

    DEFF Research Database (Denmark)

    Levring, Trine B; Kongsbak-Wismann, Martin; Rode, Anna Kathrine Obelitz

    2015-01-01

    . The aim of this study was to elucidate why activated human T cells require exogenous Cys2 in order to proliferate. We activated purified naïve human CD4+ T cells and found that glutathione (GSH) levels and DNA synthesis were dependent on Cys2 and increased in parallel with increasing concentrations of Cys...

  10. Low prevalence of DNA virus in the human endometrium og endometriosis

    DEFF Research Database (Denmark)

    Vestergaard, Anna Lindeløv; Knudsen, Ulla Breth; Munk, Torben

    2010-01-01

    biopsies and subjected to highly sensitive PCR tests detecting human papillomavirus (HPV) types, the herpes family viruses HSV-1 and -2, CMV, and EBV, and the polyomaviruses SV40, JCV, BKV, KIV, WUV, and MCV. The prevalence of pathogenic DNA viruses in the human endometrium was generally low (0...

  11. (Pheo)melanin photosensitizes UVA-induced DNA damage in cultured human melanocytes

    NARCIS (Netherlands)

    Wenczl, E.; Schans, G.P. van der; Roza, L.; Kolb, R.M.; Timmerman, A.J.; Smit, N.P.M.; Pavel, S.; Schothorst, A.A.

    1998-01-01

    The question of whether melanins are photoprotecting and/or photosensitizing in human skin cells continues to be debated. To evaluate the role of melanin upon UVA irradiation, DNA single-strand breaks (ssb) were measured in human melanocytes differing only in the amount of pigment produced by

  12. DNA polymerase kappa protects human cells against MMC-induced genotoxicity through error-free translesion DNA synthesis.

    Science.gov (United States)

    Kanemaru, Yuki; Suzuki, Tetsuya; Sassa, Akira; Matsumoto, Kyomu; Adachi, Noritaka; Honma, Masamitsu; Numazawa, Satoshi; Nohmi, Takehiko

    2017-01-01

    Interactions between genes and environment are critical factors for causing cancer in humans. The genotoxicity of environmental chemicals can be enhanced via the modulation of susceptible genes in host human cells. DNA polymerase kappa (Pol κ) is a specialized DNA polymerase that plays an important role in DNA damage tolerance through translesion DNA synthesis. To better understand the protective roles of Pol κ, we previously engineered two human cell lines either deficient in expression of Pol κ (KO) or expressing catalytically dead Pol κ (CD) in Nalm-6-MSH+ cells and examined cytotoxic sensitivity against various genotoxins. In this study, we set up several genotoxicity assays with cell lines possessing altered Pol κ activities and investigated the protective roles of Pol κ in terms of genotoxicity induced by mitomycin C (MMC), a therapeutic agent that induces bulky DNA adducts and crosslinks in DNA. We introduced a frameshift mutation in one allele of the thymidine kinase (TK) gene of the KO, CD, and wild-type Pol κ cells (WT), thereby establishing cell lines for the TK gene mutation assay, namely TK+/- cells. In addition, we formulated experimental conditions to conduct chromosome aberration (CA) and sister chromatid exchange (SCE) assays with cells. By using the WT TK+/- and KO TK+/- cells, we assayed genotoxicity of MMC. In the TK gene mutation assay, the cytotoxic and mutagenic sensitivities of KO TK+/- cells were higher than those of WT TK+/- cells. MMC induced loss of heterozygosity (LOH), base pair substitutions at CpG sites and tandem mutations at GpG sites in both cell lines. However, the frequencies of LOH and base substitutions at CpG sites were significantly higher in KO TK+/- cells than in WT TK+/- cells. MMC also induced CA and SCE in both cell lines. The KO TK+/- cells displayed higher sensitivity than that displayed by WT TK+/- cells in the SCE assay. These results suggest that Pol κ is a modulating factor for the genotoxicity of MMC and

  13. Decreased hepatotoxic bile acid composition and altered synthesis in progressive human nonalcoholic fatty liver disease

    Energy Technology Data Exchange (ETDEWEB)

    Lake, April D. [University of Arizona, Department of Pharmacology and Toxicology, Tucson, AZ 85721 (United States); Novak, Petr [Biology Centre ASCR, Institute of Plant Molecular Biology, Ceske Budejovice 37001 (Czech Republic); Shipkova, Petia; Aranibar, Nelly; Robertson, Donald; Reily, Michael D. [Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Co., Princeton, NJ 08543 (United States); Lu, Zhenqiang [The Arizona Statistical Consulting Laboratory, University of Arizona, Tucson, AZ 85721 (United States); Lehman-McKeeman, Lois D. [Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Co., Princeton, NJ 08543 (United States); Cherrington, Nathan J., E-mail: cherrington@pharmacy.arizona.edu [University of Arizona, Department of Pharmacology and Toxicology, Tucson, AZ 85721 (United States)

    2013-04-15

    Bile acids (BAs) have many physiological roles and exhibit both toxic and protective influences within the liver. Alterations in the BA profile may be the result of disease induced liver injury. Nonalcoholic fatty liver disease (NAFLD) is a prevalent form of chronic liver disease characterized by the pathophysiological progression from simple steatosis to nonalcoholic steatohepatitis (NASH). The hypothesis of this study is that the ‘classical’ (neutral) and ‘alternative’ (acidic) BA synthesis pathways are altered together with hepatic BA composition during progression of human NAFLD. This study employed the use of transcriptomic and metabolomic assays to study the hepatic toxicologic BA profile in progressive human NAFLD. Individual human liver samples diagnosed as normal, steatosis, and NASH were utilized in the assays. The transcriptomic analysis of 70 BA genes revealed an enrichment of downregulated BA metabolism and transcription factor/receptor genes in livers diagnosed as NASH. Increased mRNA expression of BAAT and CYP7B1 was observed in contrast to decreased CYP8B1 expression in NASH samples. The BA metabolomic profile of NASH livers exhibited an increase in taurine together with elevated levels of conjugated BA species, taurocholic acid (TCA) and taurodeoxycholic acid (TDCA). Conversely, cholic acid (CA) and glycodeoxycholic acid (GDCA) were decreased in NASH liver. These findings reveal a potential shift toward the alternative pathway of BA synthesis during NASH, mediated by increased mRNA and protein expression of CYP7B1. Overall, the transcriptomic changes of BA synthesis pathway enzymes together with altered hepatic BA composition signify an attempt by the liver to reduce hepatotoxicity during disease progression to NASH. - Highlights: ► Altered hepatic bile acid composition is observed in progressive NAFLD. ► Bile acid synthesis enzymes are transcriptionally altered in NASH livers. ► Increased levels of taurine and conjugated bile acids

  14. Genetic identification of missing persons: DNA analysis of human remains and compromised samples.

    Science.gov (United States)

    Alvarez-Cubero, M J; Saiz, M; Martinez-Gonzalez, L J; Alvarez, J C; Eisenberg, A J; Budowle, B; Lorente, J A

    2012-01-01

    Human identification has made great strides over the past 2 decades due to the advent of DNA typing. Forensic DNA typing provides genetic data from a variety of materials and individuals, and is applied to many important issues that confront society. Part of the success of DNA typing is the generation of DNA databases to help identify missing persons and to develop investigative leads to assist law enforcement. DNA databases house DNA profiles from convicted felons (and in some jurisdictions arrestees), forensic evidence, human remains, and direct and family reference samples of missing persons. These databases are essential tools, which are becoming quite large (for example the US Database contains 10 million profiles). The scientific, governmental and private communities continue to work together to standardize genetic markers for more effective worldwide data sharing, to develop and validate robust DNA typing kits that contain the reagents necessary to type core identity genetic markers, to develop technologies that facilitate a number of analytical processes and to develop policies to make human identity testing more effective. Indeed, DNA typing is integral to resolving a number of serious criminal and civil concerns, such as solving missing person cases and identifying victims of mass disasters and children who may have been victims of human trafficking, and provides information for historical studies. As more refined capabilities are still required, novel approaches are being sought, such as genetic testing by next-generation sequencing, mass spectrometry, chip arrays and pyrosequencing. Single nucleotide polymorphisms offer the potential to analyze severely compromised biological samples, to determine the facial phenotype of decomposed human remains and to predict the bioancestry of individuals, a new focus in analyzing this type of markers. Copyright © 2012 S. Karger AG, Basel.

  15. From Oxford to Hawaii Ecophysiological Barriers Limit Human Progression in Ten Sport Monuments

    Science.gov (United States)

    Desgorces, François-Denis; Berthelot, Geoffroy; El Helou, Nour; Thibault, Valérie; Guillaume, Marion; Tafflet, Muriel; Hermine, Olivier; Toussaint, Jean-François

    2008-01-01

    In order to understand the determinants and trends of human performance evolution, we analyzed ten outdoor events among the oldest and most popular in sports history. Best performances of the Oxford-Cambridge boat race (since 1836), the channel crossing in swimming (1875), the hour cycling record (1893), the Elfstedentocht speed skating race (1909), the cross country ski Vasaloppet (1922), the speed ski record (1930), the Streif down-hill in Kitzbühel (1947), the eastward and westward sailing transatlantic records (1960) and the triathlon Hawaii ironman (1978) all follow a similar evolutive pattern, best described through a piecewise exponential decaying model (r2 = 0.95±0.07). The oldest events present highest progression curvature during their early phase. Performance asymptotic limits predicted from the model may be achieved in fourty years (2049±32 y). Prolonged progression may be anticipated in disciplines which further rely on technology such as sailing and cycling. Human progression in outdoor sports tends to asymptotic limits depending on physiological and environmental parameters and may temporarily benefit from further technological progresses. PMID:18985149

  16. From Oxford to Hawaii ecophysiological barriers limit human progression in ten sport monuments.

    Directory of Open Access Journals (Sweden)

    François-Denis Desgorces

    Full Text Available In order to understand the determinants and trends of human performance evolution, we analyzed ten outdoor events among the oldest and most popular in sports history. Best performances of the Oxford-Cambridge boat race (since 1836, the channel crossing in swimming (1875, the hour cycling record (1893, the Elfstedentocht speed skating race (1909, the cross country ski Vasaloppet (1922, the speed ski record (1930, the Streif down-hill in Kitzbühel (1947, the eastward and westward sailing transatlantic records (1960 and the triathlon Hawaii ironman (1978 all follow a similar evolutive pattern, best described through a piecewise exponential decaying model (r(2 = 0.95+/-0.07. The oldest events present highest progression curvature during their early phase. Performance asymptotic limits predicted from the model may be achieved in fourty years (2049+/-32 y. Prolonged progression may be anticipated in disciplines which further rely on technology such as sailing and cycling. Human progression in outdoor sports tends to asymptotic limits depending on physiological and environmental parameters and may temporarily benefit from further technological progresses.

  17. Targeted sampling of cementum for recovery of nuclear DNA from human teeth and the impact of common decontamination measures

    OpenAIRE

    Higgins, Denice; Kaidonis, John; Townsend, Grant; Hughes, Toby; Austin, Jeremy J.

    2013-01-01

    Background Teeth are a valuable source of DNA for identification of fragmented and degraded human remains. While the value of dental pulp as a source of DNA is well established, the quantity and presentation of DNA in the hard dental tissues has not been extensively studied. Without this knowledge common decontamination, sampling and DNA extraction techniques may be suboptimal. Targeted sampling of specific dental tissues could maximise DNA profiling success, while minimising the need for lab...

  18. Genetic architecture of human fibrotic diseases: disease risk and disease progression

    Directory of Open Access Journals (Sweden)

    Agnès eGardet

    2013-12-01

    Full Text Available Genetic studies of human diseases have identified multiple genetic risk loci for various fibrotic diseases. This has provided insights into the myriad of biological pathways potentially involved in disease pathogenesis. These discoveries suggest that alterations in immune responses, barrier function, metabolism and telomerase activity may be implicated in the genetic risks for fibrotic diseases. In addition to genetic disease-risks, the identification of genetic disease-modifiers associated with disease complications, severity or prognosis provides crucial insights into the biological processes implicated in disease progression. Understanding the biological processes driving disease progression may be critical to delineate more effective strategies for therapeutic interventions. This review provides an overview of current knowledge and gaps regarding genetic disease-risks and genetic disease-modifiers in human fibrotic diseases.

  19. DNA flow cytometry of human spermatozoa: consistent stoichiometric staining of sperm DNA using a novel decondensation protocol.

    Science.gov (United States)

    Kovács, Tamás; Békési, Gyöngyi; Fábián, Akos; Rákosy, Zsuzsa; Horváth, Gábor; Mátyus, László; Balázs, Margit; Jenei, Attila

    2008-10-01

    Rapid flow cytometric measurement of the frequency of aneuploid human sperms is in increasing demand but development of an exploitable method is hindered by difficulties of stoichiometric staining of sperm DNA. An aggressive decondensation protocol is needed after which cell integrity still remains intact. We used flow cytometry to examine the effect of lithium diiodosalicylate (LIS, chaotropic agent) on fluorescence intensity of propidium iodide-treated human spermatozoa from 10 normozoospermic men. When flow cytometric identification of diploid spermatozoa was achieved, validation was performed after sorting by three-color FISH. In contrast with the extremely variable histograms of nondecondensed sperms, consistent identification of haploid and diploid spermatozoa was possible if samples were decondensed with LIS prior to flow cytometry. A 76-fold enrichment of diploid sperms was observed in the sorted fractions by FISH. A significant correlation was found between the proportion of sorted cells and of diploid sperms by FISH. Application of LIS during the preparation of sperm for flow cytometry appears to ensure the stoichiometric staining of sperm DNA, making quantification of aneuploid sperm percentage possible. To our knowledge this is the first report in terms of separating spermatozoa with confirmedly abnormal chromosomal content. High correlation between the proportion of cells identified as having double DNA content by flow cytometry and diploid sperm by FISH allows rapid calculation of diploidy rate. Copyright 2008 International Society for Advancement of Cytometry.

  20. CHD1L promotes hepatocellular carcinoma progression and metastasis in mice and is associated with these processes in human patients.

    Science.gov (United States)

    Chen, Leilei; Chan, Tim Hon Man; Yuan, Yun-Fei; Hu, Liang; Huang, Jun; Ma, Stephanie; Wang, Jian; Dong, Sui-Sui; Tang, Kwan Ho; Xie, Dan; Li, Yan; Guan, Xin-Yuan

    2010-04-01

    Chromodomain helicase/ATPase DNA binding protein 1-like gene (CHD1L) is a recently identified oncogene localized at 1q21, a frequently amplified region in hepatocellular carcinoma (HCC). To explore its oncogenic mechanisms, we set out to identify CHD1L-regulated genes using a chromatin immunoprecipitation-based (ChIP-based) cloning strategy in a human HCC cell line. We then further characterized 1 identified gene, ARHGEF9, which encodes a specific guanine nucleotide exchange factor (GEF) for the Rho small GTPase Cdc42. Overexpression of ARHGEF9 was detected in approximately half the human HCC samples analyzed and positively correlated with CHD1L overexpression. In vitro and in vivo functional studies in mice showed that CHD1L contributed to tumor cell migration, invasion, and metastasis by increasing cell motility and inducing filopodia formation and epithelial-mesenchymal transition (EMT) via ARHGEF9-mediated Cdc42 activation. Silencing ARHGEF9 expression by RNAi effectively abolished the invasive and metastatic abilities of CHD1L in mice. Furthermore, investigation of clinical HCC specimens showed that CHD1L and ARHGEF9 were markedly overexpressed in metastatic HCC tissue compared with healthy tissue. Increased expression of CHD1L was often observed at the invasive front of HCC tumors and correlated with venous infiltration, microsatellite tumor nodule formation, and poor disease-free survival. These findings suggest that CHD1L-ARHGEF9-Cdc42-EMT might be a novel pathway involved in HCC progression and metastasis.

  1. Quantitation of Human Papillomavirus DNA in Plasma of Oropharyngeal Carcinoma Patients

    Energy Technology Data Exchange (ETDEWEB)

    Cao Hongbin; Banh, Alice [Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA (United States); Kwok, Shirley [Department of Pathology, Stanford University School of Medicine, Stanford, CA (United States); Shi Xiaoli; Wu, Simon; Krakow, Trevor; Khong, Brian; Bavan, Brindha [Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA (United States); Bala, Rajeev; Pinsky, Benjamin A. [Department of Pathology, Stanford University School of Medicine, Stanford, CA (United States); Colevas, Dimitrios [Department of Medicine, Stanford University School of Medicine, Stanford, CA (United States); Pourmand, Nader [Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, CA (United States); Koong, Albert C. [Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA (United States); Kong, Christina S. [Department of Pathology, Stanford University School of Medicine, Stanford, CA (United States); Le, Quynh-Thu, E-mail: qle@stanford.edu [Department of Radiation Oncology, Stanford University School of Medicine, Stanford, CA (United States)

    2012-03-01

    Purpose: To determine whether human papillomavirus (HPV) DNA can be detected in the plasma of patients with HPV-positive oropharyngeal carcinoma (OPC) and to monitor its temporal change during radiotherapy. Methods and Materials: We used polymerase chain reaction to detect HPV DNA in the culture media of HPV-positive SCC90 and VU147T cells and the plasma of SCC90 and HeLa tumor-bearing mice, non-tumor-bearing controls, and those with HPV-negative tumors. We used real-time quantitative polymerase chain reaction to quantify the plasma HPV DNA in 40 HPV-positive OPC, 24 HPV-negative head-and-neck cancer patients and 10 non-cancer volunteers. The tumor HPV status was confirmed by p16{sup INK4a} staining and HPV16/18 polymerase chain reaction or HPV in situ hybridization. A total of 14 patients had serial plasma samples for HPV DNA quantification during radiotherapy. Results: HPV DNA was detectable in the plasma samples of SCC90- and HeLa-bearing mice but not in the controls. It was detected in 65% of the pretreatment plasma samples from HPV-positive OPC patients using E6/7 quantitative polymerase chain reaction. None of the HPV-negative head-and-neck cancer patients or non-cancer controls had detectable HPV DNA. The pretreatment plasma HPV DNA copy number correlated significantly with the nodal metabolic tumor volume (assessed using {sup 18}F-deoxyglucose positron emission tomography). The serial measurements in 14 patients showed a rapid decline in HPV DNA that had become undetectable at radiotherapy completion. In 3 patients, the HPV DNA level had increased to a discernable level at metastasis. Conclusions: Xenograft studies indicated that plasma HPV DNA is released from HPV-positive tumors. Circulating HPV DNA was detectable in most HPV-positive OPC patients. Thus, plasma HPV DNA might be a valuable tool for identifying relapse.

  2. Design and evaluation of Bacteroides DNA probes for the specific detection of human fecal pollution

    Energy Technology Data Exchange (ETDEWEB)

    Kreader, C.A. [Environmental Protection Agency, Cincinnati, OH (United States)

    1995-04-01

    Because Bacteroides spp. are obligate anaerobes that dominate the human fecal flora, and because some species may live only in the human intestine, these bacteria might be useful to distinguish human from nonhuman sources of fecal pollution. To test this hypothesis, PCR primers specific for 16S rRNA gene sequences of Bacteroides distasonis, B. thetaiotaomicron, and B. vulgatus were designed. Hybridization with species-specific internal probes was used to detect the intended PCR products. Extracts from 66 known Bacteroides strains, representing 10 related species, were used to confirm the specificity of these PCR-hybridization assays. To test for specificity in feces, procedures were developed to prepare DNA of sufficient purity for PCR. Extracts of feces from 9 humans and 70 nonhumans (cats, dogs, cattle, hogs, horses, sheep, goats, and chickens) were each analyzed with and without an internal positive control to verify that PCR amplification was not inhibited by substances in the extract. In addition, serial dilutions from each extract that tested positive were assayed to estimate the relative abundance of target Bacteroides spp. in the sample. Depending on the primer-probe set used, either 78 or 67% of the human fecal extracts tested had high levels of target DNA. On the other hand, only 7 to 11% of the nonhuman extracts tested had similarly high levels of target DNA. An additional 12 to 20% of the nonhuman extracts had levels of target DNA that were 100- to 1,000-fold lower than those found in humans. Although the B. vulgatus probes detected high levels of their target DNA in most of the house pets, similarly high levels of target DNA were found only in a few individuals from other groups of nonhumans. Therefore, the results indicate that these probes can distinguish human from non human feces in many cases. 50 refs., 5 figs., 2 tabs.

  3. Increasing epidermal growth factor receptor expression in human melanocytic tumor progression.

    Science.gov (United States)

    de Wit, P E; Moretti, S; Koenders, P G; Weterman, M A; van Muijen, G N; Gianotti, B; Ruiter, D J

    1992-08-01

    Different results have been reported on the expression of epidermal growth factor receptor (EGFR) in human melanocytic lesions, which may be due to different methodologic approaches. Therefore, we compared EGFR expression in six human melanoma cell lines by utilizing the monoclonal antibodies 2E9, 425, and 225, applying four immunocytochemical staining procedures. The results were compared with those obtained by a multiple point ligand binding assay. In addition, Northern blot analysis was performed. A three-step immunoperoxidase method using the monoclonal antibody 2E9 proved most sensitive. Staining intensities, estimated semiquantitatively, correlated well with the quantitative data obtained by the ligand-binding assay. Expression on the mRNA level was also in agreement with these results. Immunohistochemical staining of a large series of human cutaneous melanocytic lesions using the method selected showed differential EGFR expression in various stages of melanocytic tumor progression: 19% of common nevocellular nevi; 61% of dysplastic nevi, 89% of primary cutaneous melanomas, and 91% of melanoma metastases showed staining of the melanocytic cells. Intralesional heterogeneity of EGFR expression was present. Although the mean percentage of positive melanocytic cells in positive lesions did not increase with progression, mean staining intensity was stronger in malignant lesions compared to benign lesions. Ligand binding assays showed that EGFR expression in the highly metastasizing cell lines MV3 and BLM was at least 40 times higher than in the cell lines IF6, 530, M14, and Mel57, which do not or only sporadically metastasize after subcutaneous inoculation in nude mice. Although the differences between the various stages of progression are not absolute, we provide further evidence that EGFR expression increases in human melanocytic tumor progression.

  4. Indicators of Economic Progress: The Power of Measurement and Human Welfare

    Directory of Open Access Journals (Sweden)

    Garry Jacobs

    2010-10-01

    Full Text Available Right measurement is a powerful instrument for social progress; wrong or imprecise measurement a source of hazard and even havoc. The essential purpose of economic activity is the promotion of human development, welfare and well-being in a sustainable manner, and not growth for growth’s sake, yet we lack effective measures to monitor progress toward these objectives. Advances in understanding, theory and measurement must necessarily proceed hand in hand. A companion article in this publication sets forth the urgent need for new theory in economics. This article sets forth the complementary need for new measures. The stakes are high and the choice is ours. On one side, rising social tensions, recurring financial crises and ecological disaster; on the other, the progressive unfolding and development of human capacity in harmony with Nature. The deficiencies of GDP as a measure are well-documented by leading economists Kuznets, Tobin, Tinbergen and many others; but, unfortunately, decision-making still remains largely based on GDP, valid during 1930-70 perhaps, but certainly inappropriate today. The challenge is to derive more appropriate indicators to reflect real, sustainable economic welfare, social development and human wellbeing. The attributes that have made GDP so successful are often overlooked — it provides clear objectives for policy and decision-making. We propose new composite indicator, HEWI, which can be used to guide decision-making, which retains the strengths associated with GDP, while substantially enhancing its value as a measure of human economic development. HEWI monitors progress on factors that contribute prominently to present economic welfare — household consumption, government welfare-related expenditure, income inequality and unemployment — as well as factors that have the potential to significantly enhance long term sustainability — education, fossil fuel energy efficiency and net household savings. The index

  5. Progressing a human embryonic stem-cell-based regenerative medicine therapy towards the clinic

    OpenAIRE

    Whiting, Paul; Kerby, Julie; Coffey, Peter; da Cruz, Lyndon; McKernan, Ruth

    2015-01-01

    Since the first publication of the derivation of human embryonic stem cells in 1998, there has been hope and expectation that this technology will lead to a wave of regenerative medicine therapies with the potential to revolutionize our approach to managing certain diseases. Despite significant resources in this direction, the path to the clinic for an embryonic stem-cell-based regenerative medicine therapy has not proven straightforward, though in the past few years progress has been made. H...

  6. Aluminum oxide nanoparticles alter cell cycle progression through CCND1 and EGR1 gene expression in human mesenchymal stem cells.

    Science.gov (United States)

    Periasamy, Vaiyapuri Subbarayan; Athinarayanan, Jegan; Alshatwi, Ali A

    2016-05-01

    Aluminum oxide nanoparticles (Al2 O3 -NPs) are important ceramic materials that have been used in a variety of commercial and industrial applications. However, the impact of acute and chronic exposure to Al2 O3 -NPs on the environment and on human health has not been well studied. In this investigation, we evaluated the cytotoxic effects of Al2 O3 -NPs on human mesenchymal stem cells (hMSCs) by using a cell viability assay and observing cellular morphological changes, analyzing cell cycle progression, and monitoring the expression of cell cycle response genes (PCNA, EGR1, E2F1, CCND1, CCNC, CCNG1, and CYCD3). The Al2 O3 -NPs reduced hMSC viability in a dose- and time-dependent manner. Nuclear condensation and fragmentation, chromosomal DNA fragmentation, and cytoplasmic vacuolization were observed in Al2 O3 -NP-exposed cells. The nuclear morphological changes indicated that Al2 O3 -NPs alter cell cycle progression and gene expression. The cell cycle distribution revealed that Al2 O3 -NPs cause cell cycle arrest in the sub-G0-G1 phase, and this is associated with a reduction in the cell population in the G2/M and G0/G1 phases. Moreover, Al2 O3 -NPs induced the upregulation of cell cycle response genes, including EGR1, E2F1, and CCND1. Our results suggested that exposure to Al2 O3 -NPs could cause acute cytotoxic effects in hMSCs through cell cycle regulatory genes. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

  7. Specific chromosomal imbalances in human papillomavirus-transfected cells during progression toward immortality

    Science.gov (United States)

    Solinas-Toldo, Sabina; Dürst, Matthias; Lichter, Peter

    1997-01-01

    High risk human papillomaviruses (HPVs) known to be closely associated with cervical cancer, such as HPV16 and HPV18, have the potential to immortalize human epithelial cells in culture. Four lines of HPV-transfected keratinocytes were analyzed by comparative genomic hybridization at different time points after transfection. A number of chromosomal imbalances was found to be highly characteristic for the cultures progressing toward immortality. Whereas several of these were new and previously not found as recurrent aberrations in cervical tumors, some were identical to chromosomal changes observed during cervical carcinogenesis. The data put new emphasis on the studied cell system as a relevant model for HPV-induced pathogenesis. PMID:9108068

  8. Simian virus 40, poliovirus vaccines, and human cancer: research progress versus media and public interests

    Science.gov (United States)

    Butel, J. S.

    2000-01-01

    From 1955 through early 1963, millions of people were inadvertently exposed to simian virus 40 (SV40) as a contaminant of poliovirus vaccines; the virus had been present in the monkey kidney cultures used to prepare the vaccines and had escaped detection. SV40 was discovered in 1960 and subsequently eliminated from poliovirus vaccines. This article reviews current knowledge about SV40 and considers public responses to reports in the media. SV40 is a potent tumour virus with broad tissue tropism that induces tumours in rodents and transforms cultured cells from many species. It is also an important laboratory model for basic studies of molecular processes in eukaryotic cells and mechanisms of neoplastic transformation. SV40 neutralizing antibodies have been detected in individuals not exposed to contaminated poliovirus vaccines. There have been many reports of detection of SV40 DNA in human tumours, especially mesotheliomas, brain tumours and osteosarcomas; and DNA sequence analyses have ruled out the possibility that the viral DNA in tumours was due to laboratory contamination or that the virus had been misidentified. However, additional studies are necessary to prove that SV40 is the cause of certain human cancers. A recently published review article evaluated the status of the field and received much media attention. The public response emphasized that there is great interest in the possibility of health risks today from vaccinations received in the past.

  9. Human TFIID binds to core promoter DNA in a reorganized structural state

    Science.gov (United States)

    Cianfrocco, Michael A.; Kassavetis, George A.; Grob, Patricia; Fang, Jie; Juven-Gershon, Tamar; Kadonaga, James T.; Nogales, Eva

    2012-01-01

    A mechanistic description of metazoan transcription is essential for understanding the molecular processes that govern cellular decisions. To provide structural insights into the DNA recognition step of transcription initiation, we used single particle electron microscopy to visualize human TFIID with promoter DNA. This analysis revealed that TFIID co-exists in two predominant and distinct structural states that differ by a 100Å translocation of TFIID’s lobe A. The transition between these structural states is modulated by TFIIA, as the presence of TFIIA and promoter DNA facilitates the formation of a novel rearranged state of TFIID capable of promoter recognition and binding. DNA-labeling and footprinting, together with cryo-EM studies, mapped the locations of the TATA, Inr, MTE, and DPE promoter motifs within the TFIID-TFIIA-DNA structure. The existence of two structurally and functionally distinct forms of TFIID suggests that the different conformers may serve as specific targets for the action of regulatory factors. PMID:23332750

  10. DNA and Law Enforcement in the European Union: Tools and Human Rights Protection

    Directory of Open Access Journals (Sweden)

    Helena Soleto Muñoz

    2014-01-01

    Full Text Available Since its first successful use in criminal investigations in the 1980s, DNA has become a widely used and valuable tool to identify offenders and to acquit innocent persons. For a more beneficial use of the DNA-related data possessed, the Council of the European Union adopted Council Decisions 2008/615 and 2008/616 establishing a mechanism for a direct automated search in national EU Member States’ DNA databases. The article reveals the complications associated with the regulation on the use of DNA for criminal investigations as it is regulated by both EU and national legislation which results in a great deal of variations. It also analyses possible violations of and limitations to human rights when collecting DNA samples, as well as their analysis, use and storage.

  11. 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 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...... that is integral to a forensic science investigation and are not relevant to the breeding of animals for commercial purposes. This DNA commission was formed out of discussions at the International Society for Forensic Genetics 23rd Congress in Buenos Aires to outline recommendations on the use of non-human DNA...

  12. Human papillomavirus type 16 DNA-induced malignant transformation of NIH 3T3 cells

    Energy Technology Data Exchange (ETDEWEB)

    Yasumoto, S.; Burkhardt, A.L.; Doniger, J.; DiPaolo, J.A.

    1986-02-01

    A biological function for human papillomavirus 16 (HPV 16) DNA was demonstrated by transformation of NIH 3T3 cells. HPV 16 DNA has been found frequently in genital cancer and has been classified as a papillomavirus on the basis of DNA homology. A recombinant HPV 16 DNA (pSHPV16d), which contains a head-to-tail dimer of the full-length HPV 16 genome, induced morphologic transformation; the transformed cells were tumorigenic in nude mice. Expression of transforming activity was unique because of the long latency period (more than 4 weeks) required for induction of morphologic transformation and because the transfected DNA existed primarily in a multimeric form with some rearrangement. Furthermore, virus-specific RNAs were expressed in the transformants. The transformation of NIH 3T3 cells provides a model for analyzing the functions of HPV 16, which is associated with cervical carcinomas.

  13. Stimulation of Dmc1-mediated DNA strand exchange by the human Rad54B protein

    Science.gov (United States)

    Sarai, Naoyuki; Kagawa, Wataru; Kinebuchi, Takashi; Kagawa, Ako; Tanaka, Kozo; Miyagawa, Kiyoshi; Ikawa, Shukuko; Shibata, Takehiko; Kurumizaka, Hitoshi; Yokoyama, Shigeyuki

    2006-01-01

    The process of homologous recombination is indispensable for both meiotic and mitotic cell division, and is one of the major pathways for double-strand break (DSB) repair. The human Rad54B protein, which belongs to the SWI2/SNF2 protein family, plays a role in homologous recombination, and may function with the Dmc1 recombinase, a meiosis-specific Rad51 homolog. In the present study, we found that Rad54B enhanced the DNA strand-exchange activity of Dmc1 by stabilizing the Dmc1–single-stranded DNA (ssDNA) complex. Therefore, Rad54B may stimulate the Dmc1-mediated DNA strand exchange by stabilizing the nucleoprotein filament, which is formed on the ssDNA tails produced at DSB sites during homologous recombination. PMID:16945962

  14. An Improved Method for High Quality Metagenomics DNA Extraction from Human and Environmental Samples

    DEFF Research Database (Denmark)

    Bag, Satyabrata; Saha, Bipasa; Mehta, Ojasvi

    2016-01-01

    and quantity from culturable and uncultured microbial species living in that environment. Proper lysis of heterogeneous community microbial cells without damaging their genomes is a major challenge. In this study, we have developed an improved method for extraction of community DNA from different environmental......To explore the natural microbial community of any ecosystems by high-resolution molecular approaches including next generation sequencing, it is extremely important to develop a sensitive and reproducible DNA extraction method that facilitate isolation of microbial DNA of sufficient purity...... and human origin samples. We introduced a combination of physical, chemical and mechanical lysis methods for proper lysis of microbial inhabitants. The community microbial DNA was precipitated by using salt and organic solvent. Both the quality and quantity of isolated DNA was compared with the existing...

  15. The BER necessities: the repair of DNA damage in human-adapted bacterial pathogens.

    Science.gov (United States)

    van der Veen, Stijn; Tang, Christoph M

    2015-02-01

    During colonization and disease, bacterial pathogens must survive the onslaught of the host immune system. A key component of the innate immune response is the generation of reactive oxygen and nitrogen species by phagocytic cells, which target and disrupt pathogen molecules, particularly DNA, and the base excision repair (BER) pathway is the most important mechanism for the repair of such oxidative DNA damage. In this Review, we discuss how the human-specific pathogens Mycobacterium tuberculosis, Helicobacter pylori and Neisseria meningitidis have evolved specialized mechanisms of DNA repair, particularly their BER pathways, compared with model organisms such as Escherichia coli. This specialization in DNA repair is likely to reflect the distinct niches occupied by these important human pathogens in the host.

  16. Northern and Southern blot analysis of human RNA and DNA in autopsy material

    DEFF Research Database (Denmark)

    Larsen, S; Rygaard, K; Asnaes, S

    1992-01-01

    Fresh biopsy material for molecular biological investigations is not obtainable from all relevant normal human tissues. We studied the feasibility of using RNA and DNA from autopsies for Northern and Southern blot analysis. Tissue samples from seven organs were obtained from 10 autopsies performed...... 21-118 h postmortem. Extracted RNA and DNA were examined by Northern and Southern blot analysis using oligo-labelled human DNA probes recognizing gene transcripts of 2-5 kb. The results indicated that, in general, Northern blot analysis was feasible with the applied probes when the tissue...... was obtained less than two days postmortem. Histological examination showing slight or no autolysis and the presence of ribosomal bands after gel electrophoresis were both indicative parameters of RNA preservation. DNA was appropriate for Southern blotting when the tissue was obtained less than three to five...

  17. Circulating mitochondrial DNA as biomarker linking environmental chemical exposure to early preclinical lesions elevation of mtDNA in human serum after exposure to carcinogenic halo-alkane-based pesticides.

    Science.gov (United States)

    Budnik, Lygia T; Kloth, Stefan; Baur, Xaver; Preisser, Alexandra M; Schwarzenbach, Heidi

    2013-01-01

    There is a need for a panel of suitable biomarkers for detection of environmental chemical exposure leading to the initiation or progression of degenerative diseases or potentially, to cancer. As the peripheral blood may contain increased levels of circulating cell-free DNA in diseased individuals, we aimed to evaluate this DNA as effect biomarker recognizing vulnerability after exposure to environmental chemicals. We recruited 164 individuals presumably exposed to halo-alkane-based pesticides. Exposure evaluation was based on human biomonitoring analysis; as biomarker of exposure parent halo-methanes, -ethanes and their metabolites, as well as the hemoglobin-adducts methyl valine and hydroxyl ethyl valine in blood were used, complemented by expert evaluation of exposure and clinical intoxication symptoms as well as a questionnaire. Assessment showed exposures to halo alkanes in the concentration range being higher than non-cancer reference doses (RfD) but (mostly) lower than the occupational exposure limits. We quantified circulating DNA in serum from 86 individuals with confirmed exposure to off-gassing halo-alkane pesticides (in storage facilities or in home environment) and 30 non-exposed controls, and found that exposure was significantly associated with elevated serum levels of circulating mitochondrial DNA (in size of 79 bp, mtDNA-79, p = 0.0001). The decreased integrity of mtDNA (mtDNA-230/mtDNA-79) in exposed individuals implicates apoptotic processes (p = 0.015). The relative amounts of mtDNA-79 in serum were positively associated with the lag-time after intoxication to these chemicals (r = 0.99, pvulnerable risk groups after exposure to toxic/carcinogenic chemicals.

  18. [Maternally inherited diabetes mellitus, deafness, chronic progressive external ophthalmoplegia and myopathy as the result of A3243G mutation of mtDNA].

    Science.gov (United States)

    Gál, Anikó; Szabó, Antal; Pentelényi, Klára; Pál, Zsuzsanna

    2008-08-24

    Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome are caused mainly by the A3243G mutation of the mitochondrial genome. The A3243G substitution of mitochondrial DNA (mtDNA) is also responsible for various, other clinical phenotypes and syndromes. Here we report the case of a 33-year-old woman, with childhood onset ophthalmoplegia externa, progressive, generalised exercise intolerability, muscle weakness, hypacusis and diabetes mellitus as the symptoms of mitochondrial disease. Genetic analysis of the mitochondrial DNA revealed a heteroplasmic A to G substitution at position 3243 in the tRNS Leu(UUR) gene. In our case the classical MELAS phenotype has not yet appeared, however, some examples show in the literature that maternally inherited diabetes mellitus, progressive hypacusis, progressive ophthalmoplegia externa, exercise intolerance, and myopathy are often linked to as isolated symptoms of A3243G mutation. The phenotype in the family is consistent, the proband's daughter has ptosis, exercise intolerance, and myopathy, too. A brief summary of the different clinical phenotypes associated with A3243G mutation, and of the different mtDNA mutations which can cause chronic progressive ophthalmoplegia externa (CPEO) will also be reviewed in this case report.

  19. Mitochondrial DNA deletion associated with the reduction of adenine nucleotides in human atrium and atrial fibrillation.

    Science.gov (United States)

    Tsuboi, M; Hisatome, I; Morisaki, T; Tanaka, M; Tomikura, Y; Takeda, S; Shimoyama, M; Ohtahara, A; Ogino, K; Igawa, O; Shigemasa, C; Ohgi, S; Nanba, E

    2001-06-01

    Structural changes in the number, size, and shape of mitochondria (mt) have been observed in the atrial muscles of patients with atrial fibrillation (AF) and of animals with rapid atrial pacing, however, it is not known whether the mitochondrial function is impaired in human atrium with AF. We determined adenine nucleotides concentrations and mtDNA deletions in 26 human right atria obtained at the time of cardiac surgery, using HPLC and PCR amplification, and studied the relationship between mtDNA deletions and clinical manifestations, the haemodynamic parameters of the patients and adenine nucleotide concentrations in their atrium. The age and the prevalence of AF were significantly higher in the patients with a mtDNA deletion of 7.4 kb than in those without a deletion; there were no significant differences regarding haemodynamic parameters between the two groups. The concentrations of ATP, ADP, AMP and total adenine nucleotides in the right atrium were significantly lower in the patients with mtDNA deletions than the patients without a deletion. In a gender- and diseased-matched population, the mtDNA deletion was still significantly associated with age and a decreased concentration of adenine nucleotides in the atrium. Using quantitative PCR analysis, the proportion of mtDNA deletion to normal mtDNA of the atrium, was estimated to be 0.3-2% in four cases. These results suggest that the deletion of mtDNA associated with ageing or AF can lead to a bioenergetic deficiency due to an impaired ATP synthesis in the human atrium; however, no conclusion can be made whether mtDNA deletion were the result or the cause of an impaired ATP synthesis, ageing, hemodynamic deterioration, or AF.

  20. Human evolution in Siberia: from frozen bodies to ancient DNA

    Directory of Open Access Journals (Sweden)

    Bouakaze Caroline

    2010-01-01

    Full Text Available Abstract Background The Yakuts contrast strikingly with other populations from Siberia due to their cattle- and horse-breeding economy as well as their Turkic language. On the basis of ethnological and linguistic criteria as well as population genetic studies, it has been assumed that they originated from South Siberian populations. However, many questions regarding the origins of this intriguing population still need to be clarified (e.g. the precise origin of paternal lineages and the admixture rate with indigenous populations. This study attempts to better understand the origins of the Yakuts by performing genetic analyses on 58 mummified frozen bodies dated from the 15th to the 19th century, excavated from Yakutia (Eastern Siberia. Results High quality data were obtained for the autosomal STRs, Y-chromosomal STRs and SNPs and mtDNA due to exceptional sample preservation. A comparison with the same markers on seven museum specimens excavated 3 to 15 years ago showed significant differences in DNA quantity and quality. Direct access to ancient genetic data from these molecular markers combined with the archaeological evidence, demographical studies and comparisons with 166 contemporary individuals from the same location as the frozen bodies helped us to clarify the microevolution of this intriguing population. Conclusion We were able to trace the origins of the male lineages to a small group of horse-riders from the Cis-Baïkal area. Furthermore, mtDNA data showed that intermarriages between the first settlers with Evenks women led to the establishment of genetic characteristics during the 15th century that are still observed today.

  1. The influence of high temperature on the possibility of DNA typing in various human tissues.

    Science.gov (United States)

    Maciejewska, Agnieszka; Wlodarczyk, Renata; Pawlowski, Ryszard

    2015-01-01

    The identification of unknown victims of high temperatures (fire, terrorist attack, and other disasters) is one of the most difficult tasks faced by forensic geneticists. The main aim of this study was to in­vestigate the availability of DNA isolated from various human tissue samples exposed to high temperatures of 100–1000°C for 5 and 10 minutes. Samples of varying thickness of thigh muscle, liver, heart, adipose tissue, bone, teeth, hair and nails of 52 fresh cadavers and 59 healthy teeth of 29 volunteers were used. The study was performed using the following commercially available STR (Short Tandem Repeats) and miniSTR kits: AmpFlSTR®SGM Plus® and AmpFlSTR®MiniFilerTM. Hyper variable region I (HVI) of human mitochondrial DNA (mtDNA) was sequenced with BigDye Terminator Cycle Sequencing Kit 1.1. The PEP (Primer-Extension Preamplification) method was used for the whole human genome amplification. It was possible to obtain complete DNA profiles (AmpFlSTR®SGM Plus®, AmpFlSTR®MiniFilerTM Applied Biosystems, USA and mtDNA HVI region) for tissue samples of heart, liver and thigh muscle, exposed up to 900°C for 5 min. However, under the applied conditions, limited usefulness of hair, nails and teeth for identification purposes was shown. DNA stability in tissues subjected to incineration depends on many factors, like tissue type and its thickness, temperature and time of exposure. In the cases of human remains exposed to high temperatures, samples of soft tissues of the highest weight (thickness) provide the best chance of successful identification through the genetic analysis. In some cases of negative results, even if using mtDNA typing, application of the whole genome amplification (WGA) technique could provide the expected results for highly degraded DNA templates.

  2. Searching the Human Genome for Snail and Slug With DNA@Home

    OpenAIRE

    Zarns, Kristopher; Desell, Travis; Nechaev, Sergei; Dhasarathy, Archana

    2015-01-01

    DNA@Home is a volunteer computing project that aims to use Gibbs Sampling for the identification and location of DNA control signals on full genome-scale datasets. A fault tolerant and asynchronous implementation of Gibbs sampling using the Berkeley Open Infrastructure for Network Computing (BOINC) was used to identify the location of binding sites of the SNAI1 (Snail) and SNAI2 (Slug) transcription factors across the human genome. Genes regulated by Slug but not Snail, and genes regulated by...

  3. Does aerobic exercises induce mtDNA mutation in human blood ...

    African Journals Online (AJOL)

    The aim of this study was to determine the effect of eight weeks aerobic training on mitochondrial DNA (mtDNA) mutation in human blood leucocytes. Twenty untrained healthy students (training group: n =10, age = 20.7±1.5 yrs, weight = 67.7±10 kg, BF% = 17.5±7.35 & control group: n =10, age = 21±1.3 yrs, weight ...

  4. The pathological consequences of impaired genome integrity in humans; disorders of the DNA replication machinery.

    Science.gov (United States)

    O'Driscoll, Mark

    2017-01-01

    Accurate and efficient replication of the human genome occurs in the context of an array of constitutional barriers, including regional topological constraints imposed by chromatin architecture and processes such as transcription, catenation of the helical polymer and spontaneously generated DNA lesions, including base modifications and strand breaks. DNA replication is fundamentally important for tissue development and homeostasis; differentiation programmes are intimately linked with stem cell division. Unsurprisingly, impairments of the DNA replication machinery can have catastrophic consequences for genome stability and cell division. Functional impacts on DNA replication and genome stability have long been known to play roles in malignant transformation through a variety of complex mechanisms, and significant further insights have been gained from studying model organisms in this context. Congenital hypomorphic defects in components of the DNA replication machinery have been and continue to be identified in humans. These disorders present with a wide range of clinical features. Indeed, in some instances, different mutations in the same gene underlie different clinical presentations. Understanding the origin and molecular basis of these features opens a window onto the range of developmental impacts of suboptimal DNA replication and genome instability in humans. Here, I will briefly overview the basic steps involved in DNA replication and the key concepts that have emerged from this area of research, before switching emphasis to the pathological consequences of defects within the DNA replication network; the human disorders. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  5. Research Progress on the Risk Factors and Outcomes of Human Carotid Atherosclerotic Plaques.

    Science.gov (United States)

    Xiong, Xiang-Dong; Xiong, Wei-Dong; Xiong, Shang-Shen; Chen, Gui-Hai

    2017-03-20

    Atherosclerosis is an inflammatory process that results in complex lesions or plaques that protrude into the arterial lumen. Carotid atherosclerotic plaque rupture, with distal atheromatous debris embolization, causes cerebrovascular events. This review aimed to explore research progress on the risk factors and outcomes of human carotid atherosclerotic plaques, and the molecular and cellular mechanisms of human carotid atherosclerotic plaque vulnerability for therapeutic intervention. We searched the PubMed database for recently published research articles up to June 2016, with the key words of "risk factors", "outcomes", "blood components", "molecular mechanisms", "cellular mechanisms", and "human carotid atherosclerotic plaques". The articles, regarding the latest developments related to the risk factors and outcomes, atherosclerotic plaque composition, blood components, and consequences of human carotid atherosclerotic plaques, and the molecular and cellular mechanisms of human carotid atherosclerotic plaque vulnerability for therapeutic intervention, were selected. This review described the latest researches regarding the interactive effects of both traditional and novel risk factors for human carotid atherosclerotic plaques, novel insights into human carotid atherosclerotic plaque composition and blood components, and consequences of human carotid atherosclerotic plaque. Carotid plaque biology and serologic biomarkers of vulnerability can be used to predict the risk of cerebrovascular events. Furthermore, plaque composition, rather than lesion burden, seems to most predict rupture and subsequent thrombosis.

  6. Genome-wide DNA methylation analysis in cohesin mutant human cell lines

    Science.gov (United States)

    Liu, Jinglan; Zhang, Zhe; Bando, Masashige; Itoh, Takehiko; Deardorff, Matthew A.; Li, Jennifer R.; Clark, Dinah; Kaur, Maninder; Tatsuro, Kondo; Kline, Antonie D.; Chang, Celia; Vega, Hugo; Jackson, Laird G.; Spinner, Nancy B.; Shirahige, Katsuhiko; Krantz, Ian D.

    2010-01-01

    The cohesin complex has recently been shown to be a key regulator of eukaryotic gene expression, although the mechanisms by which it exerts its effects are poorly understood. We have undertaken a genome-wide analysis of DNA methylation in cohesin-deficient cell lines from probands with Cornelia de Lange syndrome (CdLS). Heterozygous mutations in NIPBL, SMC1A and SMC3 genes account for ∼65% of individuals with CdLS. SMC1A and SMC3 are subunits of the cohesin complex that controls sister chromatid cohesion, whereas NIPBL facilitates cohesin loading and unloading. We have examined the methylation status of 27 578 CpG dinucleotides in 72 CdLS and control samples. We have documented the DNA methylation pattern in human lymphoblastoid cell lines (LCLs) as well as identified specific differential DNA methylation in CdLS. Subgroups of CdLS probands and controls can be classified using selected CpG loci. The X chromosome was also found to have a unique DNA methylation pattern in CdLS. Cohesin preferentially binds to hypo-methylated DNA in control LCLs, whereas the differential DNA methylation alters cohesin binding in CdLS. Our results suggest that in addition to DNA methylation multiple mechanisms may be involved in transcriptional regulation in human cells and in the resultant gene misexpression in CdLS. PMID:20448023

  7. Microbial Degradation of Forensic Samples of Biological Origin: Potential Threat to Human DNA Typing.

    Science.gov (United States)

    Dash, Hirak Ranjan; Das, Surajit

    2017-12-06

    Forensic biology is a sub-discipline of biological science with an amalgam of other branches of science used in the criminal justice system. Any nucleated cell/tissue harbouring DNA, either live or dead, can be used as forensic exhibits, a source of investigation through DNA typing. These biological materials of human origin are rich source of proteins, carbohydrates, lipids, trace elements as well as water and, thus, provide a virtuous milieu for the growth of microbes. The obstinate microbial growth augments the degradation process and is amplified with the passage of time and improper storage of the biological materials. Degradation of these biological materials carriages a huge challenge in the downstream processes of forensic DNA typing technique, such as short tandem repeats (STR) DNA typing. Microbial degradation yields improper or no PCR amplification, heterozygous peak imbalance, DNA contamination from non-human sources, degradation of DNA by microbial by-products, etc. Consequently, the most precise STR DNA typing technique is nullified and definite opinion can be hardly given with degraded forensic exhibits. Thus, suitable precautionary measures should be taken for proper storage and processing of the biological exhibits to minimize their decaying process by micro-organisms.

  8. Cell-Cycle-Dependent Reconfiguration of the DNA Methylome during Terminal Differentiation of Human B Cells into Plasma Cells

    Directory of Open Access Journals (Sweden)

    Gersende Caron

    2015-11-01

    Full Text Available Molecular mechanisms underlying terminal differentiation of B cells into plasma cells are major determinants of adaptive immunity but remain only partially understood. Here we present the transcriptional and epigenomic landscapes of cell subsets arising from activation of human naive B cells and differentiation into plasmablasts. Cell proliferation of activated B cells was linked to a slight decrease in DNA methylation levels, but followed by a committal step in which an S phase-synchronized differentiation switch was associated with an extensive DNA demethylation and local acquisition of 5-hydroxymethylcytosine at enhancers and genes related to plasma cell identity. Downregulation of both TGF-β1/SMAD3 signaling and p53 pathway supported this final step, allowing the emergence of a CD23-negative subpopulation in transition from B cells to plasma cells. Remarkably, hydroxymethylation of PRDM1, a gene essential for plasma cell fate, was coupled to progression in S phase, revealing an intricate connection among cell cycle, DNA (hydroxymethylation, and cell fate determination.

  9. A novel reannotation strategy for dissecting DNA methylation patterns of human long intergenic non-coding RNAs in cancers.

    Science.gov (United States)

    Zhi, Hui; Ning, Shangwei; Li, Xiang; Li, Yuyun; Wu, Wei; Li, Xia

    2014-07-01

    Despite growing consensus that long intergenic non-coding ribonucleic acids (lincRNAs) are modulators of cancer, the knowledge about the deoxyribonucleic acid (DNA) methylation patterns of lincRNAs in cancers remains limited. In this study, we constructed DNA methylation profiles for 4629 tumors and 705 normal tissue samples from 20 different types of human cancer by reannotating data of DNA methylation arrays. We found that lincRNAs had different promoter methylation patterns in cancers. We classified 2461 lincRNAs into two categories and three subcategories, according to their promoter methylation patterns in tumors. LincRNAs with resistant methylation patterns in tumors had conserved transcriptional regulation regions and were ubiquitously expressed across normal tissues. By integrating cancer subtype data and patient clinical information, we identified lincRNAs with promoter methylation patterns that were associated with cancer status, subtype or prognosis for several cancers. Network analysis of aberrantly methylated lincRNAs in cancers showed that lincRNAs with aberrant methylation patterns might be involved in cancer development and progression. The methylated and demethylated lincRNAs identified in this study provide novel insights for developing cancer biomarkers and potential therapeutic targets. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. ADAM15 Is Functionally Associated with the Metastatic Progression of Human Bladder Cancer.

    Directory of Open Access Journals (Sweden)

    Guadalupe Lorenzatti Hiles

    Full Text Available ADAM15 is a member of a family of catalytically active disintegrin membrane metalloproteinases that function as molecular signaling switches, shed membrane bound growth factors and/or cleave and inactivate cell adhesion molecules. Aberrant metalloproteinase function of ADAM15 may contribute to tumor progression through the release of growth factors or disruption of cell adhesion. In this study, we utilized human bladder cancer tissues and cell lines to evaluate the expression and function of ADAM15 in the progression of human bladder cancer. Examination of genome and transcriptome databases revealed that ADAM15 ranked in the top 5% of amplified genes and its mRNA was significantly overexpressed in invasive and metastatic bladder cancer compared to noninvasive disease. Immunostaining of a bladder tumor tissue array designed to evaluate disease progression revealed increased ADAM15 immunoreactivity associated with increasing cancer stage and exhibited significantly stronger staining in metastatic samples. About half of the invasive tumors and the majority of the metastatic cases exhibited high ADAM15 staining index, while all low grade and noninvasive cases exhibited negative or low staining. The knockdown of ADAM15 mRNA expression significantly inhibited bladder tumor cell migration and reduced the invasive capacity of bladder tumor cells through MatrigelTM and monolayers of vascular endothelium. The knockdown of ADAM15 in a human xenograft model of bladder cancer inhibited tumor growth by 45% compared to controls. Structural modeling of the catalytic domain led to the design of a novel ADAM15-specific sulfonamide inhibitor that demonstrated bioactivity and significantly reduced the viability of bladder cancer cells in vitro and in human bladder cancer xenografts. Taken together, the results revealed an undescribed role of ADAM15 in the invasion of human bladder cancer and suggested that the ADAM15 catalytic domain may represent a viable

  11. Carriers of human mitochondrial DNA macrohaplogroup M colonized India from southeastern Asia.

    Science.gov (United States)

    Marrero, Patricia; Abu-Amero, Khaled K; Larruga, Jose M; Cabrera, Vicente M

    2016-11-10

    From a mtDNA dominant perspective, the exit from Africa of modern humans to colonize Eurasia occurred once, around 60 kya, following a southern coastal route across Arabia and India to reach Australia short after. These pioneers carried with them the currently dominant Eurasian lineages M and N. Based also on mtDNA phylogenetic and phylogeographic grounds, some authors have proposed the coeval existence of a northern route across the Levant that brought mtDNA macrohaplogroup N to Australia. To contrast both hypothesis, here we reanalyzed the phylogeography and respective ages of mtDNA haplogroups belonging to macrohaplogroup M in different regions of Eurasia and Australasia. The macrohaplogroup M has a historical implantation in West Eurasia, including the Arabian Peninsula. Founder ages of M lineages in India are significantly younger than those in East Asia, Southeast Asia and Near Oceania. Moreover, there is a significant positive correlation between the age of the M haplogroups and its longitudinal geographical distribution. These results point to a colonization of the Indian subcontinent by modern humans carrying M lineages from the east instead the west side. The existence of a northern route, previously proposed for the mtDNA macrohaplogroup N, is confirmed here for the macrohaplogroup M. Both mtDNA macrolineages seem to have differentiated in South East Asia from ancestral L3 lineages. Taking this genetic evidence and those reported by other disciplines we have constructed a new and more conciliatory model to explain the history of modern humans out of Africa.

  12. Assessing Progress towards Public Health, Human Rights, and International Development Goals Using Frontier Analysis.

    Science.gov (United States)

    Luh, Jeanne; Cronk, Ryan; Bartram, Jamie

    2016-01-01

    Indicators to measure progress towards achieving public health, human rights, and international development targets, such as 100% access to improved drinking water or zero maternal mortality ratio, generally focus on status (i.e., level of attainment or coverage) or trends in status (i.e., rates of change). However, these indicators do not account for different levels of development that countries experience, thus making it difficult to compare progress between countries. We describe a recently developed new use of frontier analysis and apply this method to calculate country performance indices in three areas: maternal mortality ratio, poverty headcount ratio, and primary school completion rate. Frontier analysis is used to identify the maximum achievable rates of change, defined by the historically best-performing countries, as a function of coverage level. Performance indices are calculated by comparing a country's rate of change against the maximum achievable rate at the same coverage level. A country's performance can be positive or negative, corresponding to progression or regression, respectively. The calculated performance indices allow countries to be compared against each other regardless of whether they have only begun to make progress or whether they have almost achieved the target. This paper is the first to use frontier analysis to determine the maximum achievable rates as a function of coverage level and to calculate performance indices for public health, human rights, and international development indicators. The method can be applied to multiple fields and settings, for example health targets such as cessation in smoking or specific vaccine immunizations, and offers both a new approach to analyze existing data and a new data source for consideration when assessing progress achieved.

  13. Increased oxidative DNA damage, 8-hydroxydeoxy- guanosine, in human pterygium.

    Science.gov (United States)

    Kau, H-C; Tsai, C-C; Lee, C-F; Kao, S-C; Hsu, W-M; Liu, J-H; Wei, Y-H

    2006-07-01

    Chronic exposure to ultraviolet (UV) light is a widely accepted aetiological factor in the development of pterygium. UV radiation may induce production of reactive oxygen species via photosensitized oxidation, thus causing oxidative damage. This study was conducted to test the hypothesis that oxidative damage to DNA is increased in pterygium. Immunohistochemical analysis employing a monoclonal antibody specific for 8-hydroxy-2'-deoxyguanosine (8-OHdG), a ubiquitous maker of oxidative stress, was performed in three patients with primary pterygium. The levels of 8-OHdG in DNA isolated from the other 29 pterygium specimens and their adjacent normal conjunctival tissues were determined using enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry of 8-OHdG showed a distinct pattern of more extensive and intense staining in the nuclei of pterygium tissue compared with that in their adjacent normal conjunctiva. ELISA also revealed that the average level of 8-OHdG in the pterygium tissues was 4.7-fold higher than that of the corresponding normal conjunctiva (P<0.001). The increased levels of 8-OHdG in the pterygium tissues indicate that oxidative stress could play a role in the development of pterygium. These findings provide new information to better understand the pathogenesis of pterygium and are useful in the prevention and treatment of this disease.

  14. Multiplex ligation-dependent probe amplification (MLPA) assay for the detection of mitochondrial DNA deletion in chronic progressive external ophthalmoplegia (CPEO).

    Science.gov (United States)

    Kwon, Min-Jung; Ki, Chang-Seok; Kim, Ji-Youn; Lee, Seung-Tae; Kim, Jong-Won; Kang, Sa-Yoon

    2011-01-01

    Chronic progressive external ophthalmoplegia (CPEO) is a mitochondrial myopathy commonly caused by deleterious changes in the mitochondrial DNA (mtDNA). We describe a 45-year-old man who was referred to us for investigation of progressive ptosis. We performed a multiplex ligation-dependent probe amplification (MLPA) assay of mtDNA from muscle tissue and peripheral blood leukocytes, and followed up with gap-polymerase chain reaction (PCR) and direct sequence analysis. Results showed a deletion of a 4,407 bp segment in the mtDNA region, ranging from nucleotide position 8,577 in the MT-ATP6 gene to nucleotide position 12,983 in the MT-ND5 gene. To the best of our knowledge, this is the first report of a CPEO patient with a large novel deletion of mtDNA genetically confirmed by MLPA assay. MLPA can be a feasible platform for clinical laboratories to detect large deletion mutations in the mtDNA for suspected cases.

  15. Human Papillomavirus and Overall Survival After Progression of Oropharyngeal Squamous Cell Carcinoma

    Science.gov (United States)

    Fakhry, Carole; Zhang, Qiang; Nguyen-Tan, Phuc Felix; Rosenthal, David; El-Naggar, Adel; Garden, Adam S.; Soulieres, Denis; Trotti, Andy; Avizonis, Vilija; Ridge, John Andrew; Harris, Jonathan; Le, Quynh-Thu; Gillison, Maura

    2014-01-01

    Purpose Risk of cancer progression is reduced for patients with human papillomavirus (HPV) –positive oropharynx cancer (OPC) relative to HPV-negative OPC, but it is unknown whether risk of death after progression is similarly reduced. Patients and Methods Patients with stage III-IV OPC enrolled onto Radiation Therapy Oncology Group trials 0129 or RTOG 0522 who had known tumor p16 status plus local, regional, and/or distant progression after receiving platinum-based chemoradiotherapy were eligible for a retrospective analysis of the association between tumor p16 status and overall survival (OS) after disease progression. Rates were estimated by Kaplan-Meier method and compared by log-rank; hazard ratios (HRs) were estimated by Cox models. Tests and models were stratified by treatment protocol. Results A total of 181 patients with p16-positive (n = 105) or p16-negative (n = 76) OPC were included in the analysis. Patterns of failure and median time to progression (8.2 v 7.3 months; P = .67) were similar for patients with p16-positive and p16-negative tumors. After a median follow-up period of 4.0 years after disease progression, patients with p16-positive OPC had significantly improved survival rates compared with p16-negative patients (2-year OS, 54.6% v 27.6%; median, 2.6 v 0.8 years; P < .001). p16-positive tumor status (HR, 0.48; 95% CI, 0.31 to 0.74) and receipt of salvage surgery (HR, 0.48; 95% CI; 0.27 to 0.84) reduced risk of death after disease progression whereas distant versus locoregional progression (HR, 1.99; 95% CI, 1.28 to 3.09) increased risk, after adjustment for tumor stage and cigarette pack-years at enrollment. Conclusion Tumor HPV status is a strong and independent predictor of OS after disease progression and should be a stratification factor for clinical trials for patients with recurrent or metastatic OPC. PMID:24958820

  16. Enhanced genetic analysis of single human bioparticles recovered by simplified micromanipulation from forensic 'touch DNA' evidence.

    Science.gov (United States)

    Farash, Katherine; Hanson, Erin K; Ballantyne, Jack

    2015-03-09

    DNA profiles can be obtained from 'touch DNA' evidence, which comprises microscopic traces of human biological material. Current methods for the recovery of trace DNA employ cotton swabs or adhesive tape to sample an area of interest. However, such a 'blind-swabbing' approach will co-sample cellular material from the different individuals, even if the individuals' cells are located in geographically distinct locations on the item. Thus, some of the DNA mixtures encountered in touch DNA samples are artificially created by the swabbing itself. In some instances, a victim's DNA may be found in significant excess thus masking any potential perpetrator's DNA. In order to circumvent the challenges with standard recovery and analysis methods, we have developed a lower cost, 'smart analysis' method that results in enhanced genetic analysis of touch DNA evidence. We describe an optimized and efficient micromanipulation recovery strategy for the collection of bio-particles present in touch DNA samples, as well as an enhanced amplification strategy involving a one-step 5 µl microvolume lysis/STR amplification to permit the recovery of STR profiles from the bio-particle donor(s). The use of individual or few (i.e., "clumps") bioparticles results in the ability to obtain single source profiles. These procedures represent alternative enhanced techniques for the isolation and analysis of single bioparticles from forensic touch DNA evidence. While not necessary in every forensic investigation, the method could be highly beneficial for the recovery of a single source perpetrator DNA profile in cases involving physical assault (e.g., strangulation) that may not be possible using standard analysis techniques. Additionally, the strategies developed here offer an opportunity to obtain genetic information at the single cell level from a variety of other non-forensic trace biological material.

  17. Probing conformational changes of human DNA polymerase lambda using mass spectrometry-based protein footprinting.

    Science.gov (United States)

    Fowler, Jason D; Brown, Jessica A; Kvaratskhelia, Mamuka; Suo, Zucai

    2009-07-17

    Crystallographic studies of the C-terminal DNA polymerase-beta-like domain of full-length human DNA polymerase lambda (fPollambda) suggested that the catalytic cycle might not involve a large protein domain rearrangement as observed with several replicative DNA polymerases and DNA polymerase beta. To examine solution-phase protein conformational changes in fPollambda, which also contains a breast cancer susceptibility gene 1 C-terminal domain and a proline-rich domain at its N-terminus, we used a mass spectrometry-based protein footprinting approach. In parallel experiments, surface accessibility maps for Arg residues were compared for the free fPollambda versus the binary complex of enzyme*gapped DNA and the ternary complex of enzyme*gapped DNA*dNTP (2'-deoxynucleotide triphosphate). These experiments suggested that fPollambda does not undergo major conformational changes during the catalysis in the solution phase. Furthermore, the mass spectrometry-based protein footprinting experiments revealed that active site residue R386 was shielded from the surface only in the presence of both a gapped DNA substrate and an incoming nucleotide. Site-directed mutagenesis and pre-steady-state kinetic studies confirmed the importance of R386 for the enzyme activity and indicated the key role for its guanidino group in stabilizing the negative charges of an incoming nucleotide and the leaving pyrophosphate product. We suggest that such interactions could be shared by and important for catalytic functions of other DNA polymerases.

  18. Flow cytometric analysis of cellular DNA content in human astrocytomas and oligodendrogliomas.

    Science.gov (United States)

    Ahyai, A

    1988-01-01

    This report presents a flow-cytometric analysis of cellular DNA in biopsies and primary cell cultures of 21 human astrocytomas and 19 oligodendrogliomas. A distinct correlation between histological dedifferentiation and pathological DNA distribution was found. Classification was made according to increasing histological anaplasia, corresponding to a four-grade scale and proliferation index (PI). Four types of gliomas were defined according to characteristic DNA patterns and proliferative activities in comparison to their histological grading: 1. purely diploid DNA patterns with low 4C (premitotic) peaks and PI values up to 10 in well-differentiated gliomas; 2. increase of tetraploid cells and PI of 10-16 in tumors with histological grades II or II-III; 3. diploid-tetraploid DNA distribution with PI values up to 30-31 and malignancy grade III; 4. polyploid and aneuploid karyograms with excessive 4C increase, emerging in grade III and especially grade III-IV of these gliomas. Varying DNA distribution during tumor development could be observed in a malignant transformation of an oligodendroglioma I to a glioblastoma after a course of 3 1/2 years. A more detailed subdivision of these tumors according to their DNA content and proliferative activity was achieved. With the exception of occasional variation in karyograms, DNA distribution usually remained stable in primary tissue cultures (PTC).

  19. Evaluation of DNA extraction kits for molecular diagnosis of human Blastocystis subtypes from fecal samples.

    Science.gov (United States)

    Yoshikawa, Hisao; Dogruman-Al, Funda; Dogruman-Ai, Funda; Turk, Songul; Kustimur, Semra; Balaban, Neriman; Sultan, Nedim

    2011-10-01

    Blastocystis sp. is now recognized as one of the most common intestinal parasite in human fecal examinations. Recently, PCR-based diagnostic methods of Blastocystis infection using direct DNA extraction from fresh fecal samples with commercially available kits are reported. Several kits have been developed, but little has been done in comparing the detective sensitivity between PCR methods using the commercial kits. In this study, we compared the detective sensitivity among five commercially available kits (MagNA Pure LC DNA Isolation Kit I, Roche; QuickGene SP Kit DNA, FujiFilm; NucleoSpin Plant II, Macherey-Nagel; QIAamp DNA Stool Mini Kit, Qiagen; ZR Fecal DNA Kit, Zymo Research) and fecal culture method. In a preliminary test, the DNA isolated with two kits (FujiFilm and Macherey-Nagel) showed negative PCR, while the other three kits showed positive PCR. Then, DNA from 50 clinical samples that was Blastocystis-positive in the examination of fecal culture method were isolated with the three kits and 1.1 kbp SSU rRNA gene was detected with PCR. The positive rates of the three kits (Roche, Qiagen, and Zymo Research) were 10, 48 and 94%, respectively. The present study indicated that there is different detective sensitivity among the commercial kits, and fecal culture method is superior in detection rate and cost performance than DNA-elution kits for diagnosis of Blastocystis sp. subtypes.

  20. Effect of Amalaki rasayana on DNA damage and repair in randomized aged human individuals.

    Science.gov (United States)

    Vishwanatha, Udupi; Guruprasad, Kanive P; Gopinath, Puthiya M; Acharya, Raviraj V; Prasanna, Bokkasa V; Nayak, Jayakrishna; Ganesh, Rajeshwari; Rao, Jayalaxmi; Shree, Rashmi; Anchan, Suchitra; Raghu, Kothanahalli S; Joshi, Manjunath B; Paladhi, Puspendu; Varier, Panniampilly M; Muraleedharan, Kollath; Muraleedharan, Thrikovil S; Satyamoorthy, Kapaettu

    2016-09-15

    Preparations from Phyllanthus emblica called Amalaki rasayana is used in the Indian traditional medicinal system of Ayurveda for healthy living in elderly. The biological effects and its mechanisms are not fully understood. Since the diminishing DNA repair is the hallmark of ageing, we tested the influence of Amalaki rasayana on recognized DNA repair activities in healthy aged individuals. Amalaki rasayana was prepared fresh and healthy aged randomized human volunteers were administrated with either rasayana or placebo for 45 days strictly as per the traditional text. The DNA repair was analyzed in peripheral blood mononuclear cells before and after rasayana administration and after 45 days post-rasayana treatment regimen. UVC-induced DNA strand break repair (DSBR) based on extent of DNA unwinding by fluorometric analysis, nucleotide excision repair (NER) by flow cytometry and constitutive base excision repair (BER) by gap filling method were analyzed. Amalaki rasayana administration stably maintained/enhanced the DSBR in aged individuals. There were no adverse side effects. Further, subjects with different body mass index showed differential DNA strand break repair capacity. No change in unscheduled DNA synthesis during NER and BER was observed between the groups. Intake of Amalaki rasayana by aged individuals showed stable maintenance of DNA strand break repair without toxic effects. However, there was no change in nucleotide and base excision repair activities. Results warrant further studies on the effects of Amalaki rasayana on DSBR activities. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  1. Alphoid satellite DNA is tightly associated with centromere antigens in human chromosomes throughout the cell cycle

    Energy Technology Data Exchange (ETDEWEB)

    Masumoto, Hiroshi; Sugimoto, Kenji; Okazaki, Tuneko (Nagoya Univ. (Japan))

    1989-03-01

    In this study, the authors have examined a DNA element specific to the centromere domain of human chromosomes. Purified HeLa chromosomes were digested with the restriction enzyme Sau3AI and fractionated by sedimentation through a sucrose gradient. Fractions showing antigenicity to anticentromere (kinetochore) serum obtained from a scleroderma CREST patient were used to construct a DNA library. From this library they found one clone which has specifically hybridized to the centromere domain of metaphase chromosomes using a biotinylated probe DNA and FITC-conjugated avidin. The clone contained a stretch of alphoid DNA dimer. To determine precisely the relative location of the alphoid DNA stretch and the centromere antigen, a method was developed to carry out in situ hybridization of DNA and indirect immunofluorescent staining of antigen on the same cell preparation. Using this method, they have found perfect overlapping of the alphoid DNA sites with the centromere antigen in both metaphase chromosomes and nuclei at various stages in the cell cycle. They have also observed this exact correlation at the attachment sites of artificially extended sister chromatids. These results suggest the possibility that alphoid DNA repeats are a key component of kinetochore structure.

  2. Crystal Structure of the Human Hsmar1-Derived Transposase Domain in the DNA Repair Enzyme Metnase

    Energy Technology Data Exchange (ETDEWEB)

    Goodwin, Kristie D.; He, Hongzhen; Imasaki, Tsuyoshi; Lee, Suk-Hee; Georgiadis, Millie M. (Indiana-Med)

    2010-08-12

    Although the human genome is littered with sequences derived from the Hsmar1 transposon, the only intact Hsmar1 transposase gene exists within a chimeric SET-transposase fusion protein referred to as Metnase or SETMAR. Metnase retains many of the transposase activities including terminal inverted repeat (TIR) specific DNA-binding activity, DNA cleavage activity, albeit uncoupled from TIR-specific binding, and the ability to form a synaptic complex. However, Metnase has evolved as a DNA repair protein that is specifically involved in nonhomologous end joining. Here, we present two crystal structures of the transposase catalytic domain of Metnase revealing a dimeric enzyme with unusual active site plasticity that may be involved in modulating metal binding. We show through characterization of a dimerization mutant, F460K, that the dimeric form of the enzyme is required for its DNA cleavage, DNA-binding, and nonhomologous end joining activities. Of significance is the conservation of F460 along with residues that we propose may be involved in the modulation of metal binding in both the predicted ancestral Hsmar1 transposase sequence as well as in the modern enzyme. The Metnase transposase has been remarkably conserved through evolution; however, there is a clustering of substitutions located in alpha helices 4 and 5 within the putative DNA-binding site, consistent with loss of transposition specific DNA cleavage activity and acquisition of DNA repair specific cleavage activity.

  3. Divergence between samples of chimpanzee and human DNA sequences is 5%, counting indels

    Science.gov (United States)

    Britten, Roy J.

    2002-01-01

    Five chimpanzee bacterial artificial chromosome (BAC) sequences (described in GenBank) have been compared with the best matching regions of the human genome sequence to assay the amount and kind of DNA divergence. The conclusion is the old saw that we share 98.5% of our DNA sequence with chimpanzee is probably in error. For this sample, a better estimate would be that 95% of the base pairs are exactly shared between chimpanzee and human DNA. In this sample of 779 kb, the divergence due to base substitution is 1.4%, and there is an additional 3.4% difference due to the presence of indels. The gaps in alignment are present in about equal amounts in the chimp and human sequences. They occur equally in repeated and nonrepeated sequences, as detected by repeatmasker (http://ftp.genome.washington.edu/RM/RepeatMasker.html). PMID:12368483

  4. Bovine leukaemia virus DNA in fresh milk and raw beef for human consumption.

    Science.gov (United States)

    Olaya-Galán, N N; Corredor-Figueroa, A P; Guzmán-Garzón, T C; Ríos-Hernandez, K S; Salas-Cárdenas, S P; Patarroyo, M A; Gutierrez, M F

    2017-11-01

    Bovine leukaemia virus (BLV) is the causative agent of enzootic bovine leucosis, which has been reported worldwide. BLV has been found recently in human tissue and it could have a significant impact on human health. A possible hypothesis regarding viral entry to humans is through the consumption of infected foodstuffs. This study was aimed at detecting the presence of BLV DNA in raw beef and fresh milk for human consumption. Nested PCR directed at the BLV gag gene (272 bp) was used as a diagnostic test. PCR products were confirmed by Sanger sequencing. Forty-nine per cent of the samples proved positive for the presence of proviral DNA. This is the first study highlighting the presence of the BLV gag gene in meat products for human consumption and confirms the presence of the viral DNA in raw milk, as in previous reports. The presence of viral DNA in food products could suggest that viral particles may also be found. Further studies are needed to confirm the presence of infected viral particles, even though the present findings could represent a first approach to BLV transmission to humans through foodstuff consumption.

  5. Different cDNA microarray patterns of gene expression reflecting changes during metastatic progression in adenoid cystic carcinoma

    Directory of Open Access Journals (Sweden)

    Yu Fan

    2003-12-01

    Full Text Available Abstract Background The metastatic ability of tumor cells is determined by level of expression of specific genes that may be identified with the aid of cDNA microarray containing thousands of genes and can be used to establish the expression profile of disease related genes in complex biological system. Materials and Methods Salivary adenoid cystic carcinoma cell line and its high metastases adenoid cystic carcinoma clone were used as model systems to reveal the gene expression alteration related to metastasis mechanism by cDNA microarray analysis. The correlation of metastatic phenotypic changes and expression levels of 4 selected genes (encoding CD98, L6, RPL29, and TSH were further validated by using RT-PCR analysis of human tumor specimens from primary adenoid cystic carcinoma and corresponding metastasis lymph nodes. Results Of the 7,675 clones of known genes and expressed sequence tags (ESTs that were analyzed, 30 showed significantly different (minimum 3 fold expression levels in two cell lines. Out of 30 genes found differentially expressed, 18 were up regulated (with ratio more than 3 and 12 down regulated (with ratio less than 1/3. Conclusion Some of these genes are known to be involved in human tumor antigen, immune surveillance, adhesion, cell signaling pathway and growth control. It is suggested that the microarray in combination with a relevant analysis facilitates rapid and simultaneous identification of multiple genes of interests and in this study it provided a profound clue to screen candidate targets for early diagnosis and intervention.

  6. Immunogenicity of bivalent human papillomavirus DNA vaccine using human endogenous retrovirus envelope-coated baculoviral vectors in mice and pigs.

    Directory of Open Access Journals (Sweden)

    Hee-Jung Lee

    Full Text Available Human papillomavirus is known to be the major pathogen of cervical cancer. Here, we report the efficacy of a bivalent human papillomavirus type 16 and 18 DNA vaccine system following repeated dosing in mice and pigs using a recombinant baculovirus bearing human endogenous retrovirus envelope protein (AcHERV as a vector. The intramuscular administration of AcHERV-based HPV16L1 and HPV18L1 DNA vaccines induced antigen-specific serum IgG, vaginal IgA, and neutralizing antibodies to levels comparable to those achieved using the commercially marketed vaccine Cervarix. Similar to Cervarix, AcHERV-based bivalent vaccinations completely blocked subsequent vaginal challenge with HPV type-specific pseudovirions. However, AcHERV-based bivalent vaccinations induced significantly higher cell-mediated immune responses than Cervarix, promoting 4.5- (HPV16L1 and 3.9-(HPV18L1 fold higher interferon-γ production in splenocytes upon stimulation with antigen type-specific pseudovirions. Repeated dosing did not affect the immunogenicity of AcHERV DNA vaccines. Three sequential immunizations with AcHERV-HP18L1 DNA vaccine followed by three repeated dosing with AcHERV-HP16L1 over 11 weeks induced an initial production of anti-HPV18L1 antibody followed by subsequent induction of anti-HPV16L1 antibody. Finally, AcHERV-based bivalent DNA vaccination induced antigen-specific serum IgG immune responses in pigs. These results support the further development of AcHERV as a bivalent human papillomavirus DNA vaccine system for use in preventing the viral infection as well as treating the infected women by inducing both humoral and cell-mediated immune responses. Moreover, the possibility of repeated dosing indicates the utility of AcHERV system for reusable vectors of other viral pathogen vaccines.

  7. Immunogenicity of bivalent human papillomavirus DNA vaccine using human endogenous retrovirus envelope-coated baculoviral vectors in mice and pigs.

    Science.gov (United States)

    Lee, Hee-Jung; Hur, Yoon-Ki; Cho, Youn-Dong; Kim, Mi-Gyeong; Lee, Hoon-Taek; Oh, Yu-Kyoung; Kim, Young Bong

    2012-01-01

    Human papillomavirus is known to be the major pathogen of cervical cancer. Here, we report the efficacy of a bivalent human papillomavirus type 16 and 18 DNA vaccine system following repeated dosing in mice and pigs using a recombinant baculovirus bearing human endogenous retrovirus envelope protein (AcHERV) as a vector. The intramuscular administration of AcHERV-based HPV16L1 and HPV18L1 DNA vaccines induced antigen-specific serum IgG, vaginal IgA, and neutralizing antibodies to levels comparable to those achieved using the commercially marketed vaccine Cervarix. Similar to Cervarix, AcHERV-based bivalent vaccinations completely blocked subsequent vaginal challenge with HPV type-specific pseudovirions. However, AcHERV-based bivalent vaccinations induced significantly higher cell-mediated immune responses than Cervarix, promoting 4.5- (HPV16L1) and 3.9-(HPV18L1) fold higher interferon-γ production in splenocytes upon stimulation with antigen type-specific pseudovirions. Repeated dosing did not affect the immunogenicity of AcHERV DNA vaccines. Three sequential immunizations with AcHERV-HP18L1 DNA vaccine followed by three repeated dosing with AcHERV-HP16L1 over 11 weeks induced an initial production of anti-HPV18L1 antibody followed by subsequent induction of anti-HPV16L1 antibody. Finally, AcHERV-based bivalent DNA vaccination induced antigen-specific serum IgG immune responses in pigs. These results support the further development of AcHERV as a bivalent human papillomavirus DNA vaccine system for use in preventing the viral infection as well as treating the infected women by inducing both humoral and cell-mediated immune responses. Moreover, the possibility of repeated dosing indicates the utility of AcHERV system for reusable vectors of other viral pathogen vaccines.

  8. A magnetic bead-based method for concentrating DNA from human urine for downstream detection.

    Directory of Open Access Journals (Sweden)

    Hali Bordelon

    Full Text Available Due to the presence of PCR inhibitors, PCR cannot be used directly on most clinical samples, including human urine, without pre-treatment. A magnetic bead-based strategy is one potential method to collect biomarkers from urine samples and separate the biomarkers from PCR inhibitors. In this report, a 1 mL urine sample was mixed within the bulb of a transfer pipette containing lyophilized nucleic acid-silica adsorption buffer and silica-coated magnetic beads. After mixing, the sample was transferred from the pipette bulb to a small diameter tube, and captured biomarkers were concentrated using magnetic entrainment of beads through pre-arrayed wash solutions separated by small air gaps. Feasibility was tested using synthetic segments of the 140 bp tuberculosis IS6110 DNA sequence spiked into pooled human urine samples. DNA recovery was evaluated by qPCR. Despite the presence of spiked DNA, no DNA was detectable in unextracted urine samples, presumably due to the presence of PCR inhibitors. However, following extraction with the magnetic bead-based method, we found that ∼50% of spiked TB DNA was recovered from human urine containing roughly 5×10(3 to 5×10(8 copies of IS6110 DNA. In addition, the DNA was concentrated approximately ten-fold into water. The final concentration of DNA in the eluate was 5×10(6, 14×10(6, and 8×10(6 copies/µL for 1, 3, and 5 mL urine samples, respectively. Lyophilized and freshly prepared reagents within the transfer pipette produced similar results, suggesting that long-term storage without refrigeration is possible. DNA recovery increased with the length of the spiked DNA segments from 10±0.9% for a 75 bp DNA sequence to 42±4% for a 100 bp segment and 58±9% for a 140 bp segment. The estimated LOD was 77 copies of DNA/µL of urine. The strategy presented here provides a simple means to achieve high nucleic acid recovery from easily obtained urine samples, which does not contain inhibitors of PCR.

  9. Three-dimensional cultures modeling premalignant progression of human breast epithelial cells: role of cysteine cathepsins.

    Science.gov (United States)

    Mullins, Stefanie R; Sameni, Mansoureth; Blum, Galia; Bogyo, Matthew; Sloane, Bonnie F; Moin, Kamiar

    2012-12-01

    The expression of the cysteine protease cathepsin B is increased in early stages of human breast cancer.To assess the potential role of cathepsin B in premalignant progression of breast epithelial cells, we employed a 3D reconstituted basement membrane overlay culture model of MCF10A human breast epithelial cells and isogenic variants that replicate the in vivo phenotypes of hyper plasia(MCF10AneoT) and atypical hyperplasia (MCF10AT1). MCF10A cells developed into polarized acinar structures with central lumens. In contrast, MCF10AneoT and MCF10AT1 cells form larger structures in which the lumens are filled with cells. CA074Me, a cell-permeable inhibitor selective for the cysteine cathepsins B and L,reduced proliferation and increased apoptosis of MCF10A, MCF10AneoT and MCF10AT1 cells in 3D culture. We detected active cysteine cathepsins in the isogenic MCF10 variants in 3D culture with GB111, a cell-permeable activity based probe, and established differential inhibition of cathepsin B in our 3D cultures. We conclude that cathepsin B promotes proliferation and premalignant progression of breast epithelial cells. These findings are consistent with studies by others showing that deletion of cathepsin B in the transgenic MMTV-PyMT mice, a murine model that is predisposed to development of mammary cancer, reduces malignant progression.

  10. A homologous subfamily of satellite III DNA on human chromosomes 14 and 22.

    OpenAIRE

    Choo, K H; Earle, E; McQuillan, C.

    1990-01-01

    We describe a new subfamily of human satellite III DNA that is represented on two different acrocentric chromosomes. This DNA is composed of a tandemly repeated array of diverged 5-base-pair monomer units of the sequence GGAAT or GGAGT. These monomers are organised into a 1.37-kilobase higher-order structure that is itself tandemly reiterated. Using a panel of somatic cell hybrids containing specific human chromosomes, this higher-order structure is demonstrated on chromosomes 14 and 22, but ...

  11. Enterobius vermicularis: ancient DNA from north and south American human coprolites

    Directory of Open Access Journals (Sweden)

    Iñiguez Alena M

    2003-01-01

    Full Text Available A molecular paleoparasitological diagnostic approach was developed for Enterobius vermicularis. Ancient DNA was extracted from 27 coprolites from archaeological sites in Chile and USA. Enzymatic amplification of human mtDNA sequences confirmed the human origin. We designed primers specific to the E. vermicularis 5S ribosomal RNA spacer region and they allowed reproducible polymerase chain reaction identification of ancient material. We suggested that the paleoparasitological microscopic identification could accompany molecular diagnosis, which also opens the possibility of sequence analysis to understand parasite-host evolution.

  12. Structure-function analysis of human enzymes initiating nucleobase repair in DNA and RNA

    OpenAIRE

    Sundheim, Ottar

    2008-01-01

    In humans, there are four known glycosylases that initiate repair of uracils in DNA. These are UNG, TDG, SMUG1, and MBD4. It was proposed that the replication independent SMUG1 was the main enzyme initiating removal of deaminated cytosine, whereas UNG2 was responsible for replication associated repair of mis-incorporated dUTP (Nilsen et al., 2001). We aimed at elucidating the specific function of the two main human uracil-DNA glycosylases in vitro and in vivo to further clarify their distinct...