P-Hint-Hunt: a deep parallelized whole genome DNA methylation detection tool.

PubMed

Peng, Shaoliang; Yang, Shunyun; Gao, Ming; Liao, Xiangke; Liu, Jie; Yang, Canqun; Wu, Chengkun; Yu, Wenqiang

2017-03-14

The increasing studies have been conducted using whole genome DNA methylation detection as one of the most important part of epigenetics research to find the significant relationships among DNA methylation and several typical diseases, such as cancers and diabetes. In many of those studies, mapping the bisulfite treated sequence to the whole genome has been the main method to study DNA cytosine methylation. However, today's relative tools almost suffer from inaccuracies and time-consuming problems. In our study, we designed a new DNA methylation prediction tool ("Hint-Hunt") to solve the problem. By having an optimal complex alignment computation and Smith-Waterman matrix dynamic programming, Hint-Hunt could analyze and predict the DNA methylation status. But when Hint-Hunt tried to predict DNA methylation status with large-scale dataset, there are still slow speed and low temporal-spatial efficiency problems. In order to solve the problems of Smith-Waterman dynamic programming and low temporal-spatial efficiency, we further design a deep parallelized whole genome DNA methylation detection tool ("P-Hint-Hunt") on Tianhe-2 (TH-2) supercomputer. To the best of our knowledge, P-Hint-Hunt is the first parallel DNA methylation detection tool with a high speed-up to process large-scale dataset, and could run both on CPU and Intel Xeon Phi coprocessors. Moreover, we deploy and evaluate Hint-Hunt and P-Hint-Hunt on TH-2 supercomputer in different scales. The experimental results illuminate our tools eliminate the deviation caused by bisulfite treatment in mapping procedure and the multi-level parallel program yields a 48 times speed-up with 64 threads. P-Hint-Hunt gain a deep acceleration on CPU and Intel Xeon Phi heterogeneous platform, which gives full play of the advantages of multi-cores (CPU) and many-cores (Phi).

Asymmetric Epigenetic Modification and Elimination of rDNA Sequences by Polyploidization in Wheat[W

PubMed Central

Guo, Xiang

2014-01-01

rRNA genes consist of long tandem repeats clustered on chromosomes, and their products are important functional components of the ribosome. In common wheat (Triticum aestivum), rDNA loci from the A and D genomes were largely lost during the evolutionary process. This biased DNA elimination may be related to asymmetric transcription and epigenetic modifications caused by the polyploid formation. Here, we observed both sets of parental nucleolus organizing regions (NORs) were expressed after hybridization, but asymmetric silencing of one parental NOR was immediately induced by chromosome doubling, and reversing the ploidy status could not reactivate silenced NORs. Furthermore, increased CHG and CHH DNA methylation on promoters was accompanied by asymmetric silencing of NORs. Enrichment of H3K27me3 and H3K9me2 modifications was also observed to be a direct response to increased DNA methylation and transcriptional inactivation of NOR loci. Both A and D genome NOR loci with these modifications started to disappear in the S4 generation and were completely eliminated by the S7 generation in synthetic tetraploid wheat. Our results indicated that asymmetric epigenetic modification and elimination of rDNA sequences between different donor genomes may lead to stable allopolyploid wheat with increased differentiation and diversity. PMID:25415973

Asymmetric epigenetic modification and elimination of rDNA sequences by polyploidization in wheat.

PubMed

Guo, Xiang; Han, Fangpu

2014-11-01

rRNA genes consist of long tandem repeats clustered on chromosomes, and their products are important functional components of the ribosome. In common wheat (Triticum aestivum), rDNA loci from the A and D genomes were largely lost during the evolutionary process. This biased DNA elimination may be related to asymmetric transcription and epigenetic modifications caused by the polyploid formation. Here, we observed both sets of parental nucleolus organizing regions (NORs) were expressed after hybridization, but asymmetric silencing of one parental NOR was immediately induced by chromosome doubling, and reversing the ploidy status could not reactivate silenced NORs. Furthermore, increased CHG and CHH DNA methylation on promoters was accompanied by asymmetric silencing of NORs. Enrichment of H3K27me3 and H3K9me2 modifications was also observed to be a direct response to increased DNA methylation and transcriptional inactivation of NOR loci. Both A and D genome NOR loci with these modifications started to disappear in the S4 generation and were completely eliminated by the S7 generation in synthetic tetraploid wheat. Our results indicated that asymmetric epigenetic modification and elimination of rDNA sequences between different donor genomes may lead to stable allopolyploid wheat with increased differentiation and diversity. © 2014 American Society of Plant Biologists. All rights reserved.

Detection of Wuchereria bancrofti DNA in paired serum and urine samples using polymerase chain reaction-based systems.

PubMed

Ximenes, Camila; Brandão, Eduardo; Oliveira, Paula; Rocha, Abraham; Rego, Tamisa; Medeiros, Rafael; Aguiar-Santos, Ana; Ferraz, João; Reis, Christian; Araujo, Paulo; Carvalho, Luiz; Melo, Fabio L

2014-12-01

The Global Program for the Elimination of Lymphatic Filariasis (GPELF) aims to eliminate this disease by the year 2020. However, the development of more specific and sensitive tests is important for the success of the GPELF. The present study aimed to standardise polymerase chain reaction (PCR)-based systems for the diagnosis of filariasis in serum and urine. Twenty paired biological urine and serum samples from individuals already known to be positive for Wuchereria bancrofti were collected during the day. Conventional PCR and semi-nested PCR assays were optimised. The detection limit of the technique for purified W. bancrofti DNA extracted from adult worms was 10 fg for the internal systems (WbF/Wb2) and 0.1 fg by using semi-nested PCR. The specificity of the primers was confirmed experimentally by amplification of 1 ng of purified genomic DNA from other species of parasites. Evaluation of the paired urine and serum samples by the semi-nested PCR technique indicated only two of the 20 tested individuals were positive, whereas the simple internal PCR system (WbF/Wb2), which has highly promising performance, revealed that all the patients were positive using both samples. This study successfully demonstrated the possibility of using the PCR technique on urine for the diagnosis of W. bancrofti infection.

A Novel Xenomonitoring Technique Using Mosquito Excreta/Feces for the Detection of Filarial Parasites and Malaria.

PubMed

Pilotte, Nils; Zaky, Weam I; Abrams, Brian P; Chadee, Dave D; Williams, Steven A

2016-04-01

Given the continued successes of the world's lymphatic filariasis (LF) elimination programs and the growing successes of many malaria elimination efforts, the necessity of low cost tools and methodologies applicable to long-term disease surveillance is greater than ever before. As many countries reach the end of their LF mass drug administration programs and a growing number of countries realize unprecedented successes in their malaria intervention efforts, the need for practical molecular xenomonitoring (MX), capable of providing surveillance for disease recrudescence in settings of decreased parasite prevalence is increasingly clear. Current protocols, however, require testing of mosquitoes in pools of 25 or fewer, making high-throughput examination a challenge. The new method we present here screens the excreta/feces from hundreds of mosquitoes per pool and provides proof-of-concept for a practical alternative to traditional methodologies resulting in significant cost and labor savings. Excreta/feces of laboratory reared Aedes aegypti or Anopheles stephensi mosquitoes provided with a Brugia malayi microfilaria-positive or Plasmodium vivax-positive blood meal respectively were tested for the presence of parasite DNA using real-time PCR. A titration of samples containing various volumes of B. malayi-negative mosquito feces mixed with positive excreta/feces was also tested to determine sensitivity of detection. Real-time PCR amplification of B. malayi and P. vivax DNA from the excreta/feces of infected mosquitoes was demonstrated, and B. malayi DNA in excreta/feces from one to two mf-positive blood meal-receiving mosquitoes was detected when pooled with volumes of feces from as many as 500 uninfected mosquitoes. While the operationalizing of excreta/feces testing may require the development of new strategies for sample collection, the high-throughput nature of this new methodology has the potential to greatly reduce MX costs. This will prove particularly useful in post-transmission-interruption settings, where this inexpensive approach to long-term surveillance will help to stretch the budgets of LF and malaria elimination programs. Furthermore, as this methodology is adaptable to the detection of both single celled (P. vivax) and multicellular eukaryotic pathogens (B. malayi), exploration of its use for the detection of various other mosquito-borne diseases including viruses should be considered. Additionally, integration strategies utilizing excreta/feces testing for the simultaneous surveillance of multiple diseases should be explored.

A Novel Xenomonitoring Technique Using Mosquito Excreta/Feces for the Detection of Filarial Parasites and Malaria

PubMed Central

Pilotte, Nils; Zaky, Weam I.; Abrams, Brian P.; Chadee, Dave D.; Williams, Steven A.

2016-01-01

Background Given the continued successes of the world’s lymphatic filariasis (LF) elimination programs and the growing successes of many malaria elimination efforts, the necessity of low cost tools and methodologies applicable to long-term disease surveillance is greater than ever before. As many countries reach the end of their LF mass drug administration programs and a growing number of countries realize unprecedented successes in their malaria intervention efforts, the need for practical molecular xenomonitoring (MX), capable of providing surveillance for disease recrudescence in settings of decreased parasite prevalence is increasingly clear. Current protocols, however, require testing of mosquitoes in pools of 25 or fewer, making high-throughput examination a challenge. The new method we present here screens the excreta/feces from hundreds of mosquitoes per pool and provides proof-of-concept for a practical alternative to traditional methodologies resulting in significant cost and labor savings. Methodology/Principal Findings Excreta/feces of laboratory reared Aedes aegypti or Anopheles stephensi mosquitoes provided with a Brugia malayi microfilaria-positive or Plasmodium vivax-positive blood meal respectively were tested for the presence of parasite DNA using real-time PCR. A titration of samples containing various volumes of B. malayi-negative mosquito feces mixed with positive excreta/feces was also tested to determine sensitivity of detection. Real-time PCR amplification of B. malayi and P. vivax DNA from the excreta/feces of infected mosquitoes was demonstrated, and B. malayi DNA in excreta/feces from one to two mf-positive blood meal-receiving mosquitoes was detected when pooled with volumes of feces from as many as 500 uninfected mosquitoes. Conclusions/Significance While the operationalizing of excreta/feces testing may require the development of new strategies for sample collection, the high-throughput nature of this new methodology has the potential to greatly reduce MX costs. This will prove particularly useful in post-transmission-interruption settings, where this inexpensive approach to long-term surveillance will help to stretch the budgets of LF and malaria elimination programs. Furthermore, as this methodology is adaptable to the detection of both single celled (P. vivax) and multicellular eukaryotic pathogens (B. malayi), exploration of its use for the detection of various other mosquito-borne diseases including viruses should be considered. Additionally, integration strategies utilizing excreta/feces testing for the simultaneous surveillance of multiple diseases should be explored. PMID:27096156

Is mass drug administration against lymphatic filariasis required in urban settings? The experience in Kano, Nigeria.

PubMed

Pam, Dung D; de Souza, Dziedzom K; D'Souza, Susan; Opoku, Millicent; Sanda, Safiya; Nazaradden, Ibrahim; Anagbogu, Ifeoma N; Okoronkwo, Chukwu; Davies, Emmanuel; Elhassan, Elisabeth; Molyneux, David H; Bockarie, Moses J; Koudou, Benjamin G

2017-10-01

The Global Programme to Eliminate Lymphatic Filariasis (GPELF), launched in 2000, has the target of eliminating the disease as a public health problem by the year 2020. The strategy adopted is mass drug administration (MDA) to all eligible individuals in endemic communities and the implementation of measures to reduce the morbidity of those suffering from chronic disease. Success has been recorded in many rural endemic communities in which elimination efforts have centered. However, implementation has been challenging in several urban African cities. The large cities of West Africa, exemplified in Nigeria in Kano are challenging for LF elimination program because reaching 65% therapeutic coverage during MDA is difficult. There is therefore a need to define a strategy which could complement MDA. Thus, in Kano State, Nigeria, while LF MDA had reached 33 of the 44 Local Government Areas (LGAs) there remained eleven 'urban' LGAs which had not been covered by MDA. Given the challenges of achieving at least 65% coverage during MDA implementation over several years in order to achieve elimination, it may be challenging to eliminate LF in such settings. In order to plan the LF control activities, this study was undertaken to confirm the LF infection prevalence in the human and mosquito populations in three urban LGAs. The prevalence of circulating filarial antigen (CFA) of Wuchereria bancrofti was assessed by an immuno-chromatography test (ICT) in 981 people in three urban LGAs of Kano state, Nigeria. Mosquitoes were collected over a period of 4 months from May to August 2015 using exit traps, gravid traps and pyrethrum knock-down spray sheet collections (PSC) in different households. A proportion of mosquitoes were analyzed for W. bancrofti, using dissection, loop-mediated isothermal amplification (LAMP) assay and conventional polymerase chain reaction (PCR). The results showed that none of the 981 subjects (constituted of <21% of children 5-10 years old) tested had detectable levels of CFA in their blood. Entomological results showed that An. gambiae s.l. had W. bancrofti DNA detectable in pools in Kano; W. bancrofti DNA was detected in between 0.96% and 6.78% and to a lesser extent in Culex mosquitoes where DNA was detected at rates of between 0.19% and 0.64%. DNA analysis showed that An. coluzzii constituted 9.9% of the collected mosquitoes and the remaining 90.1% of the mosquitoes were Culex mosquitoes. Despite detection of W. bancrofti DNA within mosquito specimens collected in three Kano urban LGAs, we were not able to find a subject with detectable level of CFA. Together with other evidence suggesting that LF transmission in urban areas in West Africa may not be of significant importance, the Federal Ministry of Health advised that two rounds of MDA be undertaken in the urban areas of Kano. It is recommended that the prevalence of W. bancrofti infection in the human and mosquito populations be re-assessed after a couple of years.

[The biological aspects of chromatin diminution].

PubMed

Akif'ev, A P; Grishanin, A K

1993-01-01

The chromatine diminution (CD), first discovered by Boveri (1887) in ascarids, represents programmed elimination of a part of genetic material in the nuclei of the somatic cells in cyclops and ascarids, and in the protist macronuclei. The CD can be considered as a macromutation sharply changing chromosomal structure, though minimally effecting the phenotype. The analysis of CD is of significance for discussing mechanisms of origin of chromosomal organization, transformation of genome molecular structure in eucaryote evolution, role of the extra DNA.

Improving the yield and quality of DNA isolated from white-rot fungi.

PubMed

Kuhad, R C; Kapoor, R K; Lal, R

2004-01-01

A new simple method used to eliminate polysaccharides that cause problems during DNA isolation was established for 6 different white-rot fungi using 1% hexadecyltrimethylammonium bromide (CTAB) as wash buffer and followed by centrifugation. Variation in the DNA yield and quality was ascertained using precipitating agents, detergents and cell-wall-hydrolyzing chitinase. Considerable amount of exopolysaccharides from fungal biomass was removed with the use of 1% CTAB wash buffer followed by centrifugation. The DNA varied in terms of yield and quality. For the DNA extraction use of 2% SDS in extraction buffer worked best for Pycnoporus cinnabarinus, Cyathus bulleri, Cyathus striatus and Cyathus stercoreus, while 2% CTAB worked best for Phanerochaete chrysosporium and Pleurotus ostreatus. Elimination of phenol and use of absolute ethanol for precipitating DNA resulted in good yield and quality of DNA. This DNA was amenable to restriction endonuclease digestion.

The Effect of Ursolic Acid on Leishmania (Leishmania) amazonensis Is Related to Programed Cell Death and Presents Therapeutic Potential in Experimental Cutaneous Leishmaniasis

PubMed Central

Yamamoto, Eduardo S.; Campos, Bruno L. S.; Jesus, Jéssica A.; Laurenti, Márcia D.; Ribeiro, Susan P.; Kallás, Esper G.; Rafael-Fernandes, Mariana; Santos-Gomes, Gabriela; Silva, Marcelo S.; Sessa, Deborah P.; Lago, João H. G.; Levy, Débora; Passero, Luiz F. D.

2015-01-01

Among neglected tropical diseases, leishmaniasis is one of the most important ones, affecting more than 12 million people worldwide. The available treatments are not well tolerated, and present diverse side effects, justifying the search for new therapeutic compounds. In the present study, the activity of ursolic acid (UA) and oleanolic acid (OA) were assayed in experimental cutaneous leishmaniasis (in vitro and in vivo). Promastigote forms of L. amazonensis were incubated with OA and UA for 24h, and effective concentration 50% (EC50) was estimated. Ultraestructural alterations in Leishmania amazonensis promastigotes after UA treatment were evaluated by transmission electron microscopy, and the possible mode of action was assayed through Annexin V and propidium iodide staining, caspase 3/7 activity, DNA fragmentation and transmembrane mitochondrial potential. The UA potential was evaluated in intracellular amastigotes, and its therapeutic potential was evaluated in L. amazonensis infected BALB/c mice. UA eliminated L. amazonensis promastigotes with an EC50 of 6.4 μg/mL, comparable with miltefosine, while OA presented only a marginal effect on promastigote forms at 100 μg/mL. The possible mechanism by which promastigotes were eliminated by UA was programmed cell death, independent of caspase 3/7, but it was highly dependent on mitochondria activity. UA was not toxic for peritoneal macrophages from BALB/c mice, and it was able to eliminate intracellular amastigotes, associated with nitric oxide (NO) production. OA did not eliminate amastigotes nor trigger NO. L. amazonensis infected BALB/c mice submitted to UA treatment presented lesser lesion size and parasitism compared to control. This study showed, for the first time, that UA eliminate promastigote forms through a mechanism associated with programed cell death, and importantly, was effective in vivo. Therefore, UA can be considered an interesting candidate for future tests as a prototype drug for the treatment of cutaneous leishmaniasis. PMID:26674781

Elimination of mitochondrial DNA is not required for herpes simplex virus 1 replication.

PubMed

Duguay, Brett A; Saffran, Holly A; Ponomarev, Alina; Duley, Shayla A; Eaton, Heather E; Smiley, James R

2014-03-01

Infection with herpes simplex virus type 1 (HSV-1) results in the rapid elimination of mitochondrial DNA (mtDNA) from host cells. It is known that a mitochondrial isoform of the viral alkaline nuclease (UL12) called UL12.5 triggers this process. However, very little is known about the impact of mtDNA depletion on viral replication or the biology of HSV-1 infections. These questions have been difficult to address because UL12.5 and UL12 are encoded by overlapping transcripts that share the same open reading frame. As a result, mutations that alter UL12.5 also affect UL12, and UL12 null mutations severely impair viral growth by interfering with the intranuclear processing of progeny viral genomes. Therefore, to specifically assess the impact of mtDNA depletion on viral replication, it is necessary to eliminate the activity of UL12.5 while preserving the nuclear functions of UL12. Previous work has shown that the human cytomegalovirus alkaline nuclease UL98 can functionally substitute for UL12 during HSV-1 replication. We found that UL98 is unable to deplete mtDNA in transfected cells and therefore generated an HSV-1 variant in which UL98 coding sequences replace the UL12/UL12.5 open reading frame. The resulting virus was severely impaired in its ability to trigger mtDNA loss but reached titers comparable to those of wild-type HSV-1 in one-step and multistep growth experiments. Together, these observations demonstrate that the elimination of mtDNA is not required for HSV-1 replication in cell culture. Herpes simplex virus types 1 and 2 destroy the DNA of host cell mitochondria, the powerhouses of cells. Epstein-Barr virus, a distantly related herpesvirus, has a similar effect, indicating that mitochondrial DNA destruction is under positive selection and thus confers a benefit to the virus. The present work shows that mitochondrial DNA destruction is not required for efficient replication of herpes simplex virus type 1 in cultured Vero kidney epithelial cells, suggesting that this activity likely benefits the virus in other cell types or in the intact human host.

Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase

PubMed Central

Ormeño, Fernando; Barrientos, Camila; Ramirez, Santiago; Ponce, Iván; Valenzuela, Lucía; Sepúlveda, Sofía; Bitar, Mainá; Kemmerling, Ulrike; Machado, Carlos Renato; Cabrera, Gonzalo; Galanti, Norbel

2016-01-01

Trypanosoma cruzi, the etiological agent of Chagas’ disease, presents three cellular forms (trypomastigotes, epimastigotes and amastigotes), all of which are submitted to oxidative species in its hosts. However, T. cruzi is able to resist oxidative stress suggesting a high efficiency of its DNA repair machinery.The Base Excision Repair (BER) pathway is one of the main DNA repair mechanisms in other eukaryotes and in T. cruzi as well. DNA glycosylases are enzymes involved in the recognition of oxidative DNA damage and in the removal of oxidized bases, constituting the first step of the BER pathway. Here, we describe the presence and activity of TcNTH1, a nuclear T. cruzi DNA glycosylase. Surprisingly, purified recombinant TcNTH1 does not remove the thymine glycol base, but catalyzes the cleavage of a probe showing an AP site. The same activity was found in epimastigote and trypomastigote homogenates suggesting that the BER pathway is not involved in thymine glycol DNA repair. TcNTH1 DNA-binding properties assayed in silico are in agreement with the absence of a thymine glycol removing function of that parasite enzyme. Over expression of TcNTH1 decrease parasite viability when transfected epimastigotes are submitted to a sustained production of H2O2.Therefore, TcNTH1 is the only known NTH1 orthologous unable to eliminate thymine glycol derivatives but that recognizes and cuts an AP site, most probably by a beta-elimination mechanism. We cannot discard that TcNTH1 presents DNA glycosylase activity on other DNA base lesions. Accordingly, a different DNA repair mechanism should be expected leading to eliminate thymine glycol from oxidized parasite DNA. Furthermore, TcNTH1 may play a role in the AP site recognition and processing. PMID:27284968

Paramecium tetraurelia chromatin assembly factor-1-like protein PtCAF-1 is involved in RNA-mediated control of DNA elimination.

PubMed

Ignarski, Michael; Singh, Aditi; Swart, Estienne C; Arambasic, Miroslav; Sandoval, Pamela Y; Nowacki, Mariusz

2014-10-29

Genome-wide DNA remodelling in the ciliate Paramecium is ensured by RNA-mediated trans-nuclear crosstalk between the germline and the somatic genomes during sexual development. The rearrangements include elimination of transposable elements, minisatellites and tens of thousands non-coding elements called internally eliminated sequences (IESs). The trans-nuclear genome comparison process employs a distinct class of germline small RNAs (scnRNAs) that are compared against the parental somatic genome to select the germline-specific subset of scnRNAs that subsequently target DNA elimination in the progeny genome. Only a handful of proteins involved in this process have been identified so far and the mechanism of DNA targeting is unknown. Here we describe chromatin assembly factor-1-like protein (PtCAF-1), which we show is required for the survival of sexual progeny and localizes first in the parental and later in the newly developing macronucleus. Gene silencing shows that PtCAF-1 is required for the elimination of transposable elements and a subset of IESs. PTCAF-1 depletion also impairs the selection of germline-specific scnRNAs during development. We identify specific histone modifications appearing during Paramecium development which are strongly reduced in PTCAF-1 depleted cells. Our results demonstrate the importance of PtCAF-1 for the epigenetic trans-nuclear cross-talk mechanism. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

28 CFR 812.4 - Collection procedures.

Code of Federal Regulations, 2011 CFR

2011-07-01

... COLUMBIA COLLECTION AND USE OF DNA INFORMATION § 812.4 Collection procedures. (a) DNA samples will be... misdemeanor pursuant to section 4(a)(5) of the DNA Analysis Backlog Elimination Act of 2000 (42 U.S.C. 14135b...

28 CFR 812.4 - Collection procedures.

Code of Federal Regulations, 2010 CFR

2010-07-01

... COLUMBIA COLLECTION AND USE OF DNA INFORMATION § 812.4 Collection procedures. (a) DNA samples will be... misdemeanor pursuant to section 4(a)(5) of the DNA Analysis Backlog Elimination Act of 2000 (42 U.S.C. 14135b...

28 CFR 812.4 - Collection procedures.

Code of Federal Regulations, 2014 CFR

2014-07-01

... COLUMBIA COLLECTION AND USE OF DNA INFORMATION § 812.4 Collection procedures. (a) DNA samples will be... misdemeanor pursuant to section 4(a)(5) of the DNA Analysis Backlog Elimination Act of 2000 (42 U.S.C. 14135b...

28 CFR 812.4 - Collection procedures.

Code of Federal Regulations, 2012 CFR

2012-07-01

... COLUMBIA COLLECTION AND USE OF DNA INFORMATION § 812.4 Collection procedures. (a) DNA samples will be... misdemeanor pursuant to section 4(a)(5) of the DNA Analysis Backlog Elimination Act of 2000 (42 U.S.C. 14135b...

28 CFR 812.4 - Collection procedures.

Code of Federal Regulations, 2013 CFR

2013-07-01

... COLUMBIA COLLECTION AND USE OF DNA INFORMATION § 812.4 Collection procedures. (a) DNA samples will be... misdemeanor pursuant to section 4(a)(5) of the DNA Analysis Backlog Elimination Act of 2000 (42 U.S.C. 14135b...

Investigating the Molecular Mechanism of TSO1 Function in Arabidopsis cell division and meristem development

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

Zhongchi Liu

2004-10-01

Unlike animals, plants are constantly exposed to environmental mutagens including ultraviolet light and reactive oxygen species. Further, plant cells are totipotent with highly plastic developmental programs. An understanding of molecular mechanisms underlying the ability of plants to monitor and repair its DNA and to eliminate damaged cells are of great importance. Previously we have identified two genes, TSO1 and TSO2, from a flowering plant Arabidopsis thaliana. Mutations in these two genes cause callus-like flowers, fasciated shoot apical meristems, and abnormal cell division, indicating that TSO1 and TSO2 may encode important cell cycle regulators. Previous funding from DOE led to themore » molecular cloning of TSO1, which was shown to encode a novel nuclear protein with two CXC domains suspected to bind DNA. This DOE grant has allowed us to characterize and isolate TSO2 that encodes the small subunit of the ribonucleotide reductase (RNR). RNR comprises two large subunits (R1) an d two small subunits (R2), catalyzes a rate-limiting step in the production of deoxyribonucleotides needed for DNA replication and repair. Previous studies in yeast and mammals indicated that defective RNR often led to cell cycle arrest, growth retardation and p53-dependent apoptosis while abnormally elevated RNR activities led to higher mutation rates. Subsequently, we identified two additional R2 genes, R2A and R2B in the Arabidopsis genome. Using reverse genetics, mutations in R2A and R2B were isolated, and double and triple mutants among the three R2 genes (TSO2, R2A and R2B) were constructed and analyzed. We showed that Arabidopsis tso2 mutants, with reduced dNTP levels, were more sensitive to UV-C. While r2a or r2b single mutants did not exhibit any phenotypes, tso2 r2b double mutants were embryonic lethal and tso2 r2a double mutants were seedling lethal indicating redundant functions among the three R2 genes. Furthermore, tso2 r2a double mutants exhibited increased DNA dam age, massive programmed cell death, and the release of transcriptional gene silencing. Our data suggests that plants can initiate programmed cell death to eliminate damaged cells despite the absence of p53 in plant genome.« less

Pdsg1 and Pdsg2, Novel Proteins Involved in Developmental Genome Remodelling in Paramecium

PubMed Central

Hoehener, Cristina; Singh, Aditi; Swart, Estienne C.; Nowacki, Mariusz

2014-01-01

The epigenetic influence of maternal cells on the development of their progeny has long been studied in various eukaryotes. Multicellular organisms usually provide their zygotes not only with nutrients but also with functional elements required for proper development, such as coding and non-coding RNAs. These maternally deposited RNAs exhibit a variety of functions, from regulating gene expression to assuring genome integrity. In ciliates, such as Paramecium these RNAs participate in the programming of large-scale genome reorganization during development, distinguishing germline-limited DNA, which is excised, from somatic-destined DNA. Only a handful of proteins playing roles in this process have been identified so far, including typical RNAi-derived factors such as Dicer-like and Piwi proteins. Here we report and characterize two novel proteins, Pdsg1 and Pdsg2 (Paramecium protein involved in Development of the Somatic Genome 1 and 2), involved in Paramecium genome reorganization. We show that these proteins are necessary for the excision of germline-limited DNA during development and the survival of sexual progeny. Knockdown of PDSG1 and PDSG2 genes affects the populations of small RNAs known to be involved in the programming of DNA elimination (scanRNAs and iesRNAs) and chromatin modification patterns during development. Our results suggest an association between RNA-mediated trans-generational epigenetic signal and chromatin modifications in the process of Paramecium genome reorganization. PMID:25397898

Pdsg1 and Pdsg2, novel proteins involved in developmental genome remodelling in Paramecium.

PubMed

Arambasic, Miroslav; Sandoval, Pamela Y; Hoehener, Cristina; Singh, Aditi; Swart, Estienne C; Nowacki, Mariusz

2014-01-01

The epigenetic influence of maternal cells on the development of their progeny has long been studied in various eukaryotes. Multicellular organisms usually provide their zygotes not only with nutrients but also with functional elements required for proper development, such as coding and non-coding RNAs. These maternally deposited RNAs exhibit a variety of functions, from regulating gene expression to assuring genome integrity. In ciliates, such as Paramecium these RNAs participate in the programming of large-scale genome reorganization during development, distinguishing germline-limited DNA, which is excised, from somatic-destined DNA. Only a handful of proteins playing roles in this process have been identified so far, including typical RNAi-derived factors such as Dicer-like and Piwi proteins. Here we report and characterize two novel proteins, Pdsg1 and Pdsg2 (Paramecium protein involved in Development of the Somatic Genome 1 and 2), involved in Paramecium genome reorganization. We show that these proteins are necessary for the excision of germline-limited DNA during development and the survival of sexual progeny. Knockdown of PDSG1 and PDSG2 genes affects the populations of small RNAs known to be involved in the programming of DNA elimination (scanRNAs and iesRNAs) and chromatin modification patterns during development. Our results suggest an association between RNA-mediated trans-generational epigenetic signal and chromatin modifications in the process of Paramecium genome reorganization.

Bimodal regulation of p21waf1 protein as function of DNA damage levels

PubMed Central

Buscemi, G; Ricci, C; Zannini, L; Fontanella, E; Plevani, P; Delia, D

2014-01-01

Human p21Waf1 protein is well known for being transcriptionally induced by p53 and activating the cell cycle checkpoint arrest in response to DNA breaks. Here we report that p21Waf1 protein undergoes a bimodal regulation, being upregulated in response to low doses of DNA damage but rapidly and transiently degraded in response to high doses of DNA lesions. Responsible for this degradation is the checkpoint kinase Chk1, which phosphorylates p21Waf1 on T145 and S146 residues and induces its proteasome-dependent proteolysis. The initial p21Waf1 degradation is then counteracted by the ATM-Chk2 pathway, which promotes the p53-dependent accumulation of p21Waf1 at any dose of damage. We also found that p21Waf1 ablation favors the activation of an apoptotic program to eliminate otherwise irreparable cells. These findings support a model in which in human cells a balance between ATM-Chk2-p53 and the ATR-Chk1 pathways modulates p21Waf1 protein levels in relation to cytostatic and cytotoxic doses of DNA damage. PMID:25486478

Spatial and temporal plasticity of chromatin during programmed DNA-reorganization in Stylonychia macronuclear development

PubMed Central

Postberg, Jan; Heyse, Katharina; Cremer, Marion; Cremer, Thomas; Lipps, Hans J

2008-01-01

Background: In this study we exploit the unique genome organization of ciliates to characterize the biological function of histone modification patterns and chromatin plasticity for the processing of specific DNA sequences during a nuclear differentiation process. Ciliates are single-cell eukaryotes containing two morphologically and functionally specialized types of nuclei, the somatic macronucleus and the germline micronucleus. In the course of sexual reproduction a new macronucleus develops from a micronuclear derivative. During this process specific DNA sequences are eliminated from the genome, while sequences that will be transcribed in the mature macronucleus are retained. Results: We show by immunofluorescence microscopy, Western analyses and chromatin immunoprecipitation (ChIP) experiments that each nuclear type establishes its specific histone modification signature. Our analyses reveal that the early macronuclear anlage adopts a permissive chromatin state immediately after the fusion of two heterochromatic germline micronuclei. As macronuclear development progresses, repressive histone modifications that specify sequences to be eliminated are introduced de novo. ChIP analyses demonstrate that permissive histone modifications are associated with sequences that will be retained in the new macronucleus. Furthermore, our data support the hypothesis that a PIWI-family protein is involved in a transnuclear cross-talk and in the RNAi-dependent control of developmental chromatin reorganization. Conclusion: Based on these data we present a comprehensive analysis of the spatial and temporal pattern of histone modifications during this nuclear differentiation process. Results obtained in this study may also be relevant for our understanding of chromatin plasticity during metazoan embryogenesis. PMID:19014664

Structure of the germline genome of Tetrahymena thermophila and relationship to the massively rearranged somatic genome

PubMed Central

Hamilton, Eileen P; Kapusta, Aurélie; Huvos, Piroska E; Bidwell, Shelby L; Zafar, Nikhat; Tang, Haibao; Hadjithomas, Michalis; Krishnakumar, Vivek; Badger, Jonathan H; Caler, Elisabet V; Russ, Carsten; Zeng, Qiandong; Fan, Lin; Levin, Joshua Z; Shea, Terrance; Young, Sarah K; Hegarty, Ryan; Daza, Riza; Gujja, Sharvari; Wortman, Jennifer R; Birren, Bruce W; Nusbaum, Chad; Thomas, Jainy; Carey, Clayton M; Pritham, Ellen J; Feschotte, Cédric; Noto, Tomoko; Mochizuki, Kazufumi; Papazyan, Romeo; Taverna, Sean D; Dear, Paul H; Cassidy-Hanley, Donna M; Xiong, Jie; Miao, Wei; Orias, Eduardo; Coyne, Robert S

2016-01-01

The germline genome of the binucleated ciliate Tetrahymena thermophila undergoes programmed chromosome breakage and massive DNA elimination to generate the somatic genome. Here, we present a complete sequence assembly of the germline genome and analyze multiple features of its structure and its relationship to the somatic genome, shedding light on the mechanisms of genome rearrangement as well as the evolutionary history of this remarkable germline/soma differentiation. Our results strengthen the notion that a complex, dynamic, and ongoing interplay between mobile DNA elements and the host genome have shaped Tetrahymena chromosome structure, locally and globally. Non-standard outcomes of rearrangement events, including the generation of short-lived somatic chromosomes and excision of DNA interrupting protein-coding regions, may represent novel forms of developmental gene regulation. We also compare Tetrahymena’s germline/soma differentiation to that of other characterized ciliates, illustrating the wide diversity of adaptations that have occurred within this phylum. DOI: http://dx.doi.org/10.7554/eLife.19090.001 PMID:27892853

Supercoiled plasmid DNA purification by integrating membrane technology with a monolithic chromatography.

PubMed

Nunes, Catherine; Sousa, Angela; Nunes, José C; Morão, António M; Sousa, Fani; Queiroz, João A

2014-06-01

The present study describes the integration of membrane technology with monolithic chromatography to obtain plasmid DNA with high quality. Isolation and clarification of plasmid DNA lysate were first conducted by a microfiltration step, by using a hydrophilic nylon microfiltration membrane, avoiding the need of centrifugation. For the total elimination of the remaining impurities, a suitable purification step is required. Monolithic stationary phases have been successfully applied as an alternative to conventional supports. Thus, the sample recovered from the membrane process was applied into a nongrafted CarbonylDiImidazole disk. Throughout the global procedure, a reduced level of impurities such as proteins and RNA was obtained, and no genomic DNA was detectable in the plasmid DNA sample. The chromatographic process demonstrated an efficient performance on supercoiled plasmid DNA purity and recovery (100 and 84.44%, respectively). Thereby, combining the membrane technology to eliminate some impurities from lysate sample with an efficient chromatographic strategy to purify the supercoiled plasmid DNA arises as a powerful approach for industrial-scale systems aiming at plasmid DNA purification. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Constructing Smart Protocells with Built-In DNA Computational Core to Eliminate Exogenous Challenge.

PubMed

Lyu, Yifan; Wu, Cuichen; Heinke, Charles; Han, Da; Cai, Ren; Teng, I-Ting; Liu, Yuan; Liu, Hui; Zhang, Xiaobing; Liu, Qiaoling; Tan, Weihong

2018-06-06

A DNA reaction network is like a biological algorithm that can respond to "molecular input signals", such as biological molecules, while the artificial cell is like a microrobot whose function is powered by the encapsulated DNA reaction network. In this work, we describe the feasibility of using a DNA reaction network as the computational core of a protocell, which will perform an artificial immune response in a concise way to eliminate a mimicked pathogenic challenge. Such a DNA reaction network (RN)-powered protocell can realize the connection of logical computation and biological recognition due to the natural programmability and biological properties of DNA. Thus, the biological input molecules can be easily involved in the molecular computation and the computation process can be spatially isolated and protected by artificial bilayer membrane. We believe the strategy proposed in the current paper, i.e., using DNA RN to power artificial cells, will lay the groundwork for understanding the basic design principles of DNA algorithm-based nanodevices which will, in turn, inspire the construction of artificial cells, or protocells, that will find a place in future biomedical research.

The chemical stability of abasic RNA compared to abasic DNA

PubMed Central

Küpfer, Pascal A.; Leumann, Christian J.

2007-01-01

We describe the synthesis of an abasic RNA phosphoramidite carrying a photocleavable 1-(2-nitrophenyl)ethyl (NPE) group at the anomeric center and a triisopropylsilyloxymethyl (TOM) group as 2′-O-protecting group together with the analogous DNA and the 2′-OMe RNA abasic building blocks. These units were incorporated into RNA-, 2′-OMe-RNA- and DNA for the purpose of studying their chemical stabilities towards backbone cleavage in a comparative way. Stability measurements were performed under basic conditions (0.1 M NaOH) and in the presence of aniline (pH 4.6) at 37°C. The kinetics and mechanisms of strand cleavage were followed by High pressure liquid chromotography and ESI-MS. Under basic conditions, strand cleavage at abasic RNA sites can occur via β,δ-elimination and 2′,3′-cyclophosphate formation. We found that β,δ-elimination was 154-fold slower compared to the same mechanism in abasic DNA. Overall strand cleavage of abasic RNA (including cyclophosphate formation) was still 16.8 times slower compared to abasic DNA. In the presence of aniline at pH 4.6, where only β,δ-elimination contributes to strand cleavage, a 15-fold reduced cleavage rate at the RNA abasic site was observed. Thus abasic RNA is significantly more stable than abasic DNA. The higher stability of abasic RNA is discussed in the context of its potential biological role. PMID:17151071

Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR–Cas system

PubMed Central

Elmore, Joshua R.; Sheppard, Nolan F.; Ramia, Nancy; Deighan, Trace; Li, Hong; Terns, Rebecca M.; Terns, Michael P.

2016-01-01

CRISPR–Cas systems eliminate nucleic acid invaders in bacteria and archaea. The effector complex of the Type III-B Cmr system cleaves invader RNAs recognized by the CRISPR RNA (crRNA ) of the complex. Here we show that invader RNAs also activate the Cmr complex to cleave DNA. As has been observed for other Type III systems, Cmr eliminates plasmid invaders in Pyrococcus furiosus by a mechanism that depends on transcription of the crRNA target sequence within the plasmid. Notably, we found that the target RNA per se induces DNA cleavage by the Cmr complex in vitro. DNA cleavage activity does not depend on cleavage of the target RNA but notably does require the presence of a short sequence adjacent to the target sequence within the activating target RNA (rPAM [RNA protospacer-adjacent motif]). The activated complex does not require a target sequence (or a PAM) in the DNA substrate. Plasmid elimination by the P. furiosus Cmr system also does not require the Csx1 (CRISPR-associated Rossman fold [CARF] superfamily) protein. Plasmid silencing depends on the HD nuclease and Palm domains of the Cmr2 (Cas10 superfamily) protein. The results establish the Cmr complex as a novel DNA nuclease activated by invader RNAs containing a crRNA target sequence and a rPAM. PMID:26848045

Repair of O6-alkylguanines in the nuclear DNA of human lymphocytes and leukaemic cells: analysis at the single-cell level.

PubMed Central

Thomale, J.; Seiler, F.; Müller, M. R.; Seeber, S.; Rajewsky, M. F.

1994-01-01

Inter-individual and cell-cell variability of repair of O6-alkylguanines (O6-AlkGua) in nuclear DNA was studied at the single-cell level in peripheral lymphocytes from healthy donors and in leukaemic cells isolated from patients with chronic lymphatic leukaemia (CLL) or acute myeloid leukaemia (AML). Cells were pulse exposed to N-ethyl- or N-(n-)butyl-N-nitrosourea in vitro, and O6-AlkGua residues in DNA were quantified using an anti-(O6-AlkGua) monoclonal antibody and electronically intensified fluorescence. The kinetics of O6-AlkGua elimination revealed considerable inter-individual differences in O6-ethylguanine (O6-EtGua) half-life (t1/2) values in DNA, ranging from 1.5 to 4.5 h (five AML patients), from 0.8 to 2.8 h (five CLL patients) and from 1.2 to 7.3 h (five healthy donors). The elimination from DNA of equimolar amounts of O6-butylguanine was generally 3-5 times slower in comparison with O6-EtGua. The t1/2 values of individual samples varied in parallel for both DNA alkylation products. Upon preincubation with O6-benzylguanine, the activity of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AT) in both lymphocytes and leukaemic blasts was reduced to < or = 1%. However, while the rate of O6-EtGua elimination from DNA was decelerated it was not abolished, suggesting the possible involvement of additional repair systems that might be co-regulated with AT. Within individual samples, no major cell subpopulations were observed whose repair kinetics would differ significantly from the remaining cells. Images Figure 1 PMID:8142257

A comprehensive assessment of lymphatic filariasis in Sri Lanka six years after cessation of mass drug administration.

PubMed

Rao, Ramakrishna U; Nagodavithana, Kumara C; Samarasekera, Sandhya D; Wijegunawardana, Asha D; Premakumara, Welmillage D Y; Perera, Samudrika N; Settinayake, Sunil; Miller, J Phillip; Weil, Gary J

2014-01-01

The Sri Lankan Anti-Filariasis Campaign conducted 5 rounds of mass drug administration (MDA) with diethycarbamazine plus albendazole between 2002 and 2006. We now report results of a comprehensive surveillance program that assessed the lymphatic filariasis (LF) situation in Sri Lanka 6 years after cessation of MDA. Transmission assessment surveys (TAS) were performed per WHO guidelines in primary school children in 11 evaluation units (EUs) in all 8 formerly endemic districts. All EUs easily satisfied WHO criteria for stopping MDA. Comprehensive surveillance was performed in 19 Public Health Inspector (PHI) areas (subdistrict health administrative units). The surveillance package included cross-sectional community surveys for microfilaremia (Mf) and circulating filarial antigenemia (CFA), school surveys for CFA and anti-filarial antibodies, and collection of Culex mosquitoes with gravid traps for detection of filarial DNA (molecular xenomonitoring, MX). Provisional target rates for interruption of LF transmission were community CFA <2%, antibody in school children <2%, and filarial DNA in mosquitoes <0.25%. Community Mf and CFA prevalence rates ranged from 0-0.9% and 0-3.4%, respectively. Infection rates were significantly higher in males and lower in people who denied prior treatment. Antibody rates in school children exceeded 2% in 10 study sites; the area that had the highest community and school CFA rates also had the highest school antibody rate (6.9%). Filarial DNA rates in mosquitoes exceeded 0.25% in 10 PHI areas. Comprehensive surveillance is feasible for some national filariasis elimination programs. Low-level persistence of LF was present in all study sites; several sites failed to meet provisional endpoint criteria for LF elimination, and follow-up testing will be needed in these areas. TAS was not sensitive for detecting low-level persistence of filariasis in Sri Lanka. We recommend use of antibody and MX testing as tools to complement TAS for post-MDA surveillance.

The DNA of ciliated protozoa.

PubMed Central

Prescott, D M

1994-01-01

Ciliates contain two types of nuclei: a micronucleus and a macronucleus. The micronucleus serves as the germ line nucleus but does not express its genes. The macronucleus provides the nuclear RNA for vegetative growth. Mating cells exchange haploid micronuclei, and a new macronucleus develops from a new diploid micronucleus. The old macronucleus is destroyed. This conversion consists of amplification, elimination, fragmentation, and splicing of DNA sequences on a massive scale. Fragmentation produces subchromosomal molecules in Tetrahymena and Paramecium cells and much smaller, gene-sized molecules in hypotrichous ciliates to which telomere sequences are added. These molecules are then amplified, some to higher copy numbers than others. rDNA is differentially amplified to thousands of copies per macronucleus. Eliminated sequences include transposonlike elements and sequences called internal eliminated sequences that interrupt gene coding regions in the micronuclear genome. Some, perhaps all, of these are excised as circular molecules and destroyed. In at least some hypotrichs, segments of some micronuclear genes are scrambled in a nonfunctional order and are recorded during macronuclear development. Vegetatively growing ciliates appear to possess a mechanism for adjusting copy numbers of individual genes, which corrects gene imbalances resulting from random distribution of DNA molecules during amitosis of the macronucleus. Other distinctive features of ciliate DNA include an altered use of the conventional stop codons. Images PMID:8078435

Flow cytometry for enrichment and titration in massively parallel DNA sequencing

PubMed Central

Sandberg, Julia; Ståhl, Patrik L.; Ahmadian, Afshin; Bjursell, Magnus K.; Lundeberg, Joakim

2009-01-01

Massively parallel DNA sequencing is revolutionizing genomics research throughout the life sciences. However, the reagent costs and labor requirements in current sequencing protocols are still substantial, although improvements are continuously being made. Here, we demonstrate an effective alternative to existing sample titration protocols for the Roche/454 system using Fluorescence Activated Cell Sorting (FACS) technology to determine the optimal DNA-to-bead ratio prior to large-scale sequencing. Our method, which eliminates the need for the costly pilot sequencing of samples during titration is capable of rapidly providing accurate DNA-to-bead ratios that are not biased by the quantification and sedimentation steps included in current protocols. Moreover, we demonstrate that FACS sorting can be readily used to highly enrich fractions of beads carrying template DNA, with near total elimination of empty beads and no downstream sacrifice of DNA sequencing quality. Automated enrichment by FACS is a simple approach to obtain pure samples for bead-based sequencing systems, and offers an efficient, low-cost alternative to current enrichment protocols. PMID:19304748

Micronuclear DNA of Oxytricha nova contains sequences with autonomously replicating activity in Saccharomyces cerevisiae.

PubMed Central

Colombo, M M; Swanton, M T; Donini, P; Prescott, D M

1984-01-01

Oxytricha nova is a hypotrichous ciliate with micronuclei and macronuclei. Micronuclei, which contain large, chromosomal-sized DNA, are genetically inert but undergo meiosis and exchange during cell mating. Macronuclei, which contain only small, gene-sized DNA molecules, provide all of the nuclear RNA needed to run the cell. After cell mating the macronucleus is derived from a micronucleus, a derivation that includes excision of the genes from chromosomes and elimination of the remaining DNA. The eliminated DNA includes all of the repetitious sequences and approximately 95% of the unique sequences. We cloned large restriction fragments from the micronucleus that confer replication ability on a replication-deficient plasmid in Saccharomyces cerevisiae. Sequences that confer replication ability are called autonomously replicating sequences. The frequency and effectiveness of autonomously replicating sequences in micronuclear DNA are similar to those reported for DNAs of other organisms introduced into yeast cells. Of the 12 micronuclear fragments with autonomously replicating sequence activity, 9 also showed homology to macronuclear DNA, indicating that they contain a macronuclear gene sequence. We conclude from this that autonomously replicating sequence activity is nonrandomly distributed throughout micronuclear DNA and is preferentially associated with those regions of micronuclear DNA that contain genes. Images PMID:6092934

Mouse embryonic stem cells undergo Charontosis, a novel programmed cell death pathway dependent upon cathepsins, p53, and EndoG, in response to etoposide treatment

PubMed Central

Tichy, Elisia D.; Stephan, Zachary A.; Osterburg, Andrew; Noel, Greg; Stambrook, Peter J.

2013-01-01

Embryonic stem cells (ESCs) are hypersensitive to many DNA damaging agents and can rapidly undergo cell death or cell differentiation following exposure. Treatment of mouse ESCs (mESCs) with etoposide (ETO), a topoisomerase II poison, followed by a recovery period resulted in massive cell death with characteristics of a programmed cell death pathway (PCD). While cell death was both caspase- and necroptosis-independent, it was partially dependent on the activity of lysosomal proteases. A role for autophagy in the cell death process was eliminated, suggesting that ETO induces a novel PCD pathway in mESCs. Inhibition of p53 either as a transcription factor by pifithrin α or in its mitochondrial role by pifithrin μ significantly reduced ESC death levels. Finally, EndoG was newly identified as a protease participating in the DNA fragmentation observed during ETO-induced PCD. We coined the term Charontosis after Charon, the ferryman of the dead in Greek mythology, to refer to the PCD signaling events induced by ETO in mESCs. PMID:23500643

Toward Elimination of Hepatitis B Virus Using Novel Drugs, Approaches, and Combined Modalities.

PubMed

Boucle, Sebastien; Bassit, Leda; Ehteshami, Maryam; Schinazi, Raymond F

2016-11-01

Hepatitis B virus (HBV) causes significant morbidity and mortality worldwide. The majority of chronically infected individuals do not achieve a functional and complete cure. Treated persons who achieve a long-term sustained virologic response (undetectable HBV DNA), are still at high risk of developing morbidity and mortality from liver complications. This review focuses on novel, mechanistically diverse anti-HBV therapeutic strategies currently in development or in clinical evaluation, and highlights new combination strategies that may contribute to full elimination of HBV DNA and covalently closed circular DNA from the infected liver, leading to a complete cure of chronic hepatitis B. Copyright © 2016 Elsevier Inc. All rights reserved.

Degradation and half-life of DNA present in biomass from a genetically-modified organism during land application.

PubMed

Halter, Mathew C; Zahn, James A

2017-02-01

White biotechnology has made a positive impact on the chemical industry by providing safer, more efficient chemical manufacturing processes that have reduced the use of toxic chemicals, harsh reaction conditions, and expensive metal catalysts, which has improved alignment with the principles of Green Chemistry. The genetically-modified (GM) biocatalysts that are utilized in these processes are typically separated from high-value products and then recycled, or eliminated. Elimination routes include disposal in sanitary landfills, incineration, use as a fuel, animal feed, or reuse as an agricultural soil amendment or other value-added products. Elimination routes that have the potential to impact the food chain or environment have been more heavily scrutinized for the fate and persistence of biological products. In this study, we developed and optimized a method for monitoring the degradation of strain-specific DNA markers from a genetically-modified organism (GMO) used for the commercial production of 1,3-propanediol. Laboratory and field tests showed that a marker for heterologous DNA in the GM organism was no longer detectable by end-point polymerase chain reaction (PCR) after 14 days. The half-life of heterologous DNA was increased by 17% (from 42.4 to 49.7 h) after sterilization of the soil from a field plot, which indicated that abiotic factors were important in degradation of DNA under field conditions. There was no evidence for horizontal transfer of DNA target sequences from the GMO to viable organisms present in the soil.

Spliced DNA Sequences in the Paramecium Germline: Their Properties and Evolutionary Potential

PubMed Central

Catania, Francesco; McGrath, Casey L.; Doak, Thomas G.; Lynch, Michael

2013-01-01

Despite playing a crucial role in germline-soma differentiation, the evolutionary significance of developmentally regulated genome rearrangements (DRGRs) has received scant attention. An example of DRGR is DNA splicing, a process that removes segments of DNA interrupting genic and/or intergenic sequences. Perhaps, best known for shaping immune-system genes in vertebrates, DNA splicing plays a central role in the life of ciliated protozoa, where thousands of germline DNA segments are eliminated after sexual reproduction to regenerate a functional somatic genome. Here, we identify and chronicle the properties of 5,286 sequences that putatively undergo DNA splicing (i.e., internal eliminated sequences [IESs]) across the genomes of three closely related species of the ciliate Paramecium (P. tetraurelia, P. biaurelia, and P. sexaurelia). The study reveals that these putative IESs share several physical characteristics. Although our results are consistent with excision events being largely conserved between species, episodes of differential IES retention/excision occur, may have a recent origin, and frequently involve coding regions. Our findings indicate interconversion between somatic—often coding—DNA sequences and noncoding IESs, and provide insights into the role of DNA splicing in creating potentially functional genetic innovation. PMID:23737328

Tip60 degradation by adenovirus relieves transcriptional repression of viral transcriptional activator EIA.

PubMed

Gupta, A; Jha, S; Engel, D A; Ornelles, D A; Dutta, A

2013-10-17

Adenoviruses are linear double-stranded DNA viruses that infect human and rodent cell lines, occasionally transform them and cause tumors in animal models. The host cell challenges the virus in multifaceted ways to restrain viral gene expression and DNA replication, and sometimes even eliminates the infected cells by programmed cell death. To combat these challenges, adenoviruses abrogate the cellular DNA damage response pathway. Tip60 is a lysine acetyltransferase that acetylates histones and other proteins to regulate gene expression, DNA damage response, apoptosis and cell cycle regulation. Tip60 is a bona fide tumor suppressor as mice that are haploid for Tip60 are predisposed to tumors. We have discovered that Tip60 is degraded by adenovirus oncoproteins EIB55K and E4orf6 by a proteasome-mediated pathway. Tip60 binds to the immediate early adenovirus promoter and suppresses adenovirus EIA gene expression, which is a master regulator of adenovirus transcription, at least partly through retention of the virally encoded repressor pVII on this promoter. Thus, degradation of Tip60 by the adenoviral early proteins is important for efficient viral early gene transcription and for changes in expression of cellular genes.

Developmentally programmed DNA splicing in Paramecium reveals short-distance crosstalk between DNA cleavage sites

PubMed Central

Gratias, Ariane; Lepère, Gersende; Garnier, Olivier; Rosa, Sarah; Duharcourt, Sandra; Malinsky, Sophie; Meyer, Eric; Bétermier, Mireille

2008-01-01

Somatic genome assembly in the ciliate Paramecium involves the precise excision of thousands of short internal eliminated sequences (IESs) that are scattered throughout the germline genome and often interrupt open reading frames. Excision is initiated by double-strand breaks centered on the TA dinucleotides that are conserved at each IES boundary, but the factors that drive cleavage site recognition remain unknown. A degenerate consensus was identified previously at IES ends and genetic analyses confirmed the participation of their nucleotide sequence in efficient excision. Even for wild-type IESs, however, variant excision patterns (excised or nonexcised) may be inherited maternally through sexual events, in a homology-dependent manner. We show here that this maternal epigenetic control interferes with the targeting of DNA breaks at IES ends. Furthermore, we demonstrate that a mutation in the TA at one end of an IES impairs DNA cleavage not only at the mutant end but also at the wild-type end. We conclude that crosstalk between both ends takes place prior to their cleavage and propose that the ability of an IES to adopt an excision-prone conformation depends on the combination of its nucleotide sequence and of additional determinants. PMID:18420657

Analysis of sequence variability in the macronuclear DNA of Paramecium tetraurelia: A somatic view of the germline

PubMed Central

Duret, Laurent; Cohen, Jean; Jubin, Claire; Dessen, Philippe; Goût, Jean-François; Mousset, Sylvain; Aury, Jean-Marc; Jaillon, Olivier; Noël, Benjamin; Arnaiz, Olivier; Bétermier, Mireille; Wincker, Patrick; Meyer, Eric; Sperling, Linda

2008-01-01

Ciliates are the only unicellular eukaryotes known to separate germinal and somatic functions. Diploid but silent micronuclei transmit the genetic information to the next sexual generation. Polyploid macronuclei express the genetic information from a streamlined version of the genome but are replaced at each sexual generation. The macronuclear genome of Paramecium tetraurelia was recently sequenced by a shotgun approach, providing access to the gene repertoire. The 72-Mb assembly represents a consensus sequence for the somatic DNA, which is produced after sexual events by reproducible rearrangements of the zygotic genome involving elimination of repeated sequences, precise excision of unique-copy internal eliminated sequences (IES), and amplification of the cellular genes to high copy number. We report use of the shotgun sequencing data (>106 reads representing 13× coverage of a completely homozygous clone) to evaluate variability in the somatic DNA produced by these developmental genome rearrangements. Although DNA amplification appears uniform, both of the DNA elimination processes produce sequence heterogeneity. The variability that arises from IES excision allowed identification of hundreds of putative new IESs, compared to 42 that were previously known, and revealed cases of erroneous excision of segments of coding sequences. We demonstrate that IESs in coding regions are under selective pressure to introduce premature termination of translation in case of excision failure. PMID:18256234

Maternal program of apoptosis activated shortly after midblastula transition by overexpression of S-adenosylmethionine decarboxylase in Xenopus early embryos.

PubMed

Shiokawa, K; Kai, M; Higo, T; Kaito, C; Yokoska, J; Yasuhiko, Y; Kajita, E; Nagano, M; Yamada, Y; Shibata, M; Muto, T; Shinga, J; Hara, H; Takayama, E; Fukamachi, H; Yaoita, Y; Igarashi, K

2000-06-01

When we studied polyamine metabolism in Xenopus embryos, we cloned the cDNA for Xenopus S-adenosylmethionine decarboxylase (SAMDC), which converts SAM (S-adenosylmethionine), the methyl donor, into decarboxylated SAM (dcSAM), the aminopropyl donor, and microinjected its in vitro transcribed mRNA into Xenopus fertilized eggs. We found here that the mRNA injection induces a SAM deficient state in early embryos due to over-function of the overexpressed SAMDC, which in turn induces inhibition of protein synthesis. Such embryos developed quite normally until blastula stage, but stopped development at the early gastrula stage, due to induction of massive cell dissociation and cell autolysis, irrespective of the dosage and stage of the mRNA injection. We found that the dissociated cells were TUNEL-positive, contained fragmented nuclei with ladder-forming DNA, and furthermore, rescued completely by coinjection of Bcl-2 mRNA. Thus, overexpression of SAMDC in Xenopus embryos appeared to switch on apoptotic program, probably via inhibition of protein synthesis. Here, we briefly review our results together with those reported from other laboratories. After discussing the general importance of this newly discovered apoptotic program, we propose that the maternal program of apoptosis serves as a surveillance mechanism to eliminate metabolically severely-damaged cells and functions as a 'fail-safe' mechanism for normal development in Xenopus embryos.

Inactivation of Pol θ and C-NHEJ eliminates off-target integration of exogenous DNA.

PubMed

Zelensky, Alex N; Schimmel, Joost; Kool, Hanneke; Kanaar, Roland; Tijsterman, Marcel

2017-07-07

Off-target or random integration of exogenous DNA hampers precise genomic engineering and presents a safety risk in clinical gene therapy strategies. Genetic definition of random integration has been lacking for decades. Here, we show that the A-family DNA polymerase θ (Pol θ) promotes random integration, while canonical non-homologous DNA end joining plays a secondary role; cells double deficient for polymerase θ and canonical non-homologous DNA end joining are devoid of any integration events, demonstrating that these two mechanisms define random integration. In contrast, homologous recombination is not reduced in these cells and gene targeting is improved to 100% efficiency. Such complete reversal of integration outcome, from predominately random integration to exclusively gene targeting, provides a rational way forward to improve the efficacy and safety of DNA delivery and gene correction approaches.Random off-target integration events can impair precise gene targeting and poses a safety risk for gene therapy. Here the authors show that repression of polymerase θ and classical non-homologous recombination eliminates random integration.

A magic bullet to specifically eliminate mutated mitochondrial genomes from patients' cells

PubMed Central

Moraes, Carlos T

2014-01-01

When mitochondrial diseases result from mutations found in the mitochondrial DNA, engineered mitochondrial-targeted nucleases such as mitochondrial-targeted zinc finger nucleases are shown to specifically eliminate the mutated molecules, leaving the wild-type mitochondrial DNA intact to replicate and restore normal copy number. In this issue, Gammage and colleagues successfully apply this improved technology on patients' cells with two types of genetic alterations responsible for neuropathy ataxia and retinitis pigmentosa (NARP) syndrome and Kearns Sayre syndrome and progressive external ophthalmoplegia (PEO). PMID:24623377

Product differentiation during continuous-flow thermal gradient PCR.

PubMed

Crews, Niel; Wittwer, Carl; Palais, Robert; Gale, Bruce

2008-06-01

A continuous-flow PCR microfluidic device was developed in which the target DNA product can be detected and identified during its amplification. This in situ characterization potentially eliminates the requirement for further post-PCR analysis. Multiple small targets have been amplified from human genomic DNA, having sizes of 108, 122, and 134 bp. With a DNA dye in the PCR mixture, the amplification and unique melting behavior of each sample is observed from a single fluorescent image. The melting behavior of the amplifying DNA, which depends on its molecular composition, occurs spatially in the thermal gradient PCR device, and can be observed with an optical resolution of 0.1 degrees C pixel(-1). Since many PCR cycles are within the field of view of the CCD camera, melting analysis can be performed at any cycle that contains a significant quantity of amplicon, thereby eliminating the cycle-selection challenges typically associated with continuous-flow PCR microfluidics.

General Overview of the ODC Elimination Effort of the RSRM Program

NASA Technical Reports Server (NTRS)

Evans, Kurt; Golde, Rick; McCool, Alex (Technical Monitor)

2001-01-01

The purpose of the ODC Elimination Program of the Space Shuttle RSRM Program is to eliminate the usage of 1, 1, 1 trichloroethane (TCA) in all RSRM (Reusable Solid Rocket Motor) manufacturing processes. This program consists of the following phases and objectives: Phase 0 - Convert to greaseless shipping of metal components. Phase 1 - Eliminate TCA vapor degreasing and usage in propellant cleaning operations. Phase 2 - Eliminate TCA usage for hand cleaning operations. Each phase reduces peak TCA consumption (about 1.4 million pounds in 1989) by about 29, 61, and 10 percent, respectively. Phase 0 was completed in 1992, Phase 1 in 1997, and Phase 2 is in progress (about 75% complete). TCA replacement objectives are accomplished by are a series of subscale, full-scale, and static testing outlined by the NASA-funded, ODC Elimination Program.

Billions of basepairs of recently expanded, repetitive sequences are eliminated from the somatic genome during copepod development.

PubMed

Sun, Cheng; Wyngaard, Grace; Walton, D Brian; Wichman, Holly A; Mueller, Rachel Lockridge

2014-03-11

Chromatin diminution is the programmed deletion of DNA from presomatic cell or nuclear lineages during development, producing single organisms that contain two different nuclear genomes. Phylogenetically diverse taxa undergo chromatin diminution--some ciliates, nematodes, copepods, and vertebrates. In cyclopoid copepods, chromatin diminution occurs in taxa with massively expanded germline genomes; depending on species, germline genome sizes range from 15 - 75 Gb, 12-74 Gb of which are lost from pre-somatic cell lineages at germline--soma differentiation. This is more than an order of magnitude more sequence than is lost from other taxa. To date, the sequences excised from copepods have not been analyzed using large-scale genomic datasets, and the processes underlying germline genomic gigantism in this clade, as well as the functional significance of chromatin diminution, have remained unknown. Here, we used high-throughput genomic sequencing and qPCR to characterize the germline and somatic genomes of Mesocyclops edax, a freshwater cyclopoid copepod with a germline genome of ~15 Gb and a somatic genome of ~3 Gb. We show that most of the excised DNA consists of repetitive sequences that are either 1) verifiable transposable elements (TEs), or 2) non-simple repeats of likely TE origin. Repeat elements in both genomes are skewed towards younger (i.e. less divergent) elements. Excised DNA is a non-random sample of the germline repeat element landscape; younger elements, and high frequency DNA transposons and LINEs, are disproportionately eliminated from the somatic genome. Our results suggest that germline genome expansion in M. edax reflects explosive repeat element proliferation, and that billions of base pairs of such repeats are deleted from the somatic genome every generation. Thus, we hypothesize that chromatin diminution is a mechanism that controls repeat element load, and that this load can evolve to be divergent between tissue types within single organisms.

Billions of basepairs of recently expanded, repetitive sequences are eliminated from the somatic genome during copepod development

PubMed Central

2014-01-01

Background Chromatin diminution is the programmed deletion of DNA from presomatic cell or nuclear lineages during development, producing single organisms that contain two different nuclear genomes. Phylogenetically diverse taxa undergo chromatin diminution — some ciliates, nematodes, copepods, and vertebrates. In cyclopoid copepods, chromatin diminution occurs in taxa with massively expanded germline genomes; depending on species, germline genome sizes range from 15 – 75 Gb, 12–74 Gb of which are lost from pre-somatic cell lineages at germline – soma differentiation. This is more than an order of magnitude more sequence than is lost from other taxa. To date, the sequences excised from copepods have not been analyzed using large-scale genomic datasets, and the processes underlying germline genomic gigantism in this clade, as well as the functional significance of chromatin diminution, have remained unknown. Results Here, we used high-throughput genomic sequencing and qPCR to characterize the germline and somatic genomes of Mesocyclops edax, a freshwater cyclopoid copepod with a germline genome of ~15 Gb and a somatic genome of ~3 Gb. We show that most of the excised DNA consists of repetitive sequences that are either 1) verifiable transposable elements (TEs), or 2) non-simple repeats of likely TE origin. Repeat elements in both genomes are skewed towards younger (i.e. less divergent) elements. Excised DNA is a non-random sample of the germline repeat element landscape; younger elements, and high frequency DNA transposons and LINEs, are disproportionately eliminated from the somatic genome. Conclusions Our results suggest that germline genome expansion in M. edax reflects explosive repeat element proliferation, and that billions of base pairs of such repeats are deleted from the somatic genome every generation. Thus, we hypothesize that chromatin diminution is a mechanism that controls repeat element load, and that this load can evolve to be divergent between tissue types within single organisms. PMID:24618421

MitoTALEN: A General Approach to Reduce Mutant mtDNA Loads and Restore Oxidative Phosphorylation Function in Mitochondrial Diseases

PubMed Central

Hashimoto, Masami; Bacman, Sandra R; Peralta, Susana; Falk, Marni J; Chomyn, Anne; Chan, David C; Williams, Sion L; Moraes, Carlos T

2015-01-01

We have designed mitochondrially targeted transcription activator-like effector nucleases or mitoTALENs to cleave specific sequences in the mitochondrial DNA (mtDNA) with the goal of eliminating mtDNA carrying pathogenic point mutations. To test the generality of the approach, we designed mitoTALENs to target two relatively common pathogenic mtDNA point mutations associated with mitochondrial diseases: the m.8344A>G tRNALys gene mutation associated with myoclonic epilepsy with ragged red fibers (MERRF) and the m.13513G>A ND5 mutation associated with MELAS/Leigh syndrome. Transmitochondrial cybrid cells harbouring the respective heteroplasmic mtDNA mutations were transfected with the respective mitoTALEN and analyzed after different time periods. MitoTALENs efficiently reduced the levels of the targeted pathogenic mtDNAs in the respective cell lines. Functional assays showed that cells with heteroplasmic mutant mtDNA were able to recover respiratory capacity and oxidative phosphorylation enzymes activity after transfection with the mitoTALEN. To improve the design in the context of the low complexity of mtDNA, we designed shorter versions of the mitoTALEN specific for the MERRF m.8344A>G mutation. These shorter mitoTALENs also eliminated the mutant mtDNA. These reductions in size will improve our ability to package these large sequences into viral vectors, bringing the use of these genetic tools closer to clinical trials. PMID:26159306

[Apoptosis: cellular and clinical aspects].

PubMed

Løvschall, H; Mosekilde, L

1997-04-01

Removal of damaged cells is essential for the maintenance of life in multicellular organisms. The process of self destruction, apoptosis, eliminates surplus or damaged cells as part of the pathophysiological defence system. Apoptosis is essential in structural and functional organogenesis during embryological development. The physiological regulation of tissue kinetics is a product of both cell proliferation and cell death. Internal and external regulatory stimuli regulate the balance between apoptosis and mitosis by genetic interaction. Apoptosis is characterized by condensation of chromatine as a result of DNA degradation, formation of blebs in the plasma and nuclear membranes, condensation of cytoplasma, formation of vesicular apoptotic bodies, and phagocytosis by neighbouring cells without inflammatory response. A number of observations indicate that programmed cell death plays an important role in the regulation of cytofunctional homeostasis and defense against accumulation of damaged cells, eg with DNA alterations. Dysregulation of the apoptotic gene program, eg by mutations, may not only lead to loss or degeneration of tissue, but also to hyperproliferative and tumorigenic disorders. New evidence indicates that apoptosis regulation is important both in aging processes and diseases such as: neuropathies, immunopathies, viral infections, cancer, etc. Pharmacological intervention designed to modulate apoptosis seems to raise new possibilities in the treatment of disease.

Mouse embryonic stem cells undergo charontosis, a novel programmed cell death pathway dependent upon cathepsins, p53, and EndoG, in response to etoposide treatment.

PubMed

Tichy, Elisia D; Stephan, Zachary A; Osterburg, Andrew; Noel, Greg; Stambrook, Peter J

2013-05-01

Embryonic stem cells (ESCs) are hypersensitive to many DNA damaging agents and can rapidly undergo cell death or cell differentiation following exposure. Treatment of mouse ESCs (mESCs) with etoposide (ETO), a topoisomerase II poison, followed by a recovery period resulted in massive cell death with characteristics of a programmed cell death pathway (PCD). While cell death was both caspase- and necroptosis-independent, it was partially dependent on the activity of lysosomal proteases. A role for autophagy in the cell death process was eliminated, suggesting that ETO induces a novel PCD pathway in mESCs. Inhibition of p53 either as a transcription factor by pifithrin α or in its mitochondrial role by pifithrin μ significantly reduced ESC death levels. Finally, EndoG was newly identified as a protease participating in the DNA fragmentation observed during ETO-induced PCD. We coined the term charontosis after Charon, the ferryman of the dead in Greek mythology, to refer to the PCD signaling events induced by ETO in mESCs. Copyright © 2013 Elsevier B.V. All rights reserved.

Point of care diagnosis of multiple schistosome parasites: Species-specific DNA detection in urine by loop-mediated isothermal amplification (LAMP).

PubMed

Lodh, Nilanjan; Mikita, Kei; Bosompem, Kwabena M; Anyan, William K; Quartey, Joseph K; Otchere, Joseph; Shiff, Clive J

2017-09-01

Schistosomes are easily transmitted and high chance of repeat infection, so if control strategies based on targeted mass drug administration (MDA) are to succeed it is essential to have a test that is sensitive, accurate and simple to use. It is known and regularly demonstrated that praziquantel does not always eliminate an infection so in spite of the successes of control programs a residual of the reservoir survives to re-infect snails. The issue of diagnostic sensitivity becomes more critical in the assessment of program effectiveness. While serology, such as antigen capture tests might improve sensitivity, it has been shown that the presence of species-specific DNA fragments will indicate, most effectively, the presence of active parasites. Polymerase chain reaction (PCR) can amplify and detect DNA from urine residue captured on Whatman No. 3 filter paper that is dried after filtration. Previously we have detected S. mansoni and S. haematobium parasite-specific small repeat DNA fragment from filtered urine on filter paper by PCR. In the current study, we assessed the efficacy of detection of 86 urine samples for either or both schistosome parasites by PCR and loop-mediated isothermal amplification (LAMP) that were collected from a low to moderate transmission area in Ghana. Two different DNA extraction methods, standard extraction kit and field usable LAMP-PURE kit were also evaluated by PCR and LAMP amplification. With S. haematobium LAMP amplification for both extractions showed similar sensitivity and specificity when compared with PCR amplification (100%) verified by gel electrophoresis. For S. mansoni sensitivity was highest for LAMP amplification (100%) for standard extraction than PCR and LAMP with LAMP-PURE (99% and 94%). The LAMP-PURE extraction produced false negatives, which require further investigation for this field usable extraction kit. Overall high positive and negative predictive values (90% - 100%) for both species demonstrated a highly robust approach. The LAMP approach is close to point of care use and equally sensitive and specific to detection of species-specific DNA by PCR. LAMP can be an effective means to detect low intensity infection due to its simplicity and minimal DNA extraction requirement. This will enhance the effectiveness of surveillance and MDA control programs of schistosomiasis. Copyright © 2017 Elsevier B.V. All rights reserved.

5 CFR Appendix to Part 720 - Guidelines for the Development of a Federal Recruitment Program To Implement 5 U.S.C. Section...

Code of Federal Regulations, 2012 CFR

2012-01-01

... program designed to eliminate underrepresentation of minority groups in specific Federal job categories... used for a program designed to eliminate such underrepresentation; 2. To make, in consultation with OPM... programs to carry out the anti-discrimination policy in a manner designed to eliminate underrepresentation...

5 CFR Appendix to Part 720 - Guidelines for the Development of a Federal Recruitment Program To Implement 5 U.S.C. Section...

Code of Federal Regulations, 2010 CFR

2010-01-01

... program designed to eliminate underrepresentation of minority groups in specific Federal job categories... used for a program designed to eliminate such underrepresentation; 2. To make, in consultation with OPM... programs to carry out the anti-discrimination policy in a manner designed to eliminate underrepresentation...

5 CFR Appendix to Part 720 - Guidelines for the Development of a Federal Recruitment Program To Implement 5 U.S.C. Section...

Code of Federal Regulations, 2011 CFR

2011-01-01

... program designed to eliminate underrepresentation of minority groups in specific Federal job categories... used for a program designed to eliminate such underrepresentation; 2. To make, in consultation with OPM... programs to carry out the anti-discrimination policy in a manner designed to eliminate underrepresentation...

5 CFR Appendix to Part 720 - Guidelines for the Development of a Federal Recruitment Program To Implement 5 U.S.C. Section...

Code of Federal Regulations, 2013 CFR

2013-01-01

... program designed to eliminate underrepresentation of minority groups in specific Federal job categories... used for a program designed to eliminate such underrepresentation; 2. To make, in consultation with OPM... programs to carry out the anti-discrimination policy in a manner designed to eliminate underrepresentation...

5 CFR Appendix to Part 720 - Guidelines for the Development of a Federal Recruitment Program To Implement 5 U.S.C. Section...

Code of Federal Regulations, 2014 CFR

2014-01-01

... program designed to eliminate underrepresentation of minority groups in specific Federal job categories... used for a program designed to eliminate such underrepresentation; 2. To make, in consultation with OPM... programs to carry out the anti-discrimination policy in a manner designed to eliminate underrepresentation...

High-fidelity target sequencing of individual molecules identified using barcode sequences: de novo detection and absolute quantitation of mutations in plasma cell-free DNA from cancer patients.

PubMed

Kukita, Yoji; Matoba, Ryo; Uchida, Junji; Hamakawa, Takuya; Doki, Yuichiro; Imamura, Fumio; Kato, Kikuya

2015-08-01

Circulating tumour DNA (ctDNA) is an emerging field of cancer research. However, current ctDNA analysis is usually restricted to one or a few mutation sites due to technical limitations. In the case of massively parallel DNA sequencers, the number of false positives caused by a high read error rate is a major problem. In addition, the final sequence reads do not represent the original DNA population due to the global amplification step during the template preparation. We established a high-fidelity target sequencing system of individual molecules identified in plasma cell-free DNA using barcode sequences; this system consists of the following two steps. (i) A novel target sequencing method that adds barcode sequences by adaptor ligation. This method uses linear amplification to eliminate the errors introduced during the early cycles of polymerase chain reaction. (ii) The monitoring and removal of erroneous barcode tags. This process involves the identification of individual molecules that have been sequenced and for which the number of mutations have been absolute quantitated. Using plasma cell-free DNA from patients with gastric or lung cancer, we demonstrated that the system achieved near complete elimination of false positives and enabled de novo detection and absolute quantitation of mutations in plasma cell-free DNA. © The Author 2015. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

The mitochondrial genome in embryo technologies.

PubMed

Hiendleder, S; Wolf, E

2003-08-01

The mammalian mitochondrial genome encodes for 37 genes which are involved in a broad range of cellular functions. The mitochondrial DNA (mtDNA) molecule is commonly assumed to be inherited through oocyte cytoplasm in a clonal manner, and apparently species-specific mechanisms have evolved to eliminate the contribution of sperm mitochondria after natural fertilization. However, recent evidence for paternal mtDNA inheritance in embryos and offspring questions the general validity of this model, particularly in the context of assisted reproduction and embryo biotechnology. In addition to normal mt DNA haplotype variation, oocytes and spermatozoa show remarkable differences in mtDNA content and may be affected by inherited or acquired mtDNA aberrations. All these parameters have been correlated with gamete quality and reproductive success rates. Nuclear transfer (NT) technology provides experimental models for studying interactions between nuclear and mitochondrial genomes. Recent studies demonstrated (i) a significant effect of mtDNA haplotype or other maternal cytoplasmic factors on the efficiency of NT; (ii) phenotypic differences between transmitochondrial clones pointing to functionally relevant nuclear-cytoplasmic interactions; and (iii) neutral or non-neutral selection of mtDNA haplotypes in heteroplasmic conditions. Mitochondria form a dynamic reticulum, enabling complementation of mitochondrial components and possibly mixing of different mtDNA populations in heteroplasmic individuals. Future directions of research on mtDNA in the context of reproductive biotechnology range from the elimination of adverse effects of artificial heteroplasmy, e.g. created by ooplasm transfer, to engineering of optimized constellations of nuclear and cytoplasmic genes for the production of superior livestock.

Effective Program Management: A Cornerstone of Malaria Elimination

PubMed Central

Gosling, Jonathan; Case, Peter; Tulloch, Jim; Chandramohan, Daniel; Wegbreit, Jennifer; Newby, Gretchen; Gueye, Cara Smith; Koita, Kadiatou; Gosling, Roly

2015-01-01

Effective program management is essential for successful elimination of malaria. In this perspective article, evidence surrounding malaria program management is reviewed by management science and malaria experts through a literature search of published and unpublished gray documents and key informant interviews. Program management in a malaria elimination setting differs from that in a malaria control setting in a number of ways, although knowledge and understanding of these distinctions are lacking. Several core features of successful health program management are critical to achieve elimination, including effective leadership and supervision at all levels, sustained political and financial commitment, reliable supply and control of physical resources, effective management of data and information, appropriate incentives, and consistent accountability. Adding to the complexity, the requirements of an elimination program may conflict with those of a control regimen. Thus, an additional challenge is successfully managing program transitions along the continuum from control to elimination to prevention of reintroduction. This article identifies potential solutions to these challenges by exploring managerial approaches that are flexible, relevant, and sustainable in various cultural and health system contexts. PMID:26013372

Biomedical Requirements for High Productivity Computing Systems

DTIC Science & Technology

2005-04-01

server at http://www.ncbi.nlm.nih.gov/BLAST/. There are many variants of BLAST, including: 1. BLASTN - Compares a DNA query to a DNA database. Searches ...database (3 reading frames from each strand of the DNA) searching . 13 4. TBLASTN - Compares a protein query to a DNA database, in the 6 possible...the molecular during this phase. After eliminating molecules that could not match the query , an atom-by-atom search for the molecules in conducted

‘Protected DNA Probes’ capable of strong hybridization without removal of base protecting groups

PubMed Central

Ohkubo, Akihiro; Kasuya, Rintaro; Sakamoto, Kazushi; Miyata, Kenichi; Taguchi, Haruhiko; Nagasawa, Hiroshi; Tsukahara, Toshifumi; Watanobe, Takuma; Maki, Yoshiyuki; Seio, Kohji; Sekine, Mitsuo

2008-01-01

We propose a new strategy called the ‘Protected DNA Probes (PDP) method’ in which appropriately protected bases selectively bind to the complementary bases without the removal of their base protecting groups. Previously, we reported that 4-N-acetylcytosine oligonucleotides (ac4C) exhibited a higher hybridization affinity for ssDNA than the unmodified oligonucleotides. For the PDP strategy, we created a modified adenine base and synthesized an N-acylated deoxyadenosine mimic having 6-N-acetyl-8-aza-7-deazaadenine (ac6az8c7A). It was found that PDP containing ac4C and ac6az8c7A exhibited higher affinity for the complementary ssDNA than the corresponding unmodified DNA probes and showed similar base recognition ability. Moreover, it should be noted that this PDP strategy could guarantee highly efficient synthesis of DNA probes on controlled pore glass (CPG) with high purity and thereby could eliminate the time-consuming procedures for isolating DNA probes. This strategy could also avoid undesired base-mediated elimination of DNA probes from CPG under basic conditions such as concentrated ammonia solution prescribed for removal of base protecting groups in the previous standard approach. Here, several successful applications of this strategy to single nucleotide polymorphism detection are also described in detail using PDPs immobilized on glass plates and those prepared on CPG plates, suggesting its potential usefulness. PMID:18272535

Translations on USSR Science and Technology, Biomedical and Behavioral Sciences, Number 33

DTIC Science & Technology

1978-07-05

7.6, 6.6 mM MgCl2, 6.6 mM 2-ß- mercaptoethanol, 2 y£ BamHI. Restriction was performed at 37°C for 15 min. We used, as marker DNA, EcoRI restrictor...linear DNA as function of molecular weight, plotted according to marker DNA to determine the molecular weights of DNA restrictors. Electron...according to the amp marker ; 3) reaction to agents that eliminate plasmids (acridine orange, ethidium bromide, sodium dodecylsulfate). The obtained

BATTLE: Biomarker-Based Approaches of Targeted Therapy for Lung Cancer Elimination

DTIC Science & Technology

2008-04-01

although a grade 3 neutropenia was dose-limiting in one importance. Th th ubstrate of the CYP3A4 isoenzyme and P-gp. Its metabolism is sensitive to...tratification in clinis Molecular Pathway Biomarkers Type of Analysis EGFR EGFR Mutation ( exons 18 to 21) DNA sequencing EGFR Increased Copy Number...polysomy/am 1plification) DNA FISH K-Ras/B-Raf K-RAS Mutation (codons 12,13, 61) DNA sequencing B-RAF Mutations ( exons 11 and 15) DNA sequencing

Developmentally programmed DNA deletion in Tetrahymena thermophila by a transposition-like reaction pathway.

PubMed Central

Saveliev, S V; Cox, M M

1996-01-01

We provide a molecular description of key intermediates in the deletion of two internal eliminated sequences (IES elements), the M and R regions, during macronuclear development in Tetrahymena thermophila. Using a variety of PCR-based methods in vivo, double-strand breaks are detected that are generated by hydrolytic cleavage and correspond closely to the observed chromosomal junctions left behind in the macronuclei. The breaks exhibit a temporal and structural relationship to the deletion reaction that provides strong evidence that they are intermediates in the deletion pathway. Breaks in the individual strands are staggered by 4 bp, producing a four nucleotide 5' extension. Evidence is presented that breaks do not occur simultaneously at both ends. The results are most consistent with a deletion mechanism featuring initiation by double-strand cleavage at one end of the deleted element, followed by transesterification to generate the macronuclear junction on one DNA strand. An adenosine residue is found at all the nucleophilic 3' ends used in the postulated transesterification step. Evidence for the transesterification step is provided by detection of a 3' hydroxyl that would be liberated by such a step at a deletion boundary where no other DNA strand ends are detected. Images PMID:8654384

Hidden Sylvatic Foci of the Main Vector of Chagas Disease Triatoma infestans: Threats to the Vector Elimination Campaign?

PubMed Central

Schachter-Broide, Judith; Dujardin, Jean-Pierre; Dotson, Ellen M.; Kitron, Uriel; Gürtler, Ricardo E.

2011-01-01

Background Establishing the sources of reinfestation after residual insecticide spraying is crucial for vector elimination programs. Triatoma infestans, traditionally considered to be limited to domestic or peridomestic (abbreviated as D/PD) habitats throughout most of its range, is the target of an elimination program that has achieved limited success in the Gran Chaco region in South America. Methodology/Principal Findings During a two-year period we conducted semi-annual searches for triatomine bugs in every D/PD site and surrounding sylvatic habitats after full-coverage spraying of pyrethroid insecticides of all houses in a well-defined rural area in northwestern Argentina. We found six low-density sylvatic foci with 24 T. infestans in fallen or standing trees located 110–2,300 m from the nearest house or infested D/PD site detected after insecticide spraying, when house infestations were rare. Analysis of two mitochondrial gene fragments of 20 sylvatic specimens confirmed their species identity as T. infestans and showed that their composite haplotypes were the same as or closely related to D/PD haplotypes. Population studies with 10 polymorphic microsatellite loci and wing geometric morphometry consistently indicated the occurrence of unrestricted gene flow between local D/PD and sylvatic populations. Mitochondrial DNA and microsatellite sibship analyses in the most abundant sylvatic colony revealed descendents from five different females. Spatial analysis showed a significant association between two sylvatic foci and the nearest D/PD bug population found before insecticide spraying. Conclusions Our study shows that, despite of its high degree of domesticity, T. infestans has sylvatic colonies with normal chromatic characters (not melanic morphs) highly connected to D/PD conspecifics in the Argentinean Chaco. Sylvatic habitats may provide a transient or permanent refuge after control interventions, and function as sources for D/PD reinfestation. The occurrence of sylvatic foci of T. infestans in the Gran Chaco may pose additional threats to ongoing vector elimination efforts. PMID:22039559

Dryden Flight Research Center Chemical Pharmacy Program

NASA Technical Reports Server (NTRS)

Davis, Bette

1997-01-01

The Dryden Flight Research Center (DFRC) Chemical Pharmacy "Crib" is a chemical sharing system which loans chemicals to users, rather than issuing them or having each individual organization or group purchasing the chemicals. This cooperative system of sharing chemicals eliminates multiple ownership of the same chemicals and also eliminates stockpiles. Chemical management duties are eliminated for each of the participating organizations. The chemical storage issues, hazards and responsibilities are eliminated. The system also ensures safe storage of chemicals and proper disposal practices. The purpose of this program is to reduce the total releases and transfers of toxic chemicals. The initial cost of the program to DFRC was $585,000. A savings of $69,000 per year has been estimated for the Center. This savings includes the reduced costs in purchasing, disposal and chemical inventory/storage responsibilities. DFRC has chemicals stored in 47 buildings and at 289 locations. When the program is fully implemented throughout the Center, there will be three chemical locations at this facility. The benefits of this program are the elimination of chemical management duties; elimination of the hazard associated with chemical storage; elimination of stockpiles; assurance of safe storage; assurance of proper disposal practices; assurance of a safer workplace; and more accurate emissions reports.

Genetic counseling in the era of molecular diagnostics.

PubMed

Traas, Anne M; Casal, Margret; Haskins, Mark; Henthorn, Paula

2006-08-01

Veterinarians with an interest in theriogenology will often be asked by small animal clients for advice concerning hereditary diseases in their breeds. Many new DNA-based tests for analysis of genetic diseases and traits (e.g. coat color) are now available for use by both breeders and veterinarians. With appropriate interpretation, these tests can be invaluable tools in a breeding program. For example, they can be used to produce animals free of specific diseases, to quickly eliminate a disease from an entire breed, or to select for specific traits in breeding stock. Selection strategies that do not take into account maintaining genetic diversity of the breed may be detrimental and reduce the potential for future improvement.

JAK2 and genomic instability in the myeloproliferative neoplasms: a case of the chicken or the egg?

PubMed Central

Scott, Linda M.; Rebel, Vivienne I.

2012-01-01

The myeloproliferative neoplasms (MPNs) are a particularly useful model for studying mutation accumulation in neoplastic and the mechanisms of the molecular cells, understanding underlying defects our current This review summarizes acquisition. present their in patients with an MPN, and the effects of mutations targeting Janus kinase 2 (JAK2)-mediated intracellular signaling on DNA damage, and on the elimination of mutation-bearing cells by programmed cell death. Moreover, we discuss findings that suggest that the acquisition of disease-initiating mutations in hematopoietic stem cells of some MPN patients may be the consequence of an inherent genomic instability that was not previously appreciated. PMID:22641564

Polyphosphate present in DNA preparations from fungal species of Collectotrichum inhibits restriction endonucleases and other enzymes

USGS Publications Warehouse

Rodriguez, R.J.

1993-01-01

During the development of a procedure for the isolation of total genomic DNA from filamentous fungi (Rodriguez, R. J., and Yoder, 0. C., Exp. Mycol. 15, 232-242, 1991) a cell fraction was isolated which inhibited the digestion of DNA by restriction enzymes. After elimination of DNA, RNA, proteins, and lipids, the active compound was purified by gel filtration to yield a single fraction capable of complete inhibition of restriction enzyme activity. The inhibitor did not absorb uv light above 220 nm, and was resistant to alkali and acid at 25°C and to temperatures as high as 100°C. More extensive analyses demonstrated that the inhibitor was also capable of inhibiting T4 DNA ligase and TaqI DNA polymerase, but not DNase or RNase. Chemical analyses indicated that the inhibitor was devoid of carbohydrates, proteins, lipids, and nucleic acids but rich in phosphorus. A combination of nuclear magnetic resonance, metachromatic shift of toluidine blue, and gel filtration indicated that the inhibitor was a polyphosphate (polyP) containing approximately 60 phosphate molecules. The mechanism of inhibition appeared to involve complexing of polyP to the enzymatic proteins. All species of Colletotrichum analyzed produced polyP equivalent in chain length and concentration. A modification to the original DNA extraction procedure is described which eliminates polyP and reduces the time necessary to obtain DNA of sufficient purity for restriction enzyme digestion and TaqI polymerase amplification.

Ultra-low background DNA cloning system.

PubMed

Goto, Kenta; Nagano, Yukio

2013-01-01

Yeast-based in vivo cloning is useful for cloning DNA fragments into plasmid vectors and is based on the ability of yeast to recombine the DNA fragments by homologous recombination. Although this method is efficient, it produces some by-products. We have developed an "ultra-low background DNA cloning system" on the basis of yeast-based in vivo cloning, by almost completely eliminating the generation of by-products and applying the method to commonly used Escherichia coli vectors, particularly those lacking yeast replication origins and carrying an ampicillin resistance gene (Amp(r)). First, we constructed a conversion cassette containing the DNA sequences in the following order: an Amp(r) 5' UTR (untranslated region) and coding region, an autonomous replication sequence and a centromere sequence from yeast, a TRP1 yeast selectable marker, and an Amp(r) 3' UTR. This cassette allowed conversion of the Amp(r)-containing vector into the yeast/E. coli shuttle vector through use of the Amp(r) sequence by homologous recombination. Furthermore, simultaneous transformation of the desired DNA fragment into yeast allowed cloning of this DNA fragment into the same vector. We rescued the plasmid vectors from all yeast transformants, and by-products containing the E. coli replication origin disappeared. Next, the rescued vectors were transformed into E. coli and the by-products containing the yeast replication origin disappeared. Thus, our method used yeast- and E. coli-specific "origins of replication" to eliminate the generation of by-products. Finally, we successfully cloned the DNA fragment into the vector with almost 100% efficiency.

Cytoplasmic transfer of heritable elements other than mtDNA from SAMP1 mice into mouse tumor cells suppresses their ability to form tumors in C57BL6 mice.

PubMed

Shimizu, Akinori; Tani, Haruna; Takibuchi, Gaku; Ishikawa, Kaori; Sakurazawa, Ryota; Inoue, Takafumi; Hashimoto, Tetsuo; Nakada, Kazuto; Takenaga, Keizo; Hayashi, Jun-Ichi

2017-11-04

In a previous study, we generated transmitochondrial P29mtSAMP1 cybrids, which had nuclear DNA from the C57BL6 (referred to as B6) mouse strain-derived P29 tumor cells and mitochondrial DNA (mtDNA) exogenously-transferred from the allogeneic strain SAMP1. Because P29mtSAMP1 cybrids did not form tumors in syngeneic B6 mice, we proposed that allogeneic SAMP1 mtDNA suppressed tumor formation of P29mtSAMP1 cybrids. To test this hypothesis, current study generated P29mt(sp)B6 cybrids carrying all genomes (nuclear DNA and mtDNA) from syngeneic B6 mice by eliminating SAMP1 mtDNA from P29mtSAMP1 cybrids and reintroducing B6 mtDNA. However, the P29mt(sp)B6 cybrids did not form tumors in B6 mice, even though they had no SAMP1 mtDNA, suggesting that SAMP1 mtDNA is not involved in tumor suppression. Then, we examined another possibility of whether SAMP1 mtDNA fragments potentially integrated into the nuclear DNA of P29mtSAMP1 cybrids are responsible for tumor suppression. We generated P29 H (sp)B6 cybrids by eliminating nuclear DNA from P29mt(sp)B6 cybrids and reintroducing nuclear DNA with no integrated SAMP1 mtDNA fragment from mtDNA-less P29 cells resistant to hygromycin in selection medium containing hygromycin. However, the P29 H (sp)B6 cybrids did not form tumors in B6 mice, even though they carried neither SAMP1 mtDNA nor nuclear DNA with integrated SAMP1 mtDNA fragments. Moreover, overproduction of reactive oxygen species (ROS) and bacterial infection were not involved in tumor suppression. These observations suggest that tumor suppression was caused not by mtDNA with polymorphic mutations or infection of cytozoic bacteria but by hypothetical heritable cytoplasmic elements other than mtDNA from SAMP1 mice. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Rapid DNA extraction protocol for detection of alpha-1 antitrypsin deficiency from dried blood spots by real-time PCR.

PubMed

Struniawski, R; Szpechcinski, A; Poplawska, B; Skronski, M; Chorostowska-Wynimko, J

2013-01-01

The dried blood spot (DBS) specimens have been successfully employed for the large-scale diagnostics of α1-antitrypsin (AAT) deficiency as an easy to collect and transport alternative to plasma/serum. In the present study we propose a fast, efficient, and cost effective protocol of DNA extraction from dried blood spot (DBS) samples that provides sufficient quantity and quality of DNA and effectively eliminates any natural PCR inhibitors, allowing for successful AAT genotyping by real-time PCR and direct sequencing. DNA extracted from 84 DBS samples from chronic obstructive pulmonary disease patients was genotyped for AAT deficiency variants by real-time PCR. The results of DBS AAT genotyping were validated by serum IEF phenotyping and AAT concentration measurement. The proposed protocol allowed successful DNA extraction from all analyzed DBS samples. Both quantity and quality of DNA were sufficient for further real-time PCR and, if necessary, for genetic sequence analysis. A 100% concordance between AAT DBS genotypes and serum phenotypes in positive detection of two major deficiency S- and Z- alleles was achieved. Both assays, DBS AAT genotyping by real-time PCR and serum AAT phenotyping by IEF, positively identified PI*S and PI*Z allele in 8 out of the 84 (9.5%) and 16 out of 84 (19.0%) patients, respectively. In conclusion, the proposed protocol noticeably reduces the costs and the hand-on-time of DBS samples preparation providing genomic DNA of sufficient quantity and quality for further real-time PCR or genetic sequence analysis. Consequently, it is ideally suited for large-scale AAT deficiency screening programs and should be method of choice.

Determinants of Malaria Program Expenditures during Elimination: Case Study Evidence from Select Provinces in the Philippines

PubMed Central

Liu, Jenny X.; Newby, Gretchen; Brackery, Aprielle; Smith Gueye, Cara; Candari, Christine J.; Escubil, Luz R.; Vestergaard, Lasse S.; Baquilod, Mario

2013-01-01

...Even though eliminating malaria from the endemic margins is a part of the Global Malaria Action Plan, little guidance exists on what resources are needed to transition from controlling malaria to eliminating it. Using Philippines as an example, this study aimed to (1) estimate the financial resources used by sub-national malaria programs in different phases during elimination and (2) understand how different environmental and organizational factors may influence expenditure levels and spending proportions. The Philippines provides an opportunity to study variations in sub-national programs because its epidemiological and ecological diversity, devolved health system, and progressive elimination strategy all allow greater flexibility for lower-level governments to direct activities, but also create challenges for coordination and resource mobilization. Through key informant interviews and archival record retrieval in four selected provinces chosen based on eco-epidemiological variation, expenditures associated with provincial malaria programs were collected for selected years (mid-1990s to 2010). Results show that expenditures per person at risk per year decrease as programs progress from a state of controlled low-endemic malaria to elimination to prevention of reintroduction regardless of whether elimination was deliberately planned. However, wide variation across provinces were found: expenditures were generally higher if mainly financed with donor grants, but were moderated by the level of economic development, the level of malaria transmission and receptivity, and the capacity of program staff. Across all provinces, strong leadership appears to be a necessary condition for maintaining progress and is vital in controlling outbreaks. While sampled provinces and years may not be representative of other sub-national malaria programs, these findings suggest that the marginal yearly cost declines with each phase during elimination. PMID:24086279

Determinants of malaria program expenditures during elimination: case study evidence from select provinces in the Philippines.

PubMed

Liu, Jenny X; Newby, Gretchen; Brackery, Aprielle; Smith Gueye, Cara; Candari, Christine J; Escubil, Luz R; Vestergaard, Lasse S; Baquilod, Mario

2013-01-01

...Even though eliminating malaria from the endemic margins is a part of the Global Malaria Action Plan, little guidance exists on what resources are needed to transition from controlling malaria to eliminating it. Using Philippines as an example, this study aimed to (1) estimate the financial resources used by sub-national malaria programs in different phases during elimination and (2) understand how different environmental and organizational factors may influence expenditure levels and spending proportions. The Philippines provides an opportunity to study variations in sub-national programs because its epidemiological and ecological diversity, devolved health system, and progressive elimination strategy all allow greater flexibility for lower-level governments to direct activities, but also create challenges for coordination and resource mobilization. Through key informant interviews and archival record retrieval in four selected provinces chosen based on eco-epidemiological variation, expenditures associated with provincial malaria programs were collected for selected years (mid-1990s to 2010). Results show that expenditures per person at risk per year decrease as programs progress from a state of controlled low-endemic malaria to elimination to prevention of reintroduction regardless of whether elimination was deliberately planned. However, wide variation across provinces were found: expenditures were generally higher if mainly financed with donor grants, but were moderated by the level of economic development, the level of malaria transmission and receptivity, and the capacity of program staff. Across all provinces, strong leadership appears to be a necessary condition for maintaining progress and is vital in controlling outbreaks. While sampled provinces and years may not be representative of other sub-national malaria programs, these findings suggest that the marginal yearly cost declines with each phase during elimination.

Hybrid male sterility is caused by mitochondrial DNA deletion.

PubMed

Hayashida, Kenji; Kohno, Shigeru

2009-07-01

Although it is known that the hybrid male mouse is sterile just like any other animal's heterogametic sex, the reason why only the male germ cells are impaired has yet to be discovered. TdT-mediated dUTP nick end labeling assay using a confocal fluorescence microscope and DNA fragmentation assay of hybrid testis indicated destruction of the mitochondrial DNA (mtDNA) rather than the nuclear DNA. Previously we reported that maternal mtDNA inheritance is through selective sperm mtDNA elimination based on the sperm factor and two egg factors, and expression of these three factors was recognized in the hybrid testis. It was thereby assumed that mtDNA destruction caused by the expression of maternal mtDNA inheritance system in male germ cells is implicated in the hybrid male sterility of mice.

Apoptosis: a mechanism contributing to remodeling of skeletal muscle in response to hindlimb unweighting

NASA Technical Reports Server (NTRS)

Allen, D. L.; Linderman, J. K.; Roy, R. R.; Bigbee, A. J.; Grindeland, R. E.; Mukku, V.; Edgerton, V. R.

1997-01-01

The role of apoptosis in the elimination of myonuclei during hindlimb unloading-induced atrophy and the inhibition of apoptosis in the prevention of muscle atrophy were examined. The number of nuclei demonstrating double-stranded DNA fragmentation seen by terminal deoxynucleotidyl transferase (TDT) histochemical staining, an indicator of apoptosis, was significantly increased after 14 days of suspension. Double staining with TDT and antilaminin immunohistochemistry revealed that some TDT-positive nuclei were within the fiber lamina and were most likely myonuclei. The number of fibers containing morphologically abnormal nuclei was also significantly greater in suspended compared with control rats. Combined treatment with growth hormone and insulin-like growth factor I (GH/ IGF-I) and resistance exercise attenuated the increase in TDT-positive nuclei (approximately 26%, P > 0.05) and significantly decreased the number of fibers with morphologically abnormal nuclei. The data suggest that 1) "programmed nuclear death" contributes to the elimination of myonuclei and/or satellite cells from atrophying fibers, and 2) GH/IGF-I administration plus muscle loading ameliorates the apoptosis associated with hindlimb unloading.

Role of DNA repair enzymes in the cellular resistance to oxidative stress.

PubMed

Laval, J

1996-01-01

Oxidative stress occurs in cells when the equilibrium between prooxidant and antioxidant species is broken in favor of the prooxidant state. It is due to reactive oxygen species (ROS) generated either by the cellular metabolism such as phagocytosis, mitochondrial respiration, xenobiotic detoxification, or by exogenous factors such as ionizing radiation or chemical compounds performing red-ox reactions. Some ROS are extremely reactive and interact with all the macromolecules including lipids, nucleic acids and proteins. Cells have numerous defence systems to counteract the deleterious effects of ROS. Proteins and small molecules specifically eliminate ROS when they are formed. There are three species of superoxyde dismutases which transform the superoxyde anion O2- in hydrogen peroxyde H2O2 which in turn will be destroyed by peroxysomal catalase or by various peroxydases. There are numerous small molecules in the cell such as glutathion, alpha-tocopherol, vitamines A and C, melanine, etc. which are antioxydant molecules. ROS escaping destruction generate various lesions in DNA such as base modifications, degradation products of deoxyribose, chain breaks. These various lesions have been characterized and it is possible to quantitate them in the DNA of cells which have been irradiated or treated by free radical generating systems. The biological properties of the bases modified by ROS have been established. For example C8-hydroxyguanine (8-oxoG) is promutagenic since, if present in DNA during replication, it leads to incorporation of dAMP residues, leading to transversion mutation (GC-->TA). Purines whose imidazole ring is opened (Fapy residues) are stops for the DNA polymerase during DNA replication and are therefore potentially lethal lesions for the cell. Oxidized pyrimidines have comparable coding properties. Efficient DNA repair mechanisms remove these oxidized bases. In Escherichia coli cells, endonuclease III (NTH protein) and endonuclease VIII (NEI protein) excise many oxidized pyrimidines, whereas the FPG protein (formamidopyrimidine-DNA-glycosylase) eliminates 8-oxoG and Fapy lesions. Besides its DNA glycosylase activity, the protein FPG has a beta-lyase activity incising DNA at abasic site by a beta-delta elimination mechanism, and a dRPase activity. The FPG protein has a zinc finger motive which is mandatory for the recognition of its substrate. Mammalian cells have similar DNA repair proteins and it should be emphazized that there is conservation of the different functions and in most cases a remarquable homology of the amino acids sequences from E. coli to man.

Eliminating fast reactions in stochastic simulations of biochemical networks: A bistable genetic switch

NASA Astrophysics Data System (ADS)

Morelli, Marco J.; Allen, Rosalind J.; Tǎnase-Nicola, Sorin; ten Wolde, Pieter Rein

2008-01-01

In many stochastic simulations of biochemical reaction networks, it is desirable to "coarse grain" the reaction set, removing fast reactions while retaining the correct system dynamics. Various coarse-graining methods have been proposed, but it remains unclear which methods are reliable and which reactions can safely be eliminated. We address these issues for a model gene regulatory network that is particularly sensitive to dynamical fluctuations: a bistable genetic switch. We remove protein-DNA and/or protein-protein association-dissociation reactions from the reaction set using various coarse-graining strategies. We determine the effects on the steady-state probability distribution function and on the rate of fluctuation-driven switch flipping transitions. We find that protein-protein interactions may be safely eliminated from the reaction set, but protein-DNA interactions may not. We also find that it is important to use the chemical master equation rather than macroscopic rate equations to compute effective propensity functions for the coarse-grained reactions.

The emerging role of nuclear viral DNA sensors.

PubMed

Diner, Benjamin A; Lum, Krystal K; Cristea, Ileana M

2015-10-30

Detecting pathogenic DNA by intracellular receptors termed "sensors" is critical toward galvanizing host immune responses and eliminating microbial infections. Emerging evidence has challenged the dogma that sensing of viral DNA occurs exclusively in sub-cellular compartments normally devoid of cellular DNA. The interferon-inducible protein IFI16 was shown to bind nuclear viral DNA and initiate immune signaling, culminating in antiviral cytokine secretion. Here, we review the newly characterized nucleus-originating immune signaling pathways, their links to other crucial host defenses, and unique mechanisms by which viruses suppress their functions. We frame these findings in the context of human pathologies associated with nuclear replicating DNA viruses. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Meticulous plasma isolation is essential to avoid false low-level viraemia in Roche Cobas HIV-1 viral load assays.

PubMed

Mortier, Virginie; Vancoillie, Leen; Dauwe, Kenny; Staelens, Delfien; Demecheleer, Els; Schauvliege, Marlies; Dinakis, Sylvie; Van Maerken, Tom; Dessilly, Géraldine; Ruelle, Jean; Verhofstede, Chris

2017-10-24

Pre-analytical sample processing is often overlooked as a potential cause of inaccurate assay results. Here we demonstrate how plasma, extracted from standard EDTA-containing blood collection tubes, may contain traces of blood cells consequently resulting in a false low-level HIV-1 viral load when using Roche Cobas HIV-1 assays. The presence of human DNA in Roche Cobas 4800 RNA extracts and in RNA extracts from the Abbott HIV-1 RealTime assay was assessed by quantifying the human albumin gene by means of quantitative PCR. RNA was extracted from plasma samples before and after an additional centrifugation and tested for viral load and DNA contamination. The relation between total DNA content and viral load was defined. Elevated concentrations of genomic DNA were detected in 28 out of 100 Cobas 4800 extracts and were significantly more frequent in samples processed outside of the AIDS Reference Laboratory. An association between genomic DNA presence and spurious low-level viraemia results was demonstrated. Supplementary centrifugation of plasma before RNA extraction eliminated the contamination and the false viraemia. Plasma isolated from standard EDTA-containing blood collection tubes may contain traces of HIV DNA leading to false viral load results above the clinical cutoff. Supplementary centrifugation of plasma before viral load analysis may eliminate the occurrence of this spurious low-level viraemia.

Confinement-induced Molecular Templating and Controlled Ligation

NASA Astrophysics Data System (ADS)

Berard, Daniel; Shayegan, Marjan; Michaud, François; Henkin, Gil; Scott, Shane; Leith, Jason; Leslie, Sabrina; Leslie Lab Team

Loading and manipulating long DNA molecules within sub-50 nm cross-section nanostructures for genomic and biochemical analyses, while retaining their structural integrity, present key technological challenges to the biotechnology sector, such as device clogging and molecular breakage. We overcome these challenges by using Convex Lens-induced Confinement (CLiC) technology to gently load DNA into nanogrooves from above. Here, we demonstrate single-fluorophore visualization of custom DNA barcodes as well as efficient top-loading of DNA into sub-50 nm nanogrooves of variable topographies. We study confinement-enhanced self-ligation of polymers loaded in circular nanogrooves. Further, we use concentric, circular nanogrooves to eliminate confinement gradient-induced drift of stretched DNA.

A universal DNA-based protein detection system.

PubMed

Tran, Thua N N; Cui, Jinhui; Hartman, Mark R; Peng, Songming; Funabashi, Hisakage; Duan, Faping; Yang, Dayong; March, John C; Lis, John T; Cui, Haixin; Luo, Dan

2013-09-25

Protein immune detection requires secondary antibodies which must be carefully selected in order to avoid interspecies cross-reactivity, and is therefore restricted by the limited availability of primary/secondary antibody pairs. Here we present a versatile DNA-based protein detection system using a universal adapter to interface between IgG antibodies and DNA-modified reporter molecules. As a demonstration of this capability, we successfully used DNA nano-barcodes, quantum dots, and horseradish peroxidase enzyme to detect multiple proteins using our DNA-based labeling system. Our system not only eliminates secondary antibodies but also serves as a novel method platform for protein detection with modularity, high capacity, and multiplexed capability.

A Universal DNA-Based Protein Detection System

PubMed Central

Tran, Thua N. N.; Cui, Jinhui; Hartman, Mark R.; Peng, Songming; Funabashi, Hisakage; Duan, Faping; Yang, Dayong; March, John C.; Lis, John T.; Cui, Haixin; Luo, Dan

2014-01-01

Protein immune detection requires secondary antibodies which must be carefully selected in order to avoid interspecies cross-reactivity, and is therefore restricted by the limited availability of primary/secondary antibody pairs. Here we present a versatile DNA-based protein detection system using a universal adapter to interface between IgG antibodies and DNA-modified reporter molecules. As a demonstration of this capability, we successfully used DNA nano-barcodes, quantum dots, and horseradish peroxidase enzyme to detect multiple proteins using our DNA-based labeling system. Our system not only eliminates secondary antibodies but also serves as a novel method platform for protein detection with modularity, high capacity, and multiplexed capability. PMID:23978265

Extreme-Depth Re-sequencing of Mitochondrial DNA Finds No Evidence of Paternal Transmission in Humans.

PubMed

Pyle, Angela; Hudson, Gavin; Wilson, Ian J; Coxhead, Jonathan; Smertenko, Tania; Herbert, Mary; Santibanez-Koref, Mauro; Chinnery, Patrick F

2015-05-01

Recent reports have questioned the accepted dogma that mammalian mitochondrial DNA (mtDNA) is strictly maternally inherited. In humans, the argument hinges on detecting a signature of inter-molecular recombination in mtDNA sequences sampled at the population level, inferring a paternal source for the mixed haplotypes. However, interpreting these data is fraught with difficulty, and direct experimental evidence is lacking. Using extreme-high depth mtDNA re-sequencing up to ~1.2 million-fold coverage, we find no evidence that paternal mtDNA haplotypes are transmitted to offspring in humans, thus excluding a simple dilution mechanism for uniparental transmission of mtDNA present in all healthy individuals. Our findings indicate that an active mechanism eliminates paternal mtDNA which likely acts at the molecular level.

Extreme-Depth Re-sequencing of Mitochondrial DNA Finds No Evidence of Paternal Transmission in Humans

PubMed Central

Pyle, Angela; Hudson, Gavin; Wilson, Ian J.; Coxhead, Jonathan; Smertenko, Tania; Herbert, Mary; Santibanez-Koref, Mauro; Chinnery, Patrick F.

2015-01-01

Recent reports have questioned the accepted dogma that mammalian mitochondrial DNA (mtDNA) is strictly maternally inherited. In humans, the argument hinges on detecting a signature of inter-molecular recombination in mtDNA sequences sampled at the population level, inferring a paternal source for the mixed haplotypes. However, interpreting these data is fraught with difficulty, and direct experimental evidence is lacking. Using extreme-high depth mtDNA re-sequencing up to ~1.2 million-fold coverage, we find no evidence that paternal mtDNA haplotypes are transmitted to offspring in humans, thus excluding a simple dilution mechanism for uniparental transmission of mtDNA present in all healthy individuals. Our findings indicate that an active mechanism eliminates paternal mtDNA which likely acts at the molecular level. PMID:25973765

DNA-dependent protein kinase is a molecular target for the development of noncytotoxic radiation-sensitizing drugs.

PubMed

Shinohara, Eric T; Geng, Ling; Tan, Jiahui; Chen, Heidi; Shir, Yu; Edwards, Eric; Halbrook, James; Kesicki, Edward A; Kashishian, Adam; Hallahan, Dennis E

2005-06-15

DNA-dependent protein kinase (DNA-PK)-defective severe combined immunodeficient (SCID) mice have a greater sensitivity to ionizing radiation compared with wild-type mice due to deficient repair of DNA double-strand break. SCID cells were therefore studied to determine whether radiosensitization by the specific inhibitor of DNA-PK, IC87361, is eliminated in the absence of functional DNA-PK. IC87361 enhanced radiation sensitivity in wild-type C57BL6 endothelial cells but not in SCID cells. The tumor vascular window model was used to assess IC87361-induced radiosensitization of SCID and wild-type tumor microvasculature. Vascular density was 5% in irradiated SCID host compared with 50% in C57BL6 mice (P < 0.05). IC87361 induced radiosensitization of tumor microvasculature in wild-type mice that resembled the radiosensitive phenotype of tumor vessels in SCID mice. Radiosensitization by IC87361 was eliminated in SCID tumor vasculature, which lack functional DNA-PK. Irradiated LLC and B16F0 tumors implanted into SCID mice showed greater tumor growth delay compared with tumors implanted into either wild-type C57BL6 or nude mice. Furthermore, LLC tumors treated with radiation and IC87361 showed tumor growth delay that was significantly greater than tumors treated with radiation alone (P < 0.01 for 3 Gy alone versus 3 Gy + IC87361). DNA-PK inhibitors induced no cytotoxicity and no toxicity in mouse normal tissues. Mouse models deficient in enzyme activity are useful to assess the specificity of novel kinase inhibitors. DNA-PK is an important target for the development of novel radiation-sensitizing drugs that have little intrinsic cytotoxicity.

The impact of bowl size, program setup, and blood hematocrit on the performance of a discontinuous autotransfusion system.

PubMed

Seyfried, Timo F; Gruber, Michael; Streithoff, Fabian; Mandle, Robert J; Pawlik, Michael T; Busse, Hendrik; Hansen, Ernil

2017-03-01

Cell salvage is an essential element in the concept of blood management. Modern devices provide different bowl sizes and sensor-directed programs to optimally adjust to varying clinical situations. In an experimental performance study, the discontinuous autotransfusion device XTRA (LivaNova/Sorin) was evaluated using fresh donor blood anticoagulated with heparin 5 U/mL and adjusted to a hematocrit of 10% or 25%, representing orthopedic or cardiac surgery. Test blood was processed with the autotransfusion device XTRA in four different bowls (55 mL, 125 mL, 175 mL, and 225 mL) and in three different program modes (a standard program, an optimized program, and an emergency program). Processing speed increased with bowl size and with the emergency program (range, 6.4-29.8 mL red blood cells [RBCs]/min). The RBC recovery rate exceeded 90% for all bowls and programs except the 55-mL bowl with the emergency program. Plasma elimination exceeded 95% for all bowls and programs except the 225-mL bowl with the emergency and standard programs. Maximal RBC recovery (range, 94.7%-97.6%) and plasma elimination (range, 98.7%-99.5%) were obtained with the medium-sized bowls (125 mL and 175 mL) and the optimized program. Elimination rates for potassium or plasma free hemoglobin were consistently lower than for protein or albumin and were highest for heparin. Increased hematocrit and RBC recovery rates are obtained with the optimized program Popt with the discontinuous autotransfusion device. The emergency program Pem speeds up the process but leads to RBC loss and reduced plasma elimination rates; therefore, it should be restricted to emergency situations. All four different sized bowls have high performance. Plasma elimination is represented best by protein or albumin elimination rates. © 2017 AABB.

Modeling the Impact and Costs of Semiannual Mass Drug Administration for Accelerated Elimination of Lymphatic Filariasis

PubMed Central

de Vlas, Sake J.; Fischer, Peter U.; Weil, Gary J.; Goldman, Ann S.

2013-01-01

The Global Program to Eliminate Lymphatic Filariasis (LF) has a target date of 2020. This program is progressing well in many countries. However, progress has been slow in some countries, and others have not yet started their mass drug administration (MDA) programs. Acceleration is needed. We studied how increasing MDA frequency from once to twice per year would affect program duration and costs by using computer simulation modeling and cost projections. We used the LYMFASIM simulation model to estimate how many annual or semiannual MDA rounds would be required to eliminate LF for Indian and West African scenarios with varied pre-control endemicity and coverage levels. Results were used to estimate total program costs assuming a target population of 100,000 eligibles, a 3% discount rate, and not counting the costs of donated drugs. A sensitivity analysis was done to investigate the robustness of these results with varied assumptions for key parameters. Model predictions suggested that semiannual MDA will require the same number of MDA rounds to achieve LF elimination as annual MDA in most scenarios. Thus semiannual MDA programs should achieve this goal in half of the time required for annual programs. Due to efficiency gains, total program costs for semiannual MDA programs are projected to be lower than those for annual MDA programs in most scenarios. A sensitivity analysis showed that this conclusion is robust. Semiannual MDA is likely to shorten the time and lower the cost required for LF elimination in countries where it can be implemented. This strategy may improve prospects for global elimination of LF by the target year 2020. PMID:23301115

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Federal Register 2010, 2011, 2012, 2013, 2014

2013-12-09

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Deletion endpoint allele-specificity in the developmentally regulated elimination of an internal sequence (IES) in Paramecium.

PubMed Central

Dubrana, K; Le Mouël, A; Amar, L

1997-01-01

Ciliated protozoa undergo thousands of site-specific DNA deletion events during the programmed development of micronuclear genomes to macronuclear genomes. Two deletion elements, W1 and W2, were identified in the Paramecium primaurelia wild-type 156 strain. Here, we report the characterization of both elements in wild-type strain 168 and show that they display variant deletion patterns when compared with those of strain 156. The W1 ( 168 ) element is defective for deletion. The W2 ( 168 ) element is excised utilizing two alternative boundaries on one side, both are different from the boundary utilized to excise the W2156 element. By crossing the 156 and 168 strains, we demonstrate that the definition of all deletion endpoints are each controlled by cis -acting determinant(s) rather than by strain-specific trans-acting factor(s). Sequence comparison of all deleted DNA segments indicates that the 5'-TA-3'terminal sequence is strictly required at their ends. Furthermore the identity of the first eight base pairs of these ends to a previously established consensus sequence correlates with the frequency of the corresponding deletion events. Our data implies the existence of an adaptive convergent evolution of these Paramecium deleted DNA segment end sequences. PMID:9171098

Mitochondrial DNA Mutation Associated with Leber's Hereditary Optic Neuropathy

NASA Astrophysics Data System (ADS)

Wallace, Douglas C.; Singh, Gurparkash; Lott, Marie T.; Hodge, Judy A.; Schurr, Theodore G.; Lezza, Angela M. S.; Elsas, Louis J.; Nikoskelainen, Eeva K.

1988-12-01

Leber's hereditary optic neuropathy is a maternally inherited disease resulting in optic nerve degeneration and cardiac dysrhythmia. A mitochondrial DNA replacement mutation was identified that correlated with this disease in multiple families. This mutation converted a highly conserved arginine to a histidine at codon 340 in the NADH dehydrogenase subunit 4 gene and eliminated an Sfa NI site, thus providing a simple diagnostic test. This finding demonstrated that a nucleotide change in a mitochondrial DNA energy production gene can result in a neurological disease.

Resolving DNA-ligand intercalation in the entropic stretching regime

NASA Astrophysics Data System (ADS)

Almaqwashi, Ali A.

Single molecule studies of DNA intercalation are typically conducted by applying stretching forces to obtain force-dependent DNA elongation measurements. The zero-force properties of DNA intercalation are determined by equilibrium and kinetic force-analysis. However, the applied stretching forces that are above the entropic regime (>5 pN) prevent DNA-DNA contact which may eliminate competitive DNA-ligand interactions. In particular, it is noted that cationic mono-intercalators investigated by single molecule force spectroscopy are mostly found to intercalate DNA with single rate, while bulk studies reported additional slower rates. Here, a proposed framework quantifies DNA intercalation by cationic ligands in competition with relatively rapid kinetic DNA-ligand aggregation. At a constant applied force in the entropic stretching regime, the analysis illustrates that DNA intercalation would be measurably optimized only within a narrow range of low ligand concentrations. As DNA intercalators are considered for potential DNA-targeted therapeutics, this analysis provides insights in tuning ligand concertation to maximize therapeutics efficiency.

40 CFR 35.710 - Purpose.

Code of Federal Regulations, 2010 CFR

2010-07-01

... operate compliance monitoring programs to prevent or eliminate unreasonable risks to health or the... Administrator is unable or not likely to take action for their prevention or elimination. (c) Associated program...

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2013-10-28

... Amending Commentary .07 to Rule 904 To Extend the Pilot Program That Eliminated the Position Limits for... that eliminated the position limits for options on SPDR S&P 500 ETF (``SPY'') (``SPY Pilot Program...- regulatory organizations (``SROs'') have adopted similar rules eliminating position limits on SPY and market...

Lymphatic filarial species differentiation using evolutionarily modified tandem repeats: generation of new genetic markers.

PubMed

Sakthidevi, Moorthy; Murugan, Vadivel; Hoti, Sugeerappa Laxmanappa; Kaliraj, Perumal

2010-05-01

Polymerase chain reaction based methods are promising tools for the monitoring and evaluation of the Global Program for the Elimination of Lymphatic Filariasis. The currently available PCR methods do not differentiate the DNA of Wuchereria bancrofti or Brugia malayi by a single PCR and hence are cumbersome. Therefore, we designed a single step PCR strategy for differentiating Bancroftian infection from Brugian infection based on a newly identified gene from the W. bancrofti genome, abundant larval transcript-2 (alt-2), which is abundantly expressed. The difference in PCR product sizes generated from the presence or absence of evolutionarily altered tandem repeats in alt-2 intron-3 differentiated W. bancrofti from B. malayi. The analysis was performed on the genomic DNA of microfilariae from a number of patient blood samples or microfilariae positive slides from different Indian geographical regions. The assay gave consistent results, differentiating the two filarial parasite species accurately. This alt-2 intron-3 based PCR assay can be a potential tool for the diagnosis and differentiation of co-infections by lymphatic filarial parasites. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Elimination of the cryptic plasmid in Leuconostoc citreum by CRISPR/Cas9 system.

PubMed

Jang, Ye-Ji; Seo, Seung-Oh; Kim, Seul-Ah; Li, Ling; Kim, Tae-Jip; Kim, Sun Chang; Jin, Yong-Su; Han, Nam Soo

2017-06-10

Leuconostoc spp. are important lactic acid bacteria for the fermentation of foods. In particular, L. citreum strains isolated from various foods have been used as host strains for genetic and metabolic engineering studies. In order to develop a food-grade genetic engineering system, L. citreum CB2567 was isolated from Kimchi. However, the isolated bacterium contained a cryptic plasmid which was difficult to eliminate. As the existence of the plasmid might hinder strain engineering, we eliminated the plasmid using an RNA-guided DNA endonuclease CRISPR/Cas9 system. We demonstrated that a plasmid-free L. citreum CB2567 host strain could be efficiently constructed through a two-step procedure: 1) transformation of the "killer" plasmid expressing Cas9 endonuclease and a guide RNA (gRNA) targeting for a specific sequence in the cryptic plasmid, and 2) serial subculture without antibiotics for curing the killer plasmid. When the crude extract of L. citreum expressing Cas9 and the guide RNA was incubated with a PCR fragment containing the specific sequence recognized by the guide RNA, the PCR fragment was cleaved. Also, the cryptic plasmid pCB42 was successfully eliminated from the host strain after transforming the plasmid harboring Cas9 and the guide RNA. The Cas9 and gRNA expression plasmid used in this study can be applied for genome engineering purposes by additionally introducing an editing DNA template to repair the double strand DNA breakage caused by Cas9 in the genome of L. citreum. This study demonstrates the feasibility of developing CRISPR/Cas9-based genetic engineering tools to develop a safe host strain and construct food-grade lactic acid bacteria without residual antibiotic markers. Copyright © 2017 Elsevier B.V. All rights reserved.

Leading-edge forensic DNA analyses and the necessity of including crime scene investigators, police officers and technicians in a DNA elimination database.

PubMed

Lapointe, Martine; Rogic, Anita; Bourgoin, Sarah; Jolicoeur, Christine; Séguin, Diane

2015-11-01

In recent years, sophisticated technology has significantly increased the sensitivity and analytical power of genetic analyses so that very little starting material may now produce viable genetic profiles. This sensitivity however, has also increased the risk of detecting unknown genetic profiles assumed to be that of the perpetrator, yet originate from extraneous sources such as from crime scene workers. These contaminants may mislead investigations, keeping criminal cases active and unresolved for long spans of time. Voluntary submission of DNA samples from crime scene workers is fairly low, therefore we have created a promotional method for our staff elimination database that has resulted in a significant increase in voluntary samples since 2011. Our database enforces privacy safeguards and allows for optional anonymity to all staff members. We also offer information sessions at various police precincts to advise crime scene workers of the importance and success of our staff elimination database. This study, a pioneer in its field, has obtained 327 voluntary submissions from crime scene workers to date, of which 46 individual profiles (14%) have been matched to 58 criminal cases. By implementing our methods and respect for individual privacy, forensic laboratories everywhere may see similar growth and success in explaining unidentified genetic profiles in stagnate criminal cases. Crown Copyright © 2015. Published by Elsevier Ireland Ltd. All rights reserved.

An Efficient Multistrategy DNA Decontamination Procedure of PCR Reagents for Hypersensitive PCR Applications

PubMed Central

Pruvost, Mélanie; Bennett, E. Andrew; Grange, Thierry; Geigl, Eva-Maria

2010-01-01

Background PCR amplification of minute quantities of degraded DNA for ancient DNA research, forensic analyses, wildlife studies and ultrasensitive diagnostics is often hampered by contamination problems. The extent of these problems is inversely related to DNA concentration and target fragment size and concern (i) sample contamination, (ii) laboratory surface contamination, (iii) carry-over contamination, and (iv) contamination of reagents. Methodology/Principal Findings Here we performed a quantitative evaluation of current decontamination methods for these last three sources of contamination, and developed a new procedure to eliminate contaminating DNA contained in PCR reagents. We observed that most current decontamination methods are either not efficient enough to degrade short contaminating DNA molecules, rendered inefficient by the reagents themselves, or interfere with the PCR when used at doses high enough to eliminate these molecules. We also show that efficient reagent decontamination can be achieved by using a combination of treatments adapted to different reagent categories. Our procedure involves γ- and UV-irradiation and treatment with a mutant recombinant heat-labile double-strand specific DNase from the Antarctic shrimp Pandalus borealis. Optimal performance of these treatments is achieved in narrow experimental conditions that have been precisely analyzed and defined herein. Conclusions/Significance There is not a single decontamination method valid for all possible contamination sources occurring in PCR reagents and in the molecular biology laboratory and most common decontamination methods are not efficient enough to decontaminate short DNA fragments of low concentration. We developed a versatile multistrategy decontamination procedure for PCR reagents. We demonstrate that this procedure allows efficient reagent decontamination while preserving the efficiency of PCR amplification of minute quantities of DNA. PMID:20927390

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Technique to eliminate computational instability in multibody simulations employing the Lagrange multiplier

NASA Technical Reports Server (NTRS)

Watts, G.

1992-01-01

A programming technique to eliminate computational instability in multibody simulations that use the Lagrange multiplier is presented. The computational instability occurs when the attached bodies drift apart and violate the constraints. The programming technique uses the constraint equation, instead of integration, to determine the coordinates that are not independent. Although the equations of motion are unchanged, a complete derivation of the incorporation of the Lagrange multiplier into the equation of motion for two bodies is presented. A listing of a digital computer program which uses the programming technique to eliminate computational instability is also presented. The computer program simulates a solid rocket booster and parachute connected by a frictionless swivel.

Engineering of Systematic Elimination of a Targeted Chromosome in Human Cells.

PubMed

Sato, Hiroshi; Kato, Hiroki; Yamaza, Haruyoshi; Masuda, Keiji; Nguyen, Huong Thi Nguyen; Pham, Thanh Thi Mai; Han, Xu; Hirofuji, Yuta; Nonaka, Kazuaki

2017-01-01

Embryonic trisomy leads to abortion or congenital genetic disorders in humans. The most common autosomal chromosome abnormalities are trisomy of chromosomes 13, 18, and 21. Although alteration of gene dosage is thought to contribute to disorders caused by extra copies of chromosomes, genes associated with specific disease phenotypes remain unclear. To generate a normal cell from a trisomic cell as a means of etiological analysis or candidate therapy for trisomy syndromes, we developed a system to eliminate a targeted chromosome from human cells. Chromosome 21 was targeted by integration of a DNA cassette in HeLa cells that harbored three copies of chromosome 21. The DNA cassette included two inverted loxP sites and a herpes simplex virus thymidine kinase (HSV-tk) gene. This system causes missegregation of chromosome 21 after expression of Cre recombinase and subsequently enables the selection of cells lacking the chromosome by culturing in a medium that includes ganciclovir (GCV). Cells harboring only two copies of chromosome 21 were efficiently induced by transfection of a Cre expression vector, indicating that this approach is useful for eliminating a targeted chromosome.

Co-immunization with DNA and protein mixture: a safe and efficacious immunotherapeutic strategy for Alzheimer's disease in PDAPP mice.

PubMed

Liu, Si; Shi, DanYang; Wang, Hai-Chao; Yu, Yun-Zhou; Xu, Qing; Sun, Zhi-Wei

2015-01-14

Active immunotherapy targeting β-amyloid (Aβ) is the most promising strategy to prevent or treat Alzheimer's disease (AD). Based on pre-clinical studies and clinical trials, a safe and effective AD vaccine requires a delicate balance between providing therapeutically adequate anti-Aβ antibodies and eliminating or suppressing unwanted adverse T cell-mediated inflammatory reactions. We describe here the immunological characterization and protective efficacy of co-immunization with a 6Aβ15-T DNA and protein mixture without adjuvant as an AD immunotherapeutic strategy. Impressively, this co-immunization induced robust Th2-polarized Aβ-specific antibodies while simultaneously suppressed unwanted inflammatory T cell reactions and avoiding Aβ42-specific T cell-mediated autoimmune responses in immunized mice. Co-immunization with the DNA + protein vaccine could overcome Aβ42-associated hypo-responsiveness and elicit long-term Aβ-specific antibody responses, which helped to maintain antibody-mediated clearance of amyloid and accordingly alleviated AD symptoms in co-immunized PDAPP mice. Our DNA and protein combined vaccine, which could induce an anti-inflammatory Th2 immune response with high level Aβ-specific antibodies and low level IFN-γ production, also demonstrated the capacity to inhibit amyloid accumulation and prevent cognitive dysfunction. Hence, co-immunization with antigen-matched DNA and protein may represent a novel and efficacious strategy for AD immunotherapy to eliminate T cell inflammatory reactions while retaining high level antibody responses.

Real-time, multiplexed electrochemical DNA detection using an active complementary metal-oxide-semiconductor biosensor array with integrated sensor electronics.

PubMed

Levine, Peter M; Gong, Ping; Levicky, Rastislav; Shepard, Kenneth L

2009-03-15

Optical biosensing based on fluorescence detection has arguably become the standard technique for quantifying extents of hybridization between surface-immobilized probes and fluorophore-labeled analyte targets in DNA microarrays. However, electrochemical detection techniques are emerging which could eliminate the need for physically bulky optical instrumentation, enabling the design of portable devices for point-of-care applications. Unlike fluorescence detection, which can function well using a passive substrate (one without integrated electronics), multiplexed electrochemical detection requires an electronically active substrate to analyze each array site and benefits from the addition of integrated electronic instrumentation to further reduce platform size and eliminate the electromagnetic interference that can result from bringing non-amplified signals off chip. We report on an active electrochemical biosensor array, constructed with a standard complementary metal-oxide-semiconductor (CMOS) technology, to perform quantitative DNA hybridization detection on chip using targets conjugated with ferrocene redox labels. A 4 x 4 array of gold working electrodes and integrated potentiostat electronics, consisting of control amplifiers and current-input analog-to-digital converters, on a custom-designed 5 mm x 3 mm CMOS chip drive redox reactions using cyclic voltammetry, sense DNA binding, and transmit digital data off chip for analysis. We demonstrate multiplexed and specific detection of DNA targets as well as real-time monitoring of hybridization, a task that is difficult, if not impossible, with traditional fluorescence-based microarrays.

Butyric Acid- and Dimethyl Disulfide-Assimilating Microorganisms in a Biofilter Treating Air Emissions from a Livestock Facility▿

PubMed Central

Kristiansen, Anja; Lindholst, Sabine; Feilberg, Anders; Nielsen, Per H.; Neufeld, Josh D.; Nielsen, Jeppe L.

2011-01-01

Biofiltration has proven an efficient tool for the elimination of volatile organic compounds (VOCs) and ammonia from livestock facilities, thereby reducing nuisance odors and ammonia emissions to the local environment. The active microbial communities comprising these filter biofilms have not been well characterized. In this study, a trickle biofilter treating air from a pig facility was investigated and proved efficient in removing carboxylic acids (>70% reduction), mainly attributed to the primary filter section within which reduced organic sulfur compounds were also depleted (up to 50%). The secondary filter eliminated several aromatic compounds: phenol (81%), p-cresol (89%), 4-ethylphenol (68%), indole (48%), and skatole (69%). The active butyric acid degrading bacterial community of an air filter sample was identified by DNA stable-isotope probing (DNA-SIP) and microautoradiography, combined with fluorescence in situ hybridization (MAR-FISH). The predominant 16S rRNA gene sequences from a clone library derived from “heavy” DNA from [13C4]butyric acid incubations were Microbacterium, Gordonia, Dietzia, Rhodococcus, Propionibacterium, and Janibacter, all from the Actinobacteria. Actinobacteria were confirmed and quantified by MAR-FISH as being the major bacterial phylum assimilating butyric acid along with several Burkholderiales-related Betaproteobacteria. The active bacterial community assimilating dimethyl disulfide (DMDS) was characterized by DNA-SIP and MAR-FISH and found to be associated with the Actinobacteria, along with a few representatives of Flavobacteria and Sphingobacteria. Interestingly, ammonia-oxidizing Betaproteobacteria were also implicated in DMDS degradation, as were fungi. Thus, multiple isotope-based methods provided complementary data, enabling high-resolution identification and quantitative assessments of odor-eliminating Actinobacteria-dominated populations of these biofilter environments. PMID:22003018

Butyric acid- and dimethyl disulfide-assimilating microorganisms in a biofilter treating air emissions from a livestock facility.

PubMed

Kristiansen, Anja; Lindholst, Sabine; Feilberg, Anders; Nielsen, Per H; Neufeld, Josh D; Nielsen, Jeppe L

2011-12-01

Biofiltration has proven an efficient tool for the elimination of volatile organic compounds (VOCs) and ammonia from livestock facilities, thereby reducing nuisance odors and ammonia emissions to the local environment. The active microbial communities comprising these filter biofilms have not been well characterized. In this study, a trickle biofilter treating air from a pig facility was investigated and proved efficient in removing carboxylic acids (>70% reduction), mainly attributed to the primary filter section within which reduced organic sulfur compounds were also depleted (up to 50%). The secondary filter eliminated several aromatic compounds: phenol (81%), p-cresol (89%), 4-ethylphenol (68%), indole (48%), and skatole (69%). The active butyric acid degrading bacterial community of an air filter sample was identified by DNA stable-isotope probing (DNA-SIP) and microautoradiography, combined with fluorescence in situ hybridization (MAR-FISH). The predominant 16S rRNA gene sequences from a clone library derived from "heavy" DNA from [(13)C(4)]butyric acid incubations were Microbacterium, Gordonia, Dietzia, Rhodococcus, Propionibacterium, and Janibacter, all from the Actinobacteria. Actinobacteria were confirmed and quantified by MAR-FISH as being the major bacterial phylum assimilating butyric acid along with several Burkholderiales-related Betaproteobacteria. The active bacterial community assimilating dimethyl disulfide (DMDS) was characterized by DNA-SIP and MAR-FISH and found to be associated with the Actinobacteria, along with a few representatives of Flavobacteria and Sphingobacteria. Interestingly, ammonia-oxidizing Betaproteobacteria were also implicated in DMDS degradation, as were fungi. Thus, multiple isotope-based methods provided complementary data, enabling high-resolution identification and quantitative assessments of odor-eliminating Actinobacteria-dominated populations of these biofilter environments.

DNA Strand Breaks in Mitotic Germ Cells of Caenorhabditis elegans Evaluated by Comet Assay

PubMed Central

Park, Sojin; Choi, Seoyun; Ahn, Byungchan

2016-01-01

DNA damage responses are important for the maintenance of genome stability and the survival of organisms. Such responses are activated in the presence of DNA damage and lead to cell cycle arrest, apoptosis, and DNA repair. In Caenorhabditis elegans, double-strand breaks induced by DNA damaging agents have been detected indirectly by antibodies against DSB recognizing proteins. In this study we used a comet assay to detect DNA strand breaks and to measure the elimination of DNA strand breaks in mitotic germline nuclei of C. elegans. We found that C. elegans brc-1 mutants were more sensitive to ionizing radiation and camptothecin than the N2 wild-type strain and repaired DNA strand breaks less efficiently than N2. This study is the first demonstration of direct measurement of DNA strand breaks in mitotic germline nuclei of C. elegans. This newly developed assay can be applied to detect DNA strand breaks in different C. elegans mutants that are sensitive to DNA damaging agents. PMID:26903030

Hofmeister series salts enhance purification of plasmid DNA by non-ionic detergents

PubMed Central

Lezin, George; Kuehn, Michael R.; Brunelli, Luca

2011-01-01

Ion-exchange chromatography is the standard technique used for plasmid DNA purification, an essential molecular biology procedure. Non-ionic detergents (NIDs) have been used for plasmid DNA purification, but it is unclear whether Hofmeister series salts (HSS) change the solubility and phase separation properties of specific NIDs, enhancing plasmid DNA purification. After scaling-up NID-mediated plasmid DNA isolation, we established that NIDs in HSS solutions minimize plasmid DNA contamination with protein. In addition, large-scale NID/HSS solutions eliminated LPS contamination of plasmid DNA more effectively than Qiagen ion-exchange columns. Large-scale NID isolation/NID purification generated increased yields of high quality DNA compared to alkali isolation/column purification. This work characterizes how HSS enhance NID-mediated plasmid DNA purification, and demonstrates that NID phase transition is not necessary for LPS removal from plasmid DNA. Specific NIDs such as IGEPAL CA-520 can be utilized for rapid, inexpensive and efficient laboratory-based large-scale plasmid DNA purification, outperforming Qiagen-based column procedures. PMID:21351074

75 FR 28554 - Elimination of Classification Requirement in the Green Technology Pilot Program

Federal Register 2010, 2011, 2012, 2013, 2014

2010-05-21

...] Elimination of Classification Requirement in the Green Technology Pilot Program AGENCY: United States Patent.... classifications to assist the USPTO to balance the workload and gauge resources needed for the program. The USPTO has determined that the classification requirement is unnecessary because the workload has been...

Integration of the Reconfigurable Self-Healing eDNA Architecture in an Embedded System

NASA Technical Reports Server (NTRS)

Boesen, Michael Reibel; Keymeulen, Didier; Madsen, Jan; Lu, Thomas; Chao, Tien-Hsin

2011-01-01

In this work we describe the first real world case study for the self-healing eDNA (electronic DNA) architecture by implementing the control and data processing of a Fourier Transform Spectrometer (FTS) on an eDNA prototype. For this purpose the eDNA prototype has been ported from a Xilinx Virtex 5 FPGA to an embedded system consisting of a PowerPC and a Xilinx Virtex 5 FPGA. The FTS instrument features a novel liquid crystal waveguide, which consequently eliminates all moving parts from the instrument. The addition of the eDNA architecture to do the control and data processing has resulted in a highly fault-tolerant FTS instrument. The case study has shown that the early stage prototype of the autonomous self-healing eDNA architecture is expensive in terms of execution time.

Helicases as Prospective Targets for Anti-Cancer Therapy

PubMed Central

Gupta, Rigu; Brosh, Robert M.

2008-01-01

It has been proposed that selective inactivation of a DNA repair pathway may enhance anti-cancer therapies that eliminate cancerous cells through the cytotoxic effects of DNA damaging agents or radiation. Given the unique and critically important roles of DNA helicases in the DNA damage response, DNA repair, and maintenance of genomic stability, a number of strategies currently being explored or in use to combat cancer may be either mediated or enhanced through the modulation of helicase function. The focus of this review will be to examine the roles of helicases in DNA repair that might be suitably targeted by cancer therapeutic approaches. Treatment of cancers with anti-cancer drugs such as small molecule compounds that modulate helicase expression or function is a viable approach to selectively kill cancer cells through the inactivation of helicase-dependent DNA repair pathways, particularly those associated with DNA recombination, replication restart, and cell cycle checkpoint. PMID:18473724

Rapid method to extract DNA from Cryptococcus neoformans.

PubMed Central

Varma, A; Kwon-Chung, K J

1991-01-01

A rapid and easy method for the extraction of total cellular DNA from Cryptococcus neoformans is described. This procedure modifies and considerably simplifies previously reported methods. Numerous steps were either eliminated or replaced, including preincubations with cell wall permeability agents such as beta-mercaptoethanol and dithiothreitol. The commercially available enzyme preparation Novozyme 234 was found to contain a potent concentration of DNases which actively degrade DNA. Degradation and loss of DNA was prevented by maintaining a high concentration of EDTA in the lysing solution. This procedure resulted in high yields (150 to 200 micrograms of DNA from 100 ml of culture) of good-quality (undegraded), high-molecular-weight DNA which was readily digested by restriction endonucleases, making it suitable for use in various molecular applications. Images PMID:1909713

A simple, fast, and inexpensive CTAB-PVP-silica based method for genomic DNA isolation from single, small insect larvae and pupae.

PubMed

Huanca-Mamani, W; Rivera-Cabello, D; Maita-Maita, J

2015-07-17

In this study, we report a modified CTAB-PVP method combined with silicon dioxide (silica) treatment for the extraction of high quality genomic DNA from a single larva or pupa. This method efficiently obtains DNA from small specimens, which is difficult and challenging because of the small amount of starting tissue. Maceration with liquid nitrogen, phenol treatment, and the ethanol precipitation step are eliminated using this methodology. The A260/A280 absorbance ratios of the isolated DNA were approximately 1.8, suggesting that the DNA is pure and can be used for further molecular analysis. The quality of the isolated DNA permits molecular applications and represents a fast, cheap, and effective alternative method for laboratories with low budgets.

Diagnosing schistosomiasis: where are we?

PubMed

Gomes, Luciana Inácia; Enk, Martin Johannes; Rabello, Ana

2014-01-01

In light of the World Health Organization's initiative to extend schistosomiasis morbidity and mortality control programs by including a disease elimination strategy in low endemic settings, this paper reviews diagnostic tools described during the last decades and provide an overview of ongoing efforts in making an efficient diagnostic tool available worldwide. A literature search on PubMed using the search criteria schistosomiasis and diagnosis within the period from 1978 to 2013 was carried out. Articles with abstract in English and that used laboratory techniques specifically developed for the detection of schistosomiasis in humans were included. Publications were categorized according to the methodology applied (parasitological, immunological, or molecular) and stage of development (in house development, limited field, or large scale field testing). The initial research generated 4,535 publications, of which only 643 met the inclusion criteria. The vast majority (537) of the publications focused on immunological techniques; 81 focused on parasitological diagnosis, and 25 focused on molecular diagnostic methods. Regarding the stage of development, 307 papers referred to in-house development, 202 referred to limited field tests, and 134 referred to large scale field testing. The data obtained show that promising new diagnostic tools, especially for Schistosoma antigen and deoxyribonucleic acid (DNA) detection, which are characterized by high sensitivity and specificity, are being developed. In combination with international funding initiatives these tools may result in a significant step forward in successful disease elimination and surveillance, which is to make efficient tests accessible and its large use self-sustainable for control programs in endemic countries.

HARP preferentially co-purifies with RPA bound to DNA-PK and blocks RPA phosphorylation.

PubMed

Quan, Jinhua; Yusufzai, Timur

2014-05-01

The HepA-related protein (HARP/SMARCAL1) is an ATP-dependent annealing helicase that is capable of rewinding DNA structures that are stably unwound due to binding of the single-stranded DNA (ssDNA)-binding protein Replication Protein A (RPA). HARP has been implicated in maintaining genome integrity through its role in DNA replication and repair, two processes that generate RPA-coated ssDNA. In addition, mutations in HARP cause a rare disease known as Schimke immuno-osseous dysplasia. In this study, we purified HARP containing complexes with the goal of identifying the predominant factors that stably associate with HARP. We found that HARP preferentially interacts with RPA molecules that are bound to the DNA-dependent protein kinase (DNA-PK). We also found that RPA is phosphorylated by DNA-PK in vitro, while the RPA-HARP complexes are not. Our results suggest that, in addition to its annealing helicase activity, which eliminates the natural binding substrate for RPA, HARP blocks the phosphorylation of RPA by DNA-PK.

Recent Insights into the Control of Human Papillomavirus (HPV) Genome Stability, Loss, and Degradation.

PubMed

Fisher, Chris

2015-01-01

Most human papillomavirus (HPV) antiviral strategies have focused upon inhibiting viral DNA replication, but it is increasingly apparent that viral DNA levels can be chemically controlled by approaches that promote its instability. HPVs and other DNA viruses have a tenuous relationship with their hosts. They must replicate and hide from the DNA damage response (DDR) and innate immune systems, which serve to protect cells from foreign or "non-self" DNA, and yet they draft these same systems to support their life cycles. DNA binding antiviral agents promoting massive viral DNA instability and elimination are reviewed. Mechanistic studies of these agents have identified genetic antiviral enhancers and repressors, antiviral sensitizers, and host cell elements that protect and stabilize HPV genomes. Viral DNA degradation appears to be an important means of controlling HPV DNA levels in some cases, but the underlying mechanisms remain poorly understood. These findings may prove useful not only for understanding viral DNA persistence but also in devising future antiviral strategies.

Impact of low-frequency hotspot mutation R282Q on the structure of p53 DNA-binding domain as revealed by crystallography at 1.54 Å resolution

PubMed Central

Tu, Chao; Tan, Yu-Hong; Shaw, Gary; Zhou, Zheng; Bai, Yawen; Luo, Ray; Ji, Xinhua

2008-01-01

Tumor suppressor p53 is a sequence-specific DNA-binding protein and its central DNA-binding domain (DBD) harbors six hotspots (Arg175, Gly245, Arg248, Arg249, Arg273 and Arg282) for human cancers. Here, the crystal structure of a low-frequency hotspot mutant, p53DBD(R282Q), is reported at 1.54 Å resolution together with the results of molecular-dynamics simulations on the basis of the structure. In addition to eliminating a salt bridge, the R282Q mutation has a significant impact on the properties of two DNA-binding loops (L1 and L3). The L1 loop is flexible in the wild type, but it is not flexible in the mutant. The L3 loop of the wild type is not flexible, whereas it assumes two conformations in the mutant. Molecular-dynamics simulations indicated that both conformations of the L3 loop are accessible under biological conditions. It is predicted that the elimination of the salt bridge and the inversion of the flexibility of L1 and L3 are directly or indirectly responsible for deactivating the tumor suppressor p53. PMID:18453682

Histone H2AFX Links Meiotic Chromosome Asynapsis to Prophase I Oocyte Loss in Mammals

PubMed Central

Cloutier, Jeffrey M.; Mahadevaiah, Shantha K.; ElInati, Elias; Nussenzweig, André; Tóth, Attila; Turner, James M. A.

2015-01-01

Chromosome abnormalities are common in the human population, causing germ cell loss at meiotic prophase I and infertility. The mechanisms driving this loss are unknown, but persistent meiotic DNA damage and asynapsis may be triggers. Here we investigate the contribution of these lesions to oocyte elimination in mice with chromosome abnormalities, e.g. Turner syndrome (XO) and translocations. We show that asynapsed chromosomes trigger oocyte elimination at diplonema, which is linked to the presence of phosphorylated H2AFX (γH2AFX). We find that DNA double-strand break (DSB) foci disappear on asynapsed chromosomes during pachynema, excluding persistent DNA damage as a likely cause, and demonstrating the existence in mammalian oocytes of a repair pathway for asynapsis-associated DNA DSBs. Importantly, deletion or point mutation of H2afx restores oocyte numbers in XO females to wild type (XX) levels. Unexpectedly, we find that asynapsed supernumerary chromosomes do not elicit prophase I loss, despite being enriched for γH2AFX and other checkpoint proteins. These results suggest that oocyte loss cannot be explained simply by asynapsis checkpoint models, but is related to the gene content of asynapsed chromosomes. A similar mechanistic basis for oocyte loss may operate in humans with chromosome abnormalities. PMID:26509888

Sub-attomole oligonucleotide and p53 cDNA determinations via a high-resolution surface plasmon resonance combined with oligonucleotide-capped gold nanoparticle signal amplification.

PubMed

Yao, Xin; Li, Xin; Toledo, Freddy; Zurita-Lopez, Cecilia; Gutova, Margarita; Momand, Jamil; Zhou, Feimeng

2006-07-15

Oligonucleotide (ODN)-capped gold nanoparticles (Au-NPs) were used in a sandwich assay of ODN or polynucleotide by a flow injection surface plasmon resonance (SPR). A carboxylated dextran film was immobilized onto the SPR sensor surface to eliminate nonspecific adsorption of ODN-capped Au-NPs. The tandem use of signal amplification via the adlayer of the ODN-capped Au-NPs and the differential signal detection by the bicell detector on the SPR resulted in a remarkable DNA detection level. A 39-mer target at a quantity as low as 2.1 x 10(-20)mol, corresponding to 1.38 fM in a 15 microl solution, can be measured. To our knowledge, both the concentration and quantity detection levels are the lowest among all the gene analyses conducted with SPR to this point. The method is shown to be reproducible (relative standard deviation values <16%) and to possess high sequence specificity. It is also demonstrated to be viable for sequence-specific p53 cDNA analysis. The successful elimination of nonspecific adsorption of, and the signal amplification by, ODN-capped Au-NPs renders the SPR attractive for cases where the DNA concentration is extremely low and the sample availability is severely limited.

Results of the rubella elimination program in Catalonia (Spain), 2002–2011

PubMed Central

Barrabeig, Irene; Torner, Nuria; Martínez, Ana; Carmona, Gloria; Ciruela, Pilar; Batalla, Joan; Costa, Josep; Hernández, Sergi; Salleras, Luis; Domínguez, Angela; Group of Catalonia, the Rubella Surveillance

2013-01-01

Rubella is usually a mild disease with nonspecific symptoms, but can cause congenital rubella syndrome (CRS) when infection occurs during pregnancy. The objective of this study was to evaluate the sensitivity and positive predictive value of different data sources used for surveillance purposes in the Rubella Elimination Program of Catalonia between 2002 and 2011. The Urgent Notification to the Statutory Disease Reporting System, the Individualized Disease Reporting System, screening for other viruses included in the Measles Elimination Program, the Microbiological Reporting System and the Minimum Hospital Discharge Data were evaluated. 100 suspected cases of postnatal rubella and 6 suspected cases of CRS were detected. For postnatal rubella, Urgent Notification had the highest sensitivity (32.5%; 95%CI 18.6–49.1), followed by the Virus screening in Measles Elimination Program (25%; 95%CI 12.7-41.2). Virus screening in the Measles Elimination Program had the highest PPV (76.9%; 95%CI 46.1–94.9), followed by the Individualized Disease Reporting System (57.1%; 95%CI 28.9–82.3). For CRS cases, the Individualized Disease Reporting System had the highest sensitivity (100%, 95%CI 29.2–100) and the highest PPV (60%; 95%CI 14.7–100). Most confirmed postnatal cases (25 cases, 48.1%) were in the 25–44 y age group followed by the 15–24 y age group (11 cases, 21.2%). The highest values of sensitivity and PPV for the detection of confirmed cases corresponded to activities that were specifically introduced in the measles and rubella elimination programs. PMID:23299566

Genetics Home Reference: adenosine deaminase deficiency

MedlinePlus

... to eliminate a molecule called deoxyadenosine, which is generated when DNA is broken down. Adenosine deaminase converts ... a substitute for professional medical care or advice. Users with questions about a personal health condition should ...

North of the 46° parallel: Obstacles and challenges to recycling in Ontario's rural and northern communities.

PubMed

Lakhan, Calvin

2015-10-01

This study examines the economic challenges of recycling in Ontario's rural and northern areas. Specifically, this study quantifies the economic and diversion impact of operating recycling programs in these regions. Using a systems based cost model, focus is placed on analyzing: (1) What would happen to provincial recycling costs and diversion levels if recycling programs were eliminated in "high cost" northern and rural communities? (2) Is it possible to increase the provincial recycling rate by focusing investments in low cost, high performance regions (while simultaneously eliminating recycling programs in rural and northern areas)? (3) How would the mix of material recovered change if recycling programs were eliminated in rural and northern areas? The results of this analysis show that eliminating recycling programs in high cost regions significantly decreased system costs without negatively impacting overall recycling rates. This study also found that it was possible to increase the provincial recycling rate while simultaneously reducing program costs by targeting specific regions for recovery. The findings of this study suggest that Ontario reevaluate whether rural and northern municipalities be legislatively required to operate household recycling programs. Copyright © 2015 Elsevier Ltd. All rights reserved.

National Mass Drug Administration Costs for Lymphatic Filariasis Elimination

PubMed Central

Goldman, Ann S.; Guisinger, Victoria H.; Aikins, Moses; Amarillo, Maria Lourdes E.; Belizario, Vicente Y.; Garshong, Bertha; Gyapong, John; Kabali, Conrad; Kamal, Hussein A.; Kanjilal, Sanjat; Kyelem, Dominique; Lizardo, Jefrey; Malecela, Mwele; Mubyazi, Godfrey; Nitièma, P. Abdoulaye; Ramzy, Reda M. R.; Streit, Thomas G.; Wallace, Aaron; Brady, Molly A.; Rheingans, Richard; Ottesen, Eric A.; Haddix, Anne C.

2007-01-01

Background Because lymphatic filariasis (LF) elimination efforts are hampered by a dearth of economic information about the cost of mass drug administration (MDA) programs (using either albendazole with diethylcarbamazine [DEC] or albendazole with ivermectin), a multicenter study was undertaken to determine the costs of MDA programs to interrupt transmission of infection with LF. Such results are particularly important because LF programs have the necessary diagnostic and treatment tools to eliminate the disease as a public health problem globally, and already by 2006, the Global Programme to Eliminate LF had initiated treatment programs covering over 400 million of the 1.3 billion people at risk. Methodology/Principal Findings To obtain annual costs to carry out the MDA strategy, researchers from seven countries developed and followed a common cost analysis protocol designed to estimate 1) the total annual cost of the LF program, 2) the average cost per person treated, and 3) the relative contributions of the endemic countries and the external partners. Costs per person treated ranged from $0.06 to $2.23. Principal reasons for the variation were 1) the age (newness) of the MDA program, 2) the use of volunteers, and 3) the size of the population treated. Substantial contributions by governments were documented – generally 60%–90% of program operation costs, excluding costs of donated medications. Conclusions/Significance MDA for LF elimination is comparatively inexpensive in relation to most other public health programs. Governments and communities make the predominant financial contributions to actual MDA implementation, not counting the cost of the drugs themselves. The results highlight the impact of the use of volunteers on program costs and provide specific cost data for 7 different countries that can be used as a basis both for modifying current programs and for developing new ones. PMID:17989784

DNA repair in mammalian mitochondria: Much more than we thought?

PubMed

Liu, Pingfang; Demple, Bruce

2010-06-01

For many years, the repair of most damage in mitochondrial DNA (mtDNA) was thought limited to short-patch base excision repair (SP-BER), which replaces a single nucleotide by the sequential action of DNA glycosylases, an apurinic/apyrimidinic (AP) endonuclease, the mitochondrial DNA polymerase gamma, an abasic lyase activity, and mitochondrial DNA ligase. However, the likely array of lesions inflicted on mtDNA by oxygen radicals and the possibility of replication errors and disruptions indicated that such a restricted repair repertoire would be inadequate. Recent studies have considerably expanded our knowledge of mtDNA repair to include long-patch base excision repair (LP-BER), mismatch repair, and homologous recombination and nonhomologous end-joining. In addition, elimination of mutagenic 8-oxodeoxyguanosine triphosphate (8-oxodGTP) helps prevent cell death due to the accumulation of this oxidation product in mtDNA. Although it was suspected for many years that irreparably damaged mtDNA might be targeted for degradation, only recently was clear evidence provided for this hypothesis. Therefore, multiple DNA repair pathways and controlled degradation of mtDNA function together to maintain the integrity of mitochondrial genome.

Repair of DNA-polypeptide crosslinks by human excision nuclease

NASA Astrophysics Data System (ADS)

Reardon, Joyce T.; Sancar, Aziz

2006-03-01

DNA-protein crosslinks are relatively common DNA lesions that form during the physiological processing of DNA by replication and recombination proteins, by side reactions of base excision repair enzymes, and by cellular exposure to bifunctional DNA-damaging agents such as platinum compounds. The mechanism by which pathological DNA-protein crosslinks are repaired in humans is not known. In this study, we investigated the mechanism of recognition and repair of protein-DNA and oligopeptide-DNA crosslinks by the human excision nuclease. Under our assay conditions, the human nucleotide excision repair system did not remove a 16-kDa protein crosslinked to DNA at a detectable level. However, 4- and 12-aa-long oligopeptides crosslinked to the DNA backbone were recognized by some of the damage recognition factors of the human excision nuclease with moderate selectivity and were excised from DNA at relatively efficient rates. Our data suggest that, if coupled with proteolytic degradation of the crosslinked protein, the human excision nuclease may be the major enzyme system for eliminating protein-DNA crosslinks from the genome. damage recognition | nucleotide excision repair

Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ

DOEpatents

Gray, Joe W.; Pinkel, Daniel

1991-01-01

A method is provided for producing single stranded non-self-complementary nucleic acid probes, and for treating target DNA for use therewith. Probe is constructed by treating DNA with a restriction enzyme and an exonuclease to form template/primers for a DNA polymerase. The digested strand is resynthesized in the presence of labeled nucleoside triphosphate precursor. Labeled single stranded fragments are separated from the resynthesized fragments to form the probe. Target DNA is treated with the same restriction enzyme used to construct the probe, and is treated with an exonuclease before application of the probe. The method significantly increases the efficiency and specificity of hybridization mixtures by increasing effective probe concentration by eliminating self-hybridization between both probe and target DNAs, and by reducing the amount of target DNA available for mismatched hybridizations.

Immobilization of DNA onto poly(dimethylsiloxane) surfaces and application to a microelectrochemical enzyme-amplified DNA hybridization assay.

PubMed

Liu, Daojun; Perdue, Robbyn K; Sun, Li; Crooks, Richard M

2004-07-06

This paper describes immobilization of DNA onto the interior walls of poly(dimethylsiloxane) (PDMS) microsystems and its application to an enzyme-amplified electrochemical DNA assay. DNA immobilization was carried out by silanization of the PDMS surface with 3-mercaptopropyltrimethoxysilane to yield a thiol-terminated surface. 5'-acrylamide-modified DNA reacts with the pendant thiol groups to yield DNA-modified PDMS. Surface-immobilized DNA oligos serve as capture probes for target DNA. Biotin-labeled target DNA hybridizes to the PDMS-immobilized capture DNA, and subsequent introduction of alkaline phosphatase (AP) conjugated to streptavidin results in attachment of the enzyme to hybridized DNA. Electrochemical detection of DNA hybridization benefits from enzyme amplification. Specifically, AP converts electroinactive p-aminophenyl phosphate to electroactive p-aminophenol, which is detected using an indium tin oxide interdigitated array (IDA) electrode. The IDA electrode eliminates the need for a reference electrode and provides a steady-state current that is related to the concentration of hybridized DNA. At present, the limit of detection of the DNA target is 1 nM in a volume of 20 nL, which corresponds to 20 attomoles of DNA.

77 FR 58830 - State Program Requirements; Application To Administer Partial National Pollutant Discharge...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-09-24

... Pollutant Discharge Elimination System (AgPDES) program pursuant to Section 402(b) of the Clean Water Act... Discharge Elimination System (NPDES) permit program under Section 402(n)(3) of the Act for all discharges of... through Friday, excluding legal holidays, at EPA Region 6, 1445 Ross Ave., Dallas, Texas 75202. A copy of...

Programmed Cell Death During Caenorhabditis elegans Development

PubMed Central

Conradt, Barbara; Wu, Yi-Chun; Xue, Ding

2016-01-01

Programmed cell death is an integral component of Caenorhabditis elegans development. Genetic and reverse genetic studies in C. elegans have led to the identification of many genes and conserved cell death pathways that are important for the specification of which cells should live or die, the activation of the suicide program, and the dismantling and removal of dying cells. Molecular, cell biological, and biochemical studies have revealed the underlying mechanisms that control these three phases of programmed cell death. In particular, the interplay of transcriptional regulatory cascades and networks involving multiple transcriptional regulators is crucial in activating the expression of the key death-inducing gene egl-1 and, in some cases, the ced-3 gene in cells destined to die. A protein interaction cascade involving EGL-1, CED-9, CED-4, and CED-3 results in the activation of the key cell death protease CED-3, which is tightly controlled by multiple positive and negative regulators. The activation of the CED-3 caspase then initiates the cell disassembly process by cleaving and activating or inactivating crucial CED-3 substrates; leading to activation of multiple cell death execution events, including nuclear DNA fragmentation, mitochondrial elimination, phosphatidylserine externalization, inactivation of survival signals, and clearance of apoptotic cells. Further studies of programmed cell death in C. elegans will continue to advance our understanding of how programmed cell death is regulated, activated, and executed in general. PMID:27516615

[Monitoring of implementation of international programs of poliomyelitis eradication and measles and rubella elimination in the Republic of Belarus].

PubMed

2012-01-01

Monitoring of implementation of international programs of poliomyelitis eradication, and measles and rubella elimination in the Republic of Belarus based on results of molecular-epidemiologic studies of 2009 - 2010. 271 viral agents isolated from children with acute flaccid paralysis syndrome, other diseases, healthy children and from sewage water within the framework of poliomyelitis control implementation were identified by serological and molecular methods. Blood sera of 528 patients with fever and rash were examined for the presence of IgM to measles and rubella virus, 418 - for the presence of IgM to parvovirus B19 and parvovirus DNA. Blood sera of 33 pregnant women and 64 children with signs of intrauterine infection were studied for IgM and IgG antibodies to rubella virus. Measles virus was isolated, N-gene sequence and phylogenetic analysis carried out. The studies performed confirmed that indigenous wild polioviruses in the country do not circulate, imported wild or vaccine-related polioviruses were also not detected. Measles and rubella morbidity in the Republic of Belarus was less than 1 in 1 000 000. 2 cases of rubella (2009) and 1 case of measles (2010) was detected during adequate control level: the rate of detection of patients with fever and rash, in whom measles and rubella diagnosis was excluded by the results of laboratory examination, was more than 2 in 100 000 of the population. The etiologic agent in more than 20% of diseases with fever and rash was parvovirus B19. A single case of measles was caused by genotype D8 virus imported from India. The data obtained give evidence to conformance of the poliomyelitis, measles, rubella, innate rubella syndrome control implemented in the Republic of Belarus to WHO recommendations; maintenance of status of country as free from poliomyelitis and achievement of main criteria of elimination of both measles and rubella by 2010.

Notification: Audit of EPA’s Eliminated Programs

EPA Pesticide Factsheets

Project #OA-FY16-0181, June 2, 2016. The EPA OIG plans to begin the preliminary research of the EPA's eliminated programs identified in the agency’s 2015 and 2016 budget submissions for congressional justification as well as plans for future years.

Triazole-linked DNA as a primer surrogate in the synthesis of first-strand cDNA.

PubMed

Fujino, Tomoko; Yasumoto, Ken-ichi; Yamazaki, Naomi; Hasome, Ai; Sogawa, Kazuhiro; Isobe, Hiroyuki

2011-11-04

A phosphate-eliminated nonnatural oligonucleotide serves as a primer surrogate in reverse transcription reaction of mRNA. Despite of the nonnatural triazole linkages in the surrogate, the reverse transcriptase effectively elongated cDNA sequences on the 3'-downstream of the primer by transcription of the complementary sequence of mRNA. A structure-activity comparison with the reference natural oligonucleotides shows the superior priming activity of the surrogate containing triazole-linkages. The nonnatural linkages also protect the transcribed cDNA from digestion reactions with 5'-exonuclease and enable us to remove noise transcripts of unknown origins. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Introduction of translation stop codons into the viral glycoprotein gene in a fish DNA vaccine eliminates induction of protective immunity

USGS Publications Warehouse

Garver, K.A.; Conway, C.M.; Kurath, G.

2006-01-01

A highly efficacious DNA vaccine against a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), was mutated to introduce two stop codons to prevent glycoprotein translation while maintaining the plasmid DNA integrity and RNA transcription ability. The mutated plasmid vaccine, denoted pIHNw-G2stop, when injected intramuscularly into fish at high doses, lacked detectable glycoprotein expression in the injection site muscle, and did not provide protection against lethal virus challenge 7 days post-vaccination. These results suggest that the G-protein itself is required to stimulate the early protective antiviral response observed after vaccination with the nonmutated parental DNA vaccine. ?? Springer Science+Business Media, Inc. 2006.

Introduction of translation stop condons into the viral glycoprotein gene in a fish DNA vaccine eliminates induction of protective immunity

USGS Publications Warehouse

Garver, Kyle A.; Conway, Carla M.; Kurath, Gael

2006-01-01

A highly efficacious DNA vaccine against a fish rhabdovirus, infectious hematopoietic necrosis virus (IHNV), was mutated to introduce two stop codons to prevent glycoprotein translation while maintaining the plasmid DNA integrity and RNA transcription ability. The mutated plasmid vaccine, denoted pIHNw-G2stop, when injected intramuscularly into fish at high doses, lacked detectable glycoprotein expression in the injection site muscle, and did not provide protection against lethal virus challenge 7 days post-vaccination. These results suggest that the G-protein itself is required to stimulate the early protective antiviral response observed after vaccination with the nonmutated parental DNA vaccine.

Detection of Z DNA binding proteins in tissue culture cells.

PubMed Central

Leith, I R; Hay, R T; Russell, W C

1988-01-01

A gel electrophoresis DNA binding assay to detect Z DNA binding proteins has been developed utilising [32P] labelled poly [d(G-C)] which was converted to the Z form by incubation in 100 microM Co(NH3)6Cl3. The parameters of the assay were established using a Z DNA antibody as a model system and then applied to extracts of Hela and BHK21 cells. Using an anti-Z DNA antibody conditions were established which allowed resolution of antibody-DNA complexes and free DNA in the presence of 100 microM Co(NH3)6Cl3. The inclusion of unlabelled complementary homopolymers eliminated non-specific binding to the labelled Z-DNA probe. Competition experiments demonstrated that the assay was highly specific for double stranded non-B DNA. Application of the technique to extracts of mammalian cells demonstrated that human and hamster cells contain Z-DNA binding proteins; further characterisation by a blotting technique indicated that a 56,000 molecular weight cell protein preferentially binds Z-DNA. Images PMID:3419919

A simple procedure for parallel sequence analysis of both strands of 5'-labeled DNA.

PubMed

Razvi, F; Gargiulo, G; Worcel, A

1983-08-01

Ligation of a 5'-labeled DNA restriction fragment results in a circular DNA molecule carrying the two 32Ps at the reformed restriction site. Double digestions of the circular DNA with the original enzyme and a second restriction enzyme cleavage near the labeled site allows direct chemical sequencing of one 5'-labeled DNA strand. Similar double digestions, using an isoschizomer that cleaves differently at the 32P-labeled site, allows direct sequencing of the now 3'-labeled complementary DNA strand. It is possible to directly sequence both strands of cloned DNA inserts by using the above protocol and a multiple cloning site vector that provides the necessary restriction sites. The simultaneous and parallel visualization of both DNA strands eliminates sequence ambiguities. In addition, the labeled circular molecules are particularly useful for single-hit DNA cleavage studies and DNA footprint analysis. As an example, we show here an analysis of the micrococcal nuclease-induced breaks on the two strands of the somatic 5S RNA gene of Xenopus borealis, which suggests that the enzyme may recognize and cleave small AT-containing palindromes along the DNA helix.

RPA coordinates DNA end resection and prevents formation of DNA hairpins.

PubMed

Chen, Huan; Lisby, Michael; Symington, Lorraine S

2013-05-23

Replication protein A (RPA) is an essential eukaryotic single-stranded DNA binding protein with a central role in DNA metabolism. RPA directly participates in DNA double-strand break repair by stimulating 5'-3' end resection by the Sgs1/BLM helicase and Dna2 endonuclease in vitro. Here we investigated the role of RPA in end resection in vivo, using a heat-inducible degron system that allows rapid conditional depletion of RPA in Saccharomyces cerevisiae. We found that RPA depletion eliminated both the Sgs1-Dna2- and Exo1-dependent extensive resection pathways and synergized with mre11Δ to prevent end resection. The short single-stranded DNA tails formed in the absence of RPA were unstable due to 3' strand loss and the formation of fold-back hairpin structures that required resection initiation and Pol32-dependent DNA synthesis. Thus, RPA is required to generate ssDNA, and also to protect ssDNA from degradation and inappropriate annealing that could lead to genome rearrangements. Copyright © 2013 Elsevier Inc. All rights reserved.

Sperm mitochondria in reproduction: good or bad and where do they go?

PubMed

Luo, Shi-Ming; Schatten, Heide; Sun, Qing-Yuan

2013-11-20

The mitochondrion is the major energy provider to power sperm motility. In mammals, aside from the nuclear genome, mitochondrial DNA (mtDNA) also contributes to oxidative phosphorylation to impact production of ATP by coding 13 polypeptides. However, the role of sperm mitochondria in fertilization and its final fate after fertilization are still controversial. The viewpoints that sperm bearing more mtDNA will have a better fertilizing capability and that sperm mtDNA is actively eliminated during early embryogenesis are widely accepted. However, this may be not true for several mammalian species, including mice and humans. Here, we review the sperm mitochondria and their mtDNA in sperm functions, and the mechanisms of maternal mitochondrial inheritance in mammals. Copyright © 2013. Published by Elsevier Ltd.

Mitochondrial DNA recombination in a free-ranging Australian lizard.

PubMed

Ujvari, Beata; Dowton, Mark; Madsen, Thomas

2007-04-22

Mitochondrial DNA (mtDNA) is the traditional workhorse for reconstructing evolutionary events. The frequent use of mtDNA in such analyses derives from the apparent simplicity of its inheritance: maternal and lacking bi-parental recombination. However, in hybrid zones, the reproductive barriers are often not completely developed, resulting in the breakdown of male mitochondrial elimination mechanisms, leading to leakage of paternal mitochondria and transient heteroplasmy, resulting in an increased possibility of recombination. Despite the widespread occurrence of heteroplasmy and the presence of the molecular machinery necessary for recombination, we know of no documented example of recombination of mtDNA in any terrestrial wild vertebrate population. By sequencing the entire mitochondrial genome (16761bp), we present evidence for mitochondrial recombination in the hybrid zone of two mitochondrial haplotypes in the Australian frillneck lizard (Chlamydosaurus kingii).

Quasi-Optimal Elimination Trees for 2D Grids with Singularities

DOE PAGES

Paszyńska, A.; Paszyński, M.; Jopek, K.; ...

2015-01-01

We consmore » truct quasi-optimal elimination trees for 2D finite element meshes with singularities. These trees minimize the complexity of the solution of the discrete system. The computational cost estimates of the elimination process model the execution of the multifrontal algorithms in serial and in parallel shared-memory executions. Since the meshes considered are a subspace of all possible mesh partitions, we call these minimizers quasi-optimal. We minimize the cost functionals using dynamic programming. Finding these minimizers is more computationally expensive than solving the original algebraic system. Nevertheless, from the insights provided by the analysis of the dynamic programming minima, we propose a heuristic construction of the elimination trees that has cost O N e log ⁡ N e , where N e is the number of elements in the mesh. We show that this heuristic ordering has similar computational cost to the quasi-optimal elimination trees found with dynamic programming and outperforms state-of-the-art alternatives in our numerical experiments.« less

Elimination sequence optimization for SPAR

NASA Technical Reports Server (NTRS)

Hogan, Harry A.

1986-01-01

SPAR is a large-scale computer program for finite element structural analysis. The program allows user specification of the order in which the joints of a structure are to be eliminated since this order can have significant influence over solution performance, in terms of both storage requirements and computer time. An efficient elimination sequence can improve performance by over 50% for some problems. Obtaining such sequences, however, requires the expertise of an experienced user and can take hours of tedious effort to affect. Thus, an automatic elimination sequence optimizer would enhance productivity by reducing the analysts' problem definition time and by lowering computer costs. Two possible methods for automating the elimination sequence specifications were examined. Several algorithms based on the graph theory representations of sparse matrices were studied with mixed results. Significant improvement in the program performance was achieved, but sequencing by an experienced user still yields substantially better results. The initial results provide encouraging evidence that the potential benefits of such an automatic sequencer would be well worth the effort.

Quasi-Optimal Elimination Trees for 2D Grids with Singularities

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

Paszyńska, A.; Paszyński, M.; Jopek, K.

We consmore » truct quasi-optimal elimination trees for 2D finite element meshes with singularities. These trees minimize the complexity of the solution of the discrete system. The computational cost estimates of the elimination process model the execution of the multifrontal algorithms in serial and in parallel shared-memory executions. Since the meshes considered are a subspace of all possible mesh partitions, we call these minimizers quasi-optimal. We minimize the cost functionals using dynamic programming. Finding these minimizers is more computationally expensive than solving the original algebraic system. Nevertheless, from the insights provided by the analysis of the dynamic programming minima, we propose a heuristic construction of the elimination trees that has cost O N e log ⁡ N e , where N e is the number of elements in the mesh. We show that this heuristic ordering has similar computational cost to the quasi-optimal elimination trees found with dynamic programming and outperforms state-of-the-art alternatives in our numerical experiments.« less

Eliminating hepatitis B by antagonizing cellular inhibitors of apoptosis.

PubMed

Ebert, Gregor; Allison, Cody; Preston, Simon; Cooney, James; Toe, Jesse G; Stutz, Michael D; Ojaimi, Samar; Baschuk, Nikola; Nachbur, Ueli; Torresi, Joseph; Silke, John; Begley, C Glenn; Pellegrini, Marc

2015-05-05

We have shown that cellular inhibitor of apoptosis proteins (cIAPs) impair clearance of hepatitis B virus (HBV) infection by preventing TNF-mediated killing/death of infected cells. A key question, with profound therapeutic implications, is whether this finding can be translated to the development of drugs that promote elimination of infected cells. Drug inhibitors of cIAPs were developed as cancer therapeutics to promote TNF-mediated tumor killing. These drugs are also known as Smac mimetics, because they mimic the action of the endogenous protein Smac/Diablo that antagonizes cIAP function. Here, we show using an immunocompetent mouse model of chronic HBV infection that birinapant and other Smac mimetics are able to rapidly reduce serum HBV DNA and serum HBV surface antigen, and they promote the elimination of hepatocytes containing HBV core antigen. The efficacy of Smac mimetics in treating HBV infection is dependent on their chemistry, host CD4(+) T cells, and TNF. Birinapant enhances the ability of entecavir, an antiviral nucleoside analog, to reduce viral DNA production in HBV-infected animals. These results indicate that birinapant and other Smac mimetics may have efficacy in treating HBV infection and perhaps, other intracellular infections.

Redefining Molecular Amphipathicity in Reversing the "Coffee-Ring Effect": Implications for Single Base Mutation Detection.

PubMed

Huang, Chi; Wang, Jie; Lv, Xiaobo; Liu, Liu; Liang, Ling; Hu, Wei; Luo, Changliang; Wang, Fubing; Yuan, Quan

2018-05-21

The "coffee ring effect" is a natural phenomenon where sessile drops leave ring-shaped structures on solid surfaces upon drying. It drives non-uniform deposition of suspended compounds on substrates, which adversely affects many processes, including surface-assisted biosensing and molecular self-assembly. In this study, we describe how the coffee ring effect can be eliminated by controlling the amphipathicity of the suspended compounds, for example DNA modified with hydrophobic dye. Specifically, nuclease digestion of the hydrophilic DNA end converts the dye-labeled molecule into an amphipathic molecule (one with comparably weighted hydrophobic and hydrophilic ends) and reverses the coffee ring effect and results in uniform disc-shaped feature deposition of the dye. The amphipathic product decreases the surface tension of the sessile drops and induces Marangoni flow, which drives the uniform distribution of the amphipathic dye-labeled product in the drops. As proof-of-concept, this strategy was used in a novel enzymatic amplification method for biosensing to eliminate the coffee ring effect on a nitrocellulose membrane and increase assay reliability and sensitivity. Importantly, the reported strategy for eliminating the coffee ring effect can be extended to other sessile drop systems for potentially improving assay reliability, and sensitivity.

"Diamond in the Rough": The Impact of a Remedial Program on College Access and Opportunity for Black Males at an Historically Black Institution

ERIC Educational Resources Information Center

Palmer, Robert T.; Davis, Ryan J.

2012-01-01

Researchers, policymakers, and administrations have shown great concern over the efficacy of college remediation, which has prompted some states to eliminate remedial programs from public 4-year institutions. However, research suggests that eliminating these programs may have unintended consequences on college access and opportunity for…

StructAlign, a Program for Alignment of Structures of DNA-Protein Complexes.

PubMed

Popov, Ya V; Galitsyna, A A; Alexeevski, A V; Karyagina, A S; Spirin, S A

2015-11-01

Comparative analysis of structures of complexes of homologous proteins with DNA is important in the analysis of DNA-protein recognition. Alignment is a necessary stage of the analysis. An alignment is a matching of amino acid residues and nucleotides of one complex to residues and nucleotides of the other. Currently, there are no programs available for aligning structures of DNA-protein complexes. We present the program StructAlign, which should fill this gap. The program inputs a pair of complexes of DNA double helix with proteins and outputs an alignment of DNA chains corresponding to the best spatial fit of the protein chains.

Frequent Statement and Dereference Elimination for Imperative and Object-Oriented Distributed Programs

PubMed Central

El-Zawawy, Mohamed A.

2014-01-01

This paper introduces new approaches for the analysis of frequent statement and dereference elimination for imperative and object-oriented distributed programs running on parallel machines equipped with hierarchical memories. The paper uses languages whose address spaces are globally partitioned. Distributed programs allow defining data layout and threads writing to and reading from other thread memories. Three type systems (for imperative distributed programs) are the tools of the proposed techniques. The first type system defines for every program point a set of calculated (ready) statements and memory accesses. The second type system uses an enriched version of types of the first type system and determines which of the ready statements and memory accesses are used later in the program. The third type system uses the information gather so far to eliminate unnecessary statement computations and memory accesses (the analysis of frequent statement and dereference elimination). Extensions to these type systems are also presented to cover object-oriented distributed programs. Two advantages of our work over related work are the following. The hierarchical style of concurrent parallel computers is similar to the memory model used in this paper. In our approach, each analysis result is assigned a type derivation (serves as a correctness proof). PMID:24892098

Recent progress in DNA origami technology.

PubMed

Endo, Masayuki; Sugiyama, Hiroshi

2011-06-01

DNA origami is an emerging technology for designing defined two-dimensional DNA nanostructures. In this review, we focus on and describe several types of DNA origami-related studies, as follows: (1) programmed DNA origami assembly, (2) DNA origami-templated molecular assembly, (3) design and construction of various three-dimensional DNA origami structures, (4) programmed functionalization of DNA origami and combination with top-down nanotechnology, (5) single molecular observation on a designed DNA origami, and (6) DNA nanomachines working on a DNA origami. © 2011 by John Wiley & Sons, Inc.

DNA Damage Repair Genes Controlling Human Papillomavirus (HPV) Episome Levels under Conditions of Stability and Extreme Instability

PubMed Central

Edwards, Terri G.; Vidmar, Thomas J.; Koeller, Kevin; Bashkin, James K.; Fisher, Chris

2013-01-01

DNA damage response (DDR) genes and pathways controlling the stability of HPV episomal DNA are reported here. We set out to understand the mechanism by which a DNA-binding, N-methylpyrrole-imidazole hairpin polyamide (PA25) acts to cause the dramatic loss of HPV DNA from cells. Southern blots revealed that PA25 alters HPV episomes within 5 hours of treatment. Gene expression arrays identified numerous DDR genes that were specifically altered in HPV16 episome-containing cells (W12E) by PA25, but not in HPV-negative (C33A) cells or in cells with integrated HPV16 (SiHa). A siRNA screen of 240 DDR genes was then conducted to identify enhancers and repressors of PA25 activity. Serendipitously, the screen also identified many novel genes, such as TDP1 and TDP2, regulating normal HPV episome stability. MRN and 9-1-1 complexes emerged as important for PA25-mediated episome destruction and were selected for follow-up studies. Mre11, along with other homologous recombination and dsDNA break repair genes, was among the highly significant PA25 repressors. The Mre11 inhibitor Mirin was found to sensitize HPV episomes to PA25 resulting in a ∼5-fold reduction of the PA25 IC50. A novel assay that couples end-labeling of DNA to Q-PCR showed that PA25 causes strand breaks within HPV DNA, and that Mirin greatly enhances this activity. The 9-1-1 complex member Rad9, a representative PA25 enhancer, was transiently phosphorylated in response to PA25 treatment suggesting that it has a role in detecting and signaling episome damage by PA25 to the cell. These results establish that DNA-targeted compounds enter cells and specifically target the HPV episome. This action leads to the activation of numerous DDR pathways and the massive elimination of episomal DNA from cells. Our findings demonstrate that viral episomes can be targeted for elimination from cells by minor groove binding agents, and implicate DDR pathways as important mediators of this process. PMID:24098381

Potentiometric Aptasensing of Vibrio alginolyticus Based on DNA Nanostructure-Modified Magnetic Beads.

PubMed

Zhao, Guangtao; Ding, Jiawang; Yu, Han; Yin, Tanji; Qin, Wei

2016-12-02

A potentiometric aptasensing assay that couples the DNA nanostructure-modified magnetic beads with a solid-contact polycation-sensitive membrane electrode for the detection of Vibrio alginolyticus is herein described. The DNA nanostructure-modified magnetic beads are used for amplification of the potential response and elimination of the interfering effect from a complex sample matrix. The solid-contact polycation-sensitive membrane electrode using protamine as an indicator is employed to chronopotentiometrically detect the change in the charge or DNA concentration on the magnetic beads, which is induced by the interaction between Vibrio alginolyticus and the aptamer on the DNA nanostructures. The present potentiometric aptasensing method shows a linear range of 10-100 CFU mL -1 with a detection limit of 10 CFU mL -1 , and a good specificity for the detection of Vibrio alginolyticus . This proposed strategy can be used for the detection of other microorganisms by changing the aptamers in the DNA nanostructures.

Method of preparing and applying single stranded DNA probes to double stranded target DNAs in situ

DOEpatents

Gray, J.W.; Pinkel, D.

1991-07-02

A method is provided for producing single stranded non-self-complementary nucleic acid probes, and for treating target DNA for use therewith. The probe is constructed by treating DNA with a restriction enzyme and an exonuclease to form template/primers for a DNA polymerase. The digested strand is resynthesized in the presence of labeled nucleoside triphosphate precursor. Labeled single stranded fragments are separated from the resynthesized fragments to form the probe. Target DNA is treated with the same restriction enzyme used to construct the probe, and is treated with an exonuclease before application of the probe. The method significantly increases the efficiency and specificity of hybridization mixtures by increasing effective probe concentration by eliminating self-hybridization between both probe and target DNAs, and by reducing the amount of target DNA available for mismatched hybridizations. No Drawings

Development of Three PCR Assays for the Differentiation between Echinococcus shiquicus, E. granulosus (G1 genotype), and E. multilocularis DNA in the Co-Endemic Region of Qinghai-Tibet plateau, China

PubMed Central

Boufana, Belgees; Umhang, Gérald; Qiu, Jiamin; Chen, Xingwang; Lahmar, Samia; Boué, Franck; Jenkins, David; Craig, Philip

2013-01-01

To investigate echinococcosis in co-endemic regions, three polymerase chain reaction (PCR) assays based on the amplification of a fragment within the NADH dehydrogenase subunit 1 (ND1) mitochondrial gene were optimized for the detection of Echinococcus shiquicus, Echinococcus granulosus G1, and Echinococcus multilocularis DNA derived from parasite tissue or canid fecal samples. Specificity using parasite tissue-derived DNA was found to be 100% except for E. shiquicus primers that faintly detected E. equinus DNA. Sensitivity of the three assays for DNA detection was between 2 and 10 pg. Ethanol precipitation of negative PCR fecal samples was used to eliminate false negatives and served to increase sensitivity as exemplified by an increase in detection from 0% to 89% of E. shiquicus coproDNA using necropsy-positive fox samples. PMID:23438764

Preparation and biomedical applications of programmable and multifunctional DNA nanoflowers

PubMed Central

Lv, Yifan; Hu, Rong; Zhu, Guizhi; Zhang, Xiaobing; Mei, Lei; Liu, Qiaoling; Qiu, Liping; Wu, Cuichen; Tan, Weihong

2016-01-01

We describe a comprehensive protocol for the preparation of multifunctional DNA nanostructures termed nanoflowers (NFs), which are self-assembled from long DNA building blocks generated via rolling-circle replication (RCR) of a designed template. NF assembly is driven by liquid crystallization and dense packaging of building blocks, which eliminates the need for conventional Watson-Crick base pairing. As a result of dense DNA packaging, NFs are resistant to nuclease degradation, denaturation or dissociation at extremely low concentrations. By manually changing the template sequence, many different functional moieties including aptamers, bioimaging agents and drug-loading sites could be easily integrated into NF particles, making NFs ideal candidates for a variety of applications in biomedicine. In this protocol, the preparation of multifunctional DNA NFs with highly tunable sizes is described for applications in cell targeting, intracellular imaging and drug delivery. Preparation and characterization of functional DNA NFs takes ~5 d; the following biomedical applications take ~10 d. PMID:26357007

Product differentiation by analysis of DNA melting curves during the polymerase chain reaction.

PubMed

Ririe, K M; Rasmussen, R P; Wittwer, C T

1997-02-15

A microvolume fluorometer integrated with a thermal cycler was used to acquire DNA melting curves during polymerase chain reaction by fluorescence monitoring of the double-stranded DNA specific dye SYBR Green I. Plotting fluorescence as a function of temperature as the thermal cycler heats through the dissociation temperature of the product gives a DNA melting curve. The shape and position of this DNA melting curve are functions of the GC/AT ratio, length, and sequence and can be used to differentiate amplification products separated by less than 2 degrees C in melting temperature. Desired products can be distinguished from undesirable products, in many cases eliminating the need for gel electrophoresis. Analysis of melting curves can extend the dynamic range of initial template quantification when amplification is monitored with double-stranded DNA specific dyes. Complete amplification and analysis of products can be performed in less than 15 min.

A Novel Low Energy Electron Microscope for DNA Sequencing and Surface Analysis

PubMed Central

Mankos, M.; Shadman, K.; Persson, H.H.J.; N’Diaye, A.T.; Schmid, A.K.; Davis, R.W.

2014-01-01

Monochromatic, aberration-corrected, dual-beam low energy electron microscopy (MAD-LEEM) is a novel technique that is directed towards imaging nanostructures and surfaces with sub-nanometer resolution. The technique combines a monochromator, a mirror aberration corrector, an energy filter, and dual beam illumination in a single instrument. The monochromator reduces the energy spread of the illuminating electron beam, which significantly improves spectroscopic and spatial resolution. Simulation results predict that the novel aberration corrector design will eliminate the second rank chromatic and third and fifth order spherical aberrations, thereby improving the resolution into the sub-nanometer regime at landing energies as low as one hundred electron-Volts. The energy filter produces a beam that can extract detailed information about the chemical composition and local electronic states of non-periodic objects such as nanoparticles, interfaces, defects, and macromolecules. The dual flood illumination eliminates charging effects that are generated when a conventional LEEM is used to image insulating specimens. A potential application for MAD-LEEM is in DNA sequencing, which requires high resolution to distinguish the individual bases and high speed to reduce the cost. The MAD-LEEM approach images the DNA with low electron impact energies, which provides nucleobase contrast mechanisms without organometallic labels. Furthermore, the micron-size field of view when combined with imaging on the fly provides long read lengths, thereby reducing the demand on assembling the sequence. Experimental results from bulk specimens with immobilized single-base oligonucleotides demonstrate that base specific contrast is available with reflected, photo-emitted, and Auger electrons. Image contrast simulations of model rectangular features mimicking the individual nucleotides in a DNA strand have been developed to translate measurements of contrast on bulk DNA to the detectability of individual DNA bases in a sequence. PMID:24524867

A novel low energy electron microscope for DNA sequencing and surface analysis.

PubMed

Mankos, M; Shadman, K; Persson, H H J; N'Diaye, A T; Schmid, A K; Davis, R W

2014-10-01

Monochromatic, aberration-corrected, dual-beam low energy electron microscopy (MAD-LEEM) is a novel technique that is directed towards imaging nanostructures and surfaces with sub-nanometer resolution. The technique combines a monochromator, a mirror aberration corrector, an energy filter, and dual beam illumination in a single instrument. The monochromator reduces the energy spread of the illuminating electron beam, which significantly improves spectroscopic and spatial resolution. Simulation results predict that the novel aberration corrector design will eliminate the second rank chromatic and third and fifth order spherical aberrations, thereby improving the resolution into the sub-nanometer regime at landing energies as low as one hundred electron-Volts. The energy filter produces a beam that can extract detailed information about the chemical composition and local electronic states of non-periodic objects such as nanoparticles, interfaces, defects, and macromolecules. The dual flood illumination eliminates charging effects that are generated when a conventional LEEM is used to image insulating specimens. A potential application for MAD-LEEM is in DNA sequencing, which requires high resolution to distinguish the individual bases and high speed to reduce the cost. The MAD-LEEM approach images the DNA with low electron impact energies, which provides nucleobase contrast mechanisms without organometallic labels. Furthermore, the micron-size field of view when combined with imaging on the fly provides long read lengths, thereby reducing the demand on assembling the sequence. Experimental results from bulk specimens with immobilized single-base oligonucleotides demonstrate that base specific contrast is available with reflected, photo-emitted, and Auger electrons. Image contrast simulations of model rectangular features mimicking the individual nucleotides in a DNA strand have been developed to translate measurements of contrast on bulk DNA to the detectability of individual DNA bases in a sequence. Copyright © 2014 Elsevier B.V. All rights reserved.

A novel low energy electron microscope for DNA sequencing and surface analysis

DOE PAGES

Mankos, M.; Shadman, K.; Persson, H. H. J.; ...

2014-01-31

Monochromatic, aberration-corrected, dual-beam low energy electron microscopy (MAD-LEEM) is a novel technique that is directed towards imaging nanostructures and surfaces with sub-nanometer resolution. The technique combines a monochromator, a mirror aberration corrector, an energy filter, and dual beam illumination in a single instrument. The monochromator reduces the energy spread of the illuminating electron beam, which significantly improves spectroscopic and spatial resolution. Simulation results predict that the novel aberration corrector design will eliminate the second rank chromatic and third and fifth order spherical aberrations, thereby improving the resolution into the sub-nanometer regime at landing energies as low as one hundred electron-Volts.more » The energy filter produces a beam that can extract detailed information about the chemical composition and local electronic states of non-periodic objects such as nanoparticles, interfaces, defects, and macromolecules. The dual flood illumination eliminates charging effects that are generated when a conventional LEEM is used to image insulating specimens. A potential application for MAD-LEEM is in DNA sequencing, which requires high resolution to distinguish the individual bases and high speed to reduce the cost. The MAD-LEEM approach images the DNA with low electron impact energies, which provides nucleobase contrast mechanisms without organometallic labels. Furthermore, the micron-size field of view when combined with imaging on the fly provides long read lengths, thereby reducing the demand on assembling the sequence. Finally, experimental results from bulk specimens with immobilized single-base oligonucleotides demonstrate that base specific contrast is available with reflected, photo-emitted, and Auger electrons. Image contrast simulations of model rectangular features mimicking the individual nucleotides in a DNA strand have been developed to translate measurements of contrast on bulk DNA to the detectability of individual DNA bases in a sequence.« less

MyD88/CD40 Genetic Adjuvant Function in Cutaneous Atypical Antigen-Presenting Cells Contributes to DNA Vaccine Immunogenicity

PubMed Central

Slawin, Kevin M.; Levitt, Jonathan M.; Spencer, David M.

2016-01-01

Therapeutic DNA-based vaccines aim to prime an adaptive host immune response against tumor-associated antigens, eliminating cancer cells primarily through CD8+ cytotoxic T cell-mediated destruction. To be optimally effective, immunological adjuvants are required for the activation of tumor-specific CD8+ T cells responses by DNA vaccination. Here, we describe enhanced anti-tumor efficacy of an in vivo electroporation-delivered DNA vaccine by inclusion of a genetically encoded chimeric MyD88/CD40 (MC) adjuvant, which integrates both innate and adaptive immune signaling pathways. When incorporated into a DNA vaccine, signaling by the MC adjuvant increased antigen-specific CD8+ T cells and promoted elimination of pre-established tumors. Interestingly, MC-enhanced vaccine efficacy did not require direct-expression of either antigen or adjuvant by local antigen-presenting cells, but rather our data supports a key role for MC function in “atypical” antigen-presenting cells of skin. In particular, MC adjuvant-modified keratinocytes increased inflammatory cytokine secretion, upregulated surface MHC class I, and were able to increase in vitro and in vivo priming of antigen-specific CD8+ T cells. Furthermore, in the absence of critical CD8α+/CD103+ cross-priming dendritic cells, MC was still able to promote immune priming in vivo, albeit at a reduced level. Altogether, our data support a mechanism by which MC signaling activates an inflammatory phenotype in atypical antigen-presenting cells within the cutaneous vaccination site, leading to an enhanced CD8+ T cell response against DNA vaccine-encoded antigens, through both CD8α+/CD103+ dendritic cell-dependent and independent pathways. PMID:27741278

Powering the programmed nanostructure and function of gold nanoparticles with catenated DNA machines

NASA Astrophysics Data System (ADS)

Elbaz, Johann; Cecconello, Alessandro; Fan, Zhiyuan; Govorov, Alexander O.; Willner, Itamar

2013-06-01

DNA nanotechnology is a rapidly developing research area in nanoscience. It includes the development of DNA machines, tailoring of DNA nanostructures, application of DNA nanostructures for computing, and more. Different DNA machines were reported in the past and DNA-guided assembly of nanoparticles represents an active research effort in DNA nanotechnology. Several DNA-dictated nanoparticle structures were reported, including a tetrahedron, a triangle or linear nanoengineered nanoparticle structures; however, the programmed, dynamic reversible switching of nanoparticle structures and, particularly, the dictated switchable functions emerging from the nanostructures, are missing elements in DNA nanotechnology. Here we introduce DNA catenane systems (interlocked DNA rings) as molecular DNA machines for the programmed, reversible and switchable arrangement of different-sized gold nanoparticles. We further demonstrate that the machine-powered gold nanoparticle structures reveal unique emerging switchable spectroscopic features, such as plasmonic coupling or surface-enhanced fluorescence.

78 FR 13339 - State Program Requirements; Approval of Maine's National Pollutant Discharge Elimination System...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-02-27

... NPDES program under the Clean Water Act in the state, including the territories of the Aroostook Band of... Elimination System (NPDES) permitting program in the state under the Clean Water Act (CWA). 33 U.S.C. 1251, et... federal Clean Water Act. Second, in Aroostook Band of Micmacs v. Ryan, 484 F.3d 41 (2007) the court held...

Induction and repair of DNA double-strand breaks in rat cerebellar cortex exposed to 60Co γ-rays

NASA Astrophysics Data System (ADS)

Bulanova, T. S.; Zadneprianetc, M. G.; Ježková, L.; Kruglyakova, E. A.; Smirnova, E. V.; Boreyko, A. V.

2018-01-01

The induction and repair of DNA double-strand breaks are studied using the immunohistochemical staining procedure of paraffin-embedded rat cerebellum tissues after exposure to γ-rays of 60Co. The dose dependence of radiation-induced colocalized γH2AX/53BP1 foci is studied and its linear character is established. It is shown that these foci are efficiently eliminated 24 h after irradiation.

Introduction to the Natural Anticipator and the Artificial Anticipator

NASA Astrophysics Data System (ADS)

Dubois, Daniel M.

2010-11-01

This short communication deals with the introduction of the concept of anticipator, which is one who anticipates, in the framework of computing anticipatory systems. The definition of anticipation deals with the concept of program. Indeed, the word program, comes from "pro-gram" meaning "to write before" by anticipation, and means a plan for the programming of a mechanism, or a sequence of coded instructions that can be inserted into a mechanism, or a sequence of coded instructions, as genes or behavioural responses, that is part of an organism. Any natural or artificial programs are thus related to anticipatory rewriting systems, as shown in this paper. All the cells in the body, and the neurons in the brain, are programmed by the anticipatory genetic code, DNA, in a low-level language with four signs. The programs in computers are also computing anticipatory systems. It will be shown, at one hand, that the genetic code DNA is a natural anticipator. As demonstrated by Nobel laureate McClintock [8], genomes are programmed. The fundamental program deals with the DNA genetic code. The properties of the DNA consist in self-replication and self-modification. The self-replicating process leads to reproduction of the species, while the self-modifying process leads to new species or evolution and adaptation in existing ones. The genetic code DNA keeps its instructions in memory in the DNA coding molecule. The genetic code DNA is a rewriting system, from DNA coding to DNA template molecule. The DNA template molecule is a rewriting system to the Messenger RNA molecule. The information is not destroyed during the execution of the rewriting program. On the other hand, it will be demonstrated that Turing machine is an artificial anticipator. The Turing machine is a rewriting system. The head reads and writes, modifying the content of the tape. The information is destroyed during the execution of the program. This is an irreversible process. The input data are lost.

Automated genomic DNA purification options in agricultural applications using MagneSil paramagnetic particles

NASA Astrophysics Data System (ADS)

Bitner, Rex M.; Koller, Susan C.

2002-06-01

The automated high throughput purification of genomic DNA form plant materials can be performed using MagneSil paramagnetic particles on the Beckman-Coulter FX, BioMek 2000, and the Tecan Genesis robot. Similar automated methods are available for DNA purifications from animal blood. These methods eliminate organic extractions, lengthy incubations and cumbersome filter plates. The DNA is suitable for applications such as PCR and RAPD analysis. Methods are described for processing traditionally difficult samples such as those containing large amounts of polyphenolics or oils, while still maintaining a high level of DNA purity. The robotic protocols have ben optimized for agricultural applications such as marker assisted breeding, seed-quality testing, and SNP discovery and scoring. In addition to high yield purification of DNA from plant samples or animal blood, the use of Promega's DNA-IQ purification system is also described. This method allows for the purification of a narrow range of DNA regardless of the amount of additional DNA that is present in the initial sample. This simultaneous Isolation and Quantification of DNA allows the DNA to be used directly in applications such as PCR, SNP analysis, and RAPD, without the need for separate quantitation of the DNA.

Generation of Gene-Engineered Chimeric DNA Molecules for Specific Therapy of Autoimmune Diseases

PubMed Central

Gesheva, Vera; Szekeres, Zsuzsanna; Mihaylova, Nikolina; Dimitrova, Iliyana; Nikolova, Maria; Erdei, Anna; Prechl, Jozsef

2012-01-01

Abstract Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the development of self-reactive B and T cells and autoantibody production. In particular, double-stranded DNA-specific B cells play an important role in lupus progression, and their selective elimination is a reasonable approach for effective therapy of SLE. DNA-based vaccines aim at the induction of immune response against the vector-encoded antigen. Here, we are exploring, as a new DNA-based therapy of SLE, a chimeric DNA molecule encoding a DNA-mimotope peptide, and the Fv but not the immunogenic Fc fragment of an FcγRIIb-specific monoclonal antibody. This DNA construct was inserted in the expression vector pNut and used as a naked DNA vaccine in a mouse model of lupus. The chimeric DNA molecule can be expressed in eukaryotic cells and cross-links cell surface receptors on DNA-specific B cells, delivering an inhibitory intracellular signal. Intramuscular administration of the recombinant DNA molecule to lupus-prone MRL/lpr mice prevented increase in IgG anti-DNA antibodies and was associated with a low degree of proteinuria, modulation of cytokine profile, and suppression of lupus nephritis. PMID:23075110

Impedimetric DNA biosensor based on a nanoporous alumina membrane for the detection of the specific oligonucleotide sequence of dengue virus.

PubMed

Deng, Jiajia; Toh, Chee-Seng

2013-06-17

A novel and integrated membrane sensing platform for DNA detection is developed based on an anodic aluminum oxide (AAO) membrane. Platinum electrodes (~50-100 nm thick) are coated directly on both sides of the alumina membrane to eliminate the solution resistance outside the nanopores. The electrochemical impedance technique is employed to monitor the impedance changes within the nanopores upon DNA binding. Pore resistance (Rp) linearly increases in response towards the increasing concentration of the target DNA in the range of 1 × 10⁻¹² to 1 × 10⁻⁶ M. Moreover, the biosensor selectively differentiates the complementary sequence from single base mismatched (MM-1) strands and non-complementary strands. This study reveals a simple, selective and sensitive method to fabricate a label-free DNA biosensor.

Elimination of HIV-1 Genomes from Human T-lymphoid Cells by CRISPR/Cas9 Gene Editing

PubMed Central

Kaminski, Rafal; Chen, Yilan; Fischer, Tracy; Tedaldi, Ellen; Napoli, Alessandro; Zhang, Yonggang; Karn, Jonathan; Hu, Wenhui; Khalili, Kamel

2016-01-01

We employed an RNA-guided CRISPR/Cas9 DNA editing system to precisely remove the entire HIV-1 genome spanning between 5′ and 3′ LTRs of integrated HIV-1 proviral DNA copies from latently infected human CD4+ T-cells. Comprehensive assessment of whole-genome sequencing of HIV-1 eradicated cells ruled out any off-target effects by our CRISPR/Cas9 technology that might compromise the integrity of the host genome and further showed no effect on several cell health indices including viability, cell cycle and apoptosis. Persistent co-expression of Cas9 and the specific targeting guide RNAs in HIV-1-eradicated T-cells protected them against new infection by HIV-1. Lentivirus-delivered CRISPR/Cas9 significantly diminished HIV-1 replication in infected primary CD4+ T-cell cultures and drastically reduced viral load in ex vivo culture of CD4+ T-cells obtained from HIV-1 infected patients. Thus, gene editing using CRISPR/Cas9 may provide a new therapeutic path for eliminating HIV-1 DNA from CD4+ T-cells and potentially serve as a novel and effective platform toward curing AIDS. PMID:26939770

Using hazard maps to identify and eliminate workplace hazards: a union-led health and safety training program.

PubMed

Anderson, Joe; Collins, Michele; Devlin, John; Renner, Paul

2012-01-01

The Institute for Sustainable Work and Environment and the Utility Workers Union of America worked with a professional evaluator to design, implement, and evaluate the results of a union-led system of safety-based hazard identification program that trained workers to use hazard maps to identify workplace hazards and target them for elimination. The evaluation documented program implementation and impact using data collected from both qualitative interviews and an on-line survey from worker trainers, plant managers, and health and safety staff. Managers and workers reported that not only were many dangerous hazards eliminated as a result of hazard mapping, some of which were long-standing, difficult-to-resolve issues, but the evaluation also documented improved communication between union members and management that both workers and managers agreed resulted in better, more sustainable hazard elimination.

Study on a Luminol-based Electrochemiluminescent Sensor for Label-Free DNA Sensing

PubMed Central

Chu, Hai-Hong; Yan, Ji-Lin; Tu, Yi-Feng

2010-01-01

Automatic, inexpensive, simple and sensitive methods for DNA sensing and quantification are highly desirable for biomedical research. The rapid development of both the fundamentals and applications of electrochemiluminescence (ECL) over the past years has demonstrated its potential for analytical and bio-analytical chemistry. This paper reports the quenching effect of DNA on the ECL of luminol and the further development of a DNA sensing device. With the pre-functionalization by a composite of carbon nano-tubes (CNTs) and Au nanoparticles (AuNPs), the sensor provides a novel and valuable label-free approach for DNA sensing. Here the ECL intensity was remarkably decreased when more than 1.0 × 10−12 molar of DNA were adsorbed on the sensor. Linearity of the DNA amount with the reciprocal of ECL intensity was observed. A saturated sensor caused a 92.8% quenching effect. The research also proposes the mechanism for the quenching effect which could be attributed to the interaction between luminol and DNA and the elimination of reactive oxygen species (ROSs) by DNA. PMID:22163421

Integrated digital error suppression for improved detection of circulating tumor DNA

PubMed Central

Kurtz, David M.; Chabon, Jacob J.; Scherer, Florian; Stehr, Henning; Liu, Chih Long; Bratman, Scott V.; Say, Carmen; Zhou, Li; Carter, Justin N.; West, Robert B.; Sledge, George W.; Shrager, Joseph B.; Loo, Billy W.; Neal, Joel W.; Wakelee, Heather A.; Diehn, Maximilian; Alizadeh, Ash A.

2016-01-01

High-throughput sequencing of circulating tumor DNA (ctDNA) promises to facilitate personalized cancer therapy. However, low quantities of cell-free DNA (cfDNA) in the blood and sequencing artifacts currently limit analytical sensitivity. To overcome these limitations, we introduce an approach for integrated digital error suppression (iDES). Our method combines in silico elimination of highly stereotypical background artifacts with a molecular barcoding strategy for the efficient recovery of cfDNA molecules. Individually, these two methods each improve the sensitivity of cancer personalized profiling by deep sequencing (CAPP-Seq) by ~3 fold, and synergize when combined to yield ~15-fold improvements. As a result, iDES-enhanced CAPP-Seq facilitates noninvasive variant detection across hundreds of kilobases. Applied to clinical non-small cell lung cancer (NSCLC) samples, our method enabled biopsy-free profiling of EGFR kinase domain mutations with 92% sensitivity and 96% specificity and detection of ctDNA down to 4 in 105 cfDNA molecules. We anticipate that iDES will aid the noninvasive genotyping and detection of ctDNA in research and clinical settings. PMID:27018799

RPA Stabilization of Single-Stranded DNA Is Critical for Break-Induced Replication.

PubMed

Ruff, Patrick; Donnianni, Roberto A; Glancy, Eleanor; Oh, Julyun; Symington, Lorraine S

2016-12-20

DNA double-strand breaks (DSBs) are cytotoxic lesions that must be accurately repaired to maintain genome stability. Replication protein A (RPA) plays an important role in homology-dependent repair of DSBs by protecting the single-stranded DNA (ssDNA) intermediates formed by end resection and by facilitating Rad51 loading. We found that hypomorphic mutants of RFA1 that support intra-chromosomal homologous recombination are profoundly defective for repair processes involving long tracts of DNA synthesis, in particular break-induced replication (BIR). The BIR defects of the rfa1 mutants could be partially suppressed by eliminating the Sgs1-Dna2 resection pathway, suggesting that Dna2 nuclease attacks the ssDNA formed during end resection when not fully protected by RPA. Overexpression of Rad51 was also found to suppress the rfa1 BIR defects. We suggest that Rad51 binding to the ssDNA formed by excessive end resection and during D-loop migration can partially compensate for dysfunctional RPA. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Should mandatory energy reporting be eliminated

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

Kendall, R.

1981-08-01

Energy users report problems with the Industrial Reporting Program (IRP), a joint voluntary energy-efficiency program which began in 1975. Since energy-intensive industries were already organizing voluntary reporting, the new program required unnecessary expenditures. The Reagan administration's plan to eliminate the program by not funding it is opposed by many energy managers who want the data collected to help them with their management programs. Their interest is primarily in a data base because energy costs provide adequate incentives to conserve. They also want interaction (preferably voluntary) between industry and goverment on efficiency-improvement opportunities. (DCK)

Specific interaction of the nonstructural protein NS1 of minute virus of mice (MVM) with [ACCA](2) motifs in the centre of the right-end MVM DNA palindrome induces hairpin-primed viral DNA replication.

PubMed

Willwand, Kurt; Moroianu, Adela; Hörlein, Rita; Stremmel, Wolfgang; Rommelaere, Jean

2002-07-01

The linear single-stranded DNA genome of minute virus of mice (MVM) is replicated via a double-stranded replicative form (RF) intermediate DNA. Amplification of viral RF DNA requires the structural transition of the right-end palindrome from a linear duplex into a double-hairpin structure, which serves for the repriming of unidirectional DNA synthesis. This conformational transition was found previously to be induced by the MVM nonstructural protein NS1. Elimination of the cognate NS1-binding sites, [ACCA](2), from the central region of the right-end palindrome next to the axis of symmetry was shown to markedly reduce the efficiency of hairpin-primed DNA replication, as measured in a reconstituted in vitro replication system. Thus, [ACCA](2) sequence motifs are essential as NS1-binding elements in the context of the structural transition of the right-end MVM palindrome.

[Real-time PCR kits for the detection of the African Swine Fever virus].

PubMed

Latyshev, O E; Eliseeva, O V; Grebennikova, T V; Verkhovskiĭ, O A; Tsibezov, V V; Chernykh, O Iu; Dzhailidi, G A; Aliper, T I

2014-01-01

The results obtained using the diagnostic kit based on real-time polymerase chain reaction to detect the DNA of the African Swine Fever in the pathological material, as well as in the culture fluid, are presented. A high sensitivity and specificity for detection of the DNA in the organs and tissues of animals was shown to be useful for detection in the European Union referentiality reagent kits for DNA detection by real time PCR of ASFV. More rapid and effective method of DNA extraction using columns mini spin Quick gDNA(TM) MiniPrep was suggested and compared to the method of DNA isolation on the inorganic sorbent. High correlation of the results of the DNA detection of ASFV by real-time PCR and antigen detection results ASFV by competitive ELISA obtained with the ELISA SEROTEST/INGEZIM COMRAC PPA was demonstrated. The kit can be used in the veterinary services for effective monitoring of ASFV to contain, eliminate and prevent further spread of the disease.

Structure and mechanism of the UvrA-UvrB DNA damage sensor

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

Pakotiprapha, Danaya; Samuels, Martin; Shen, Koning

2012-04-17

Nucleotide excision repair (NER) is used by all organisms to eliminate DNA lesions. We determined the structure of the Geobacillus stearothermophilus UvrA-UvrB complex, the damage-sensor in bacterial NER and a new structure of UvrA. We observe that the DNA binding surface of UvrA, previously found in an open shape that binds damaged DNA, also exists in a closed groove shape compatible with native DNA only. The sensor contains two UvrB molecules that flank the UvrA dimer along the predicted path for DNA, ~80 Å from the lesion. We show that the conserved signature domain II of UvrA mediates a nexusmore » of contacts among UvrA, UvrB and DNA. Further, in our new structure of UvrA, this domain adopts an altered conformation while an adjacent nucleotide binding site is vacant. Our findings raise unanticipated questions about NER and also suggest a revised picture of its early stages.« less

Multiple ways to prevent transmission of paternal mitochondrial DNA for maternal inheritance in animals.

PubMed

Sato, Ken; Sato, Miyuki

2017-10-01

Mitochondria contain their own DNA (mtDNA). In most sexually reproducing organisms, mtDNA is inherited maternally (uniparentally); this type of inheritance is thus referred to as 'maternal (uniparental) inheritance'. Recent studies have revealed various mechanisms to prevent the transmission of sperm-derived paternal mtDNA to the offspring, thereby ensuring maternal inheritance of mtDNA. In the nematode Caenorhabditis elegans, paternal mitochondria and their mtDNA degenerate almost immediately after fertilization and are selectively degraded by autophagy, which is referred to as 'allophagy' (allogeneic [non-self] organelle autophagy). In the fruit fly Drosophila melanogaster, paternal mtDNA is largely eliminated by an endonuclease G-mediated mechanism. Paternal mitochondria are subsequently removed by endocytic and autophagic pathways after fertilization. In many mammals, including humans, paternal mitochondria enter fertilized eggs. However, the fate of paternal mitochondria and their mtDNA in mammals is still a matter of debate. In this review, we will summarize recent knowledge on the molecular mechanisms underlying the prevention of paternal mtDNA transmission, which ensures maternal mtDNA inheritance in animals. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Extraction of DNA from forensic-type sexual assault specimens using simple, rapid sonication procedures.

PubMed

Crouse, C A; Ban, J D; D'Alessio, J K

1993-10-01

Sonication procedures for the extraction of DNA from forensic-type semen specimens have been developed, which, when compared to currently utilized sperm DNA extraction techniques, are simple, rapid and result in comparable DNA yields. Sperm DNA extraction by sonication was performed on whole semen, seminal stains, buccal swabs and post-coital specimens. Ultrasound disruption of sperm cells and their ultimate release of cellular DNA has been conducted in the presence of sperm wash buffers followed by organic extraction or Chelex 100 with little or no compromise to DNA quality, quantity or amplifiability. Two advantages of sonication over currently used forensic techniques to extract sperm DNA include 1) sperm DNA extraction that occurs within five minutes of sonication compared with an hour or greater for water bath incubations in classic enzyme digestion DNA extractions and 2) one less preparatory step with the Chelex/sonication protocol and three less steps with the sonication/organic protocol compared with other procedures thus eliminating potential sample-to-sample cross-contamination. Sperm DNA extracted by optimum sonication procedures was used for forensic HLA DQ alpha typing and restriction fragment length polymorphisms analysis without any adverse effects on typing results.

Cell-surface glycosaminoglycans inhibit intranuclear uptake but promote post-nuclear processes of polyamidoamine dendrimer-pDNA transfection.

PubMed

Ziraksaz, Zarrintaj; Nomani, Alireza; Ruponen, Marika; Soleimani, Masoud; Tabbakhian, Majid; Haririan, Ismaeil

2013-01-23

Interaction of cell-surface glycosaminoglycans (GAGs) with non-viral vectors seems to be an important factor which modifies the intracellular destination of the gene complexes. Intracellular kinetics of polyamidoamine (PAMAM) dendrimer as a non-viral vector in cellular uptake, intranuclear delivery and transgene expression of plasmid DNA with regard to the cell-surface GAGs has not been investigated until now. The physicochemical properties of the PAMAM-pDNA complexes were characterized by photon correlation spectroscopy, atomic force microscopy, zeta measurement and agarose gel electrophoresis. The transfection efficiency and toxicity of the complexes at different nitrogen to phosphate (N:P) ratios were determined using various in vitro cell models such as human embryonic kidney cells, chinese hamster ovary cells and its mutants lacking cell-surface GAGs or heparan sulphate proteoglycans (HSPGs). Cellular uptake, nuclear uptake and transfection efficiency of the complexes were determined using flow cytometry and optimized cell-nuclei isolation with quantitative real-time PCR and luciferase assay. Physicochemical studies showed that PAMAM dendrimer binds pDNA efficiently, forms small complexes with high positive zeta potential and transfects cells properly at N:P ratios around 5 and higher. The cytotoxicity could be a problem at N:Ps higher than 10. GAGs elimination caused nearly one order of magnitude higher pDNA nuclear uptake and more than 2.6-fold higher transfection efficiency than CHO parent cells. However, neither AUC of nuclear uptake, nor AUC of transfection affected significantly by only cell-surface HSPGs elimination and interesting data related to the effect of GAGs on intranuclear pDNA using PAMAM as delivery vector have been reported in this study. Presented data shows that the rate-limiting step of PAMAM-pDNA complexes transfection is located after delivery to the cell nucleus and GAGs are regarded as an inhibitor of the intranuclear delivery step, while slightly promotes transgene expression. Copyright © 2012 Elsevier B.V. All rights reserved.

The Paramecium germline genome provides a niche for intragenic parasitic DNA: evolutionary dynamics of internal eliminated sequences.

PubMed

Arnaiz, Olivier; Mathy, Nathalie; Baudry, Céline; Malinsky, Sophie; Aury, Jean-Marc; Denby Wilkes, Cyril; Garnier, Olivier; Labadie, Karine; Lauderdale, Benjamin E; Le Mouël, Anne; Marmignon, Antoine; Nowacki, Mariusz; Poulain, Julie; Prajer, Malgorzata; Wincker, Patrick; Meyer, Eric; Duharcourt, Sandra; Duret, Laurent; Bétermier, Mireille; Sperling, Linda

2012-01-01

Insertions of parasitic DNA within coding sequences are usually deleterious and are generally counter-selected during evolution. Thanks to nuclear dimorphism, ciliates provide unique models to study the fate of such insertions. Their germline genome undergoes extensive rearrangements during development of a new somatic macronucleus from the germline micronucleus following sexual events. In Paramecium, these rearrangements include precise excision of unique-copy Internal Eliminated Sequences (IES) from the somatic DNA, requiring the activity of a domesticated piggyBac transposase, PiggyMac. We have sequenced Paramecium tetraurelia germline DNA, establishing a genome-wide catalogue of -45,000 IESs, in order to gain insight into their evolutionary origin and excision mechanism. We obtained direct evidence that PiggyMac is required for excision of all IESs. Homology with known P. tetraurelia Tc1/mariner transposons, described here, indicates that at least a fraction of IESs derive from these elements. Most IES insertions occurred before a recent whole-genome duplication that preceded diversification of the P. aurelia species complex, but IES invasion of the Paramecium genome appears to be an ongoing process. Once inserted, IESs decay rapidly by accumulation of deletions and point substitutions. Over 90% of the IESs are shorter than 150 bp and present a remarkable size distribution with a -10 bp periodicity, corresponding to the helical repeat of double-stranded DNA and suggesting DNA loop formation during assembly of a transpososome-like excision complex. IESs are equally frequent within and between coding sequences; however, excision is not 100% efficient and there is selective pressure against IES insertions, in particular within highly expressed genes. We discuss the possibility that ancient domestication of a piggyBac transposase favored subsequent propagation of transposons throughout the germline by allowing insertions in coding sequences, a fraction of the genome in which parasitic DNA is not usually tolerated.

The Paramecium Germline Genome Provides a Niche for Intragenic Parasitic DNA: Evolutionary Dynamics of Internal Eliminated Sequences

PubMed Central

Arnaiz, Olivier; Mathy, Nathalie; Baudry, Céline; Malinsky, Sophie; Aury, Jean-Marc; Denby Wilkes, Cyril; Garnier, Olivier; Labadie, Karine; Lauderdale, Benjamin E.; Le Mouël, Anne; Marmignon, Antoine; Nowacki, Mariusz; Poulain, Julie; Prajer, Malgorzata; Wincker, Patrick; Meyer, Eric; Duharcourt, Sandra; Duret, Laurent; Bétermier, Mireille; Sperling, Linda

2012-01-01

Insertions of parasitic DNA within coding sequences are usually deleterious and are generally counter-selected during evolution. Thanks to nuclear dimorphism, ciliates provide unique models to study the fate of such insertions. Their germline genome undergoes extensive rearrangements during development of a new somatic macronucleus from the germline micronucleus following sexual events. In Paramecium, these rearrangements include precise excision of unique-copy Internal Eliminated Sequences (IES) from the somatic DNA, requiring the activity of a domesticated piggyBac transposase, PiggyMac. We have sequenced Paramecium tetraurelia germline DNA, establishing a genome-wide catalogue of ∼45,000 IESs, in order to gain insight into their evolutionary origin and excision mechanism. We obtained direct evidence that PiggyMac is required for excision of all IESs. Homology with known P. tetraurelia Tc1/mariner transposons, described here, indicates that at least a fraction of IESs derive from these elements. Most IES insertions occurred before a recent whole-genome duplication that preceded diversification of the P. aurelia species complex, but IES invasion of the Paramecium genome appears to be an ongoing process. Once inserted, IESs decay rapidly by accumulation of deletions and point substitutions. Over 90% of the IESs are shorter than 150 bp and present a remarkable size distribution with a ∼10 bp periodicity, corresponding to the helical repeat of double-stranded DNA and suggesting DNA loop formation during assembly of a transpososome-like excision complex. IESs are equally frequent within and between coding sequences; however, excision is not 100% efficient and there is selective pressure against IES insertions, in particular within highly expressed genes. We discuss the possibility that ancient domestication of a piggyBac transposase favored subsequent propagation of transposons throughout the germline by allowing insertions in coding sequences, a fraction of the genome in which parasitic DNA is not usually tolerated. PMID:23071448

A DNA sequence analysis package for the IBM personal computer.

PubMed Central

Lagrimini, L M; Brentano, S T; Donelson, J E

1984-01-01

We present here a collection of DNA sequence analysis programs, called "PC Sequence" (PCS), which are designed to run on the IBM Personal Computer (PC). These programs are written in IBM PC compiled BASIC and take full advantage of the IBM PC's speed, error handling, and graphics capabilities. For a modest initial expense in hardware any laboratory can use these programs to quickly perform computer analysis on DNA sequences. They are written with the novice user in mind and require very little training or previous experience with computers. Also provided are a text editing program for creating and modifying DNA sequence files and a communications program which enables the PC to communicate with and collect information from mainframe computers and DNA sequence databases. PMID:6546433

The Economics of a Successful Raccoon Rabies Elimination Program on Long Island, New York

PubMed Central

Elser, Julie L.; Bigler, Laura L.; Anderson, Aaron M.; Maki, Joanne L.; Lein, Donald H.; Shwiff, Stephanie A.

2016-01-01

Raccoon rabies is endemic in the eastern U.S.; however, an epizootic had not been confirmed on Long Island, New York until 2004. An oral rabies vaccination (ORV) program was initiated soon after the first rabies-positive raccoon was discovered, and continued until raccoon rabies was eliminated from the vaccination zone. The cost-effectiveness and economic impact of this rabies control program were unknown. A public health surveillance data set was evaluated following the ORV program on Long Island, and is used here as a case study in the health economics of rabies prevention and control efforts. A benefit-cost analysis was performed to determine the cost-effectiveness of the program, and a regional economic model was used to estimate the macroeconomic impacts of raccoon rabies elimination to New York State. The cost of the program, approximately $2.6 million, was recovered within eight years by reducing costs associated with post-exposure prophylaxis (PEP) and veterinary diagnostic testing of rabies suspect animals. By 2019, the State of New York is projected to benefit from the ORV program by almost $27 million. The benefit-cost ratio will reach 1.71 in 2019, meaning that for every dollar spent on the program $1.71 will be saved. Regional economic modeling estimated employment growth of over 100 jobs and a Gross Domestic Product (GDP) increase of $9.2 million through 2019. This analysis suggests that baiting to eliminate rabies in a geographically constrained area can provide positive economic returns. PMID:27935946

The Economics of a Successful Raccoon Rabies Elimination Program on Long Island, New York.

PubMed

Elser, Julie L; Bigler, Laura L; Anderson, Aaron M; Maki, Joanne L; Lein, Donald H; Shwiff, Stephanie A

2016-12-01

Raccoon rabies is endemic in the eastern U.S.; however, an epizootic had not been confirmed on Long Island, New York until 2004. An oral rabies vaccination (ORV) program was initiated soon after the first rabies-positive raccoon was discovered, and continued until raccoon rabies was eliminated from the vaccination zone. The cost-effectiveness and economic impact of this rabies control program were unknown. A public health surveillance data set was evaluated following the ORV program on Long Island, and is used here as a case study in the health economics of rabies prevention and control efforts. A benefit-cost analysis was performed to determine the cost-effectiveness of the program, and a regional economic model was used to estimate the macroeconomic impacts of raccoon rabies elimination to New York State. The cost of the program, approximately $2.6 million, was recovered within eight years by reducing costs associated with post-exposure prophylaxis (PEP) and veterinary diagnostic testing of rabies suspect animals. By 2019, the State of New York is projected to benefit from the ORV program by almost $27 million. The benefit-cost ratio will reach 1.71 in 2019, meaning that for every dollar spent on the program $1.71 will be saved. Regional economic modeling estimated employment growth of over 100 jobs and a Gross Domestic Product (GDP) increase of $9.2 million through 2019. This analysis suggests that baiting to eliminate rabies in a geographically constrained area can provide positive economic returns.

Purification of Single-Stranded cDNA Based on RNA Degradation Treatment and Adsorption Chromatography.

PubMed

Trujillo-Esquivel, Elías; Franco, Bernardo; Flores-Martínez, Alberto; Ponce-Noyola, Patricia; Mora-Montes, Héctor M

2016-08-02

Analysis of gene expression is a common research tool to study networks controlling gene expression, the role of genes with unknown function, and environmentally induced responses of organisms. Most of the analytical tools used to analyze gene expression rely on accurate cDNA synthesis and quantification to obtain reproducible and quantifiable results. Thus far, most commercial kits for isolation and purification of cDNA target double-stranded molecules, which do not accurately represent the abundance of transcripts. In the present report, we provide a simple and fast method to purify single-stranded cDNA, exhibiting high purity and yield. This method is based on the treatment with RNase H and RNase A after cDNA synthesis, followed by separation in silica spin-columns and ethanol precipitation. In addition, our method avoids the use of DNase I to eliminate genomic DNA from RNA preparations, which improves cDNA yield. As a case report, our method proved to be useful in the purification of single-stranded cDNA from the pathogenic fungus Sporothrix schenckii.

Improved methods of DNA extraction from human spermatozoa that mitigate experimentally-induced oxidative DNA damage.

PubMed

Xavier, Miguel J; Nixon, Brett; Roman, Shaun D; Aitken, Robert John

2018-01-01

Current approaches for DNA extraction and fragmentation from mammalian spermatozoa provide several challenges for the investigation of the oxidative stress burden carried in the genome of male gametes. Indeed, the potential introduction of oxidative DNA damage induced by reactive oxygen species, reducing agents (dithiothreitol or beta-mercaptoethanol), and DNA shearing techniques used in the preparation of samples for chromatin immunoprecipitation and next-generation sequencing serve to cofound the reliability and accuracy of the results obtained. Here we report optimised methodology that minimises, or completely eliminates, exposure to DNA damaging compounds during extraction and fragmentation procedures. Specifically, we show that Micrococcal nuclease (MNase) digestion prior to cellular lysis generates a greater DNA yield with minimal collateral oxidation while randomly fragmenting the entire paternal genome. This modified methodology represents a significant improvement over traditional fragmentation achieved via sonication in the preparation of genomic DNA from human spermatozoa for downstream applications, such as next-generation sequencing. We also present a redesigned bioinformatic pipeline framework adjusted to correctly analyse this form of data and detect statistically relevant targets of oxidation.

Reversible Hydrolysis Reaction with the Spore Photoproduct under Alkaline Conditions.

PubMed

Adhikari, Surya; Lin, Gengjie; Li, Lei

2016-09-16

DNA lesions may reduce the electron density at the nucleobases, making them prone to further modifications upon the alkaline treatment. The dominant DNA photolesion found in UV-irradiated bacterial endospores is a thymine dimer, 5-thyminyl-5,6-dihydrothymine, i.e., the spore photoproduct (SP). Here we report a stepwise addition/elimination reaction in the SP hydrolysis product under strong basic conditions where a ureido group is added to the carboxyl moiety to form a cyclic amide, regenerating SP after eliminating a hydroxide ion. Direct amidation of carboxylic acids by reaction with amines in the presence of a catalyst is well documented; however, it is very rare for an amidation reaction to occur without activation. This uncatalyzed SP reverse reaction in aqueous solution is even more surprising because the carboxyl moiety is not a good electrophile due to the negative charge it carries. Examination of the base-catalyzed hydrolyses of two other saturated pyrimidine lesions, 5,6-dihydro-2'-deoxyuridine and pyrimidine (6-4) pyrimidone photoproduct, reveals that neither reaction is reversible even though all three hydrolysis reactions may share the same gem-diol intermediate. Therefore, the SP structure where the two thymine residues maintain a stacked conformation likely provides the needed framework enabling this highly unusual carboxyl addition/elimination reaction.

The price of performance: a cost and performance analysis of the implementation of cell-free fetal DNA testing for Down syndrome in Ontario, Canada.

PubMed

Okun, N; Teitelbaum, M; Huang, T; Dewa, C S; Hoch, J S

2014-04-01

To examine the cost and performance implications of introducing cell-free fetal DNA (cffDNA) testing within modeled scenarios in a publicly funded Canadian provincial Down syndrome (DS) prenatal screening program. Two clinical algorithms were created: the first to represent the current screening program and the second to represent one that incorporates cffDNA testing. From these algorithms, eight distinct scenarios were modeled to examine: (1) the current program (no cffDNA), (2) the current program with first trimester screening (FTS) as the nuchal translucency-based primary screen (no cffDNA), (3) a program substituting current screening with primary cffDNA, (4) contingent cffDNA with current FTS performance, (5) contingent cffDNA at a fixed price to result in overall cost neutrality,(6) contingent cffDNA with an improved detection rate (DR) of FTS, (7) contingent cffDNA with higher uptake of FTS, and (8) contingent cffDNA with optimized FTS (higher uptake and improved DR). This modeling study demonstrates that introducing contingent cffDNA testing improves performance by increasing the number of cases of DS detected prenatally, and reducing the number of amniocenteses performed and concomitant iatrogenic pregnancy loss of pregnancies not affected by DS. Costs are modestly increased, although the cost per case of DS detected is decreased with contingent cffDNA testing. Contingent models of cffDNA testing can improve overall screening performance while maintaining the provision of an 11- to 13-week scan. Costs are modestly increased, but cost per prenatally detected case of DS is decreased. © 2013 John Wiley & Sons, Ltd.

Modification of gDNA extraction from soil for PCR designed for the routine examination of soil samples contaminated with Toxocara spp. eggs.

PubMed

Borecka, A; Gawor, J

2008-06-01

A modification of gDNA extraction was developed for the polymerase chain reaction (PCR) technique, intended for the detection and differentiation of Toxocara spp. eggs in soil or sediments. Sand samples from sandpits confirmed as being contaminated with Toxocara spp. eggs by the flotation technique were analysed by PCR. The use of proteinase K made it possible to obtain genomic DNA from the sample without needing to isolate eggs using flotation or to inactivate PCR inhibitors present in the sand. Specific primers in the PCR reaction allowed discrimination between T. canis and T. cati eggs. The modification simplified the procedure, thanks to eliminating the step of gDNA isolation from eggs, which is both laborious and difficult.

Affinity of yeast nucleotide excision repair factor 2, consisting of the Rad4 and Rad23 proteins, for ultraviolet damaged DNA.

PubMed

Guzder, S N; Sung, P; Prakash, L; Prakash, S

1998-11-20

Saccharomyces cerevisiae Rad4 and Rad23 proteins are required for the nucleotide excision repair of UV light-damaged DNA. Previous studies have indicated that these two DNA repair proteins are associated in a tight complex, which we refer to as nucleotide excision repair factor 2 (NEF2). In a reconstituted nucleotide excision repair reaction, incision of UV-damaged DNA is dependent on NEF2, indicating a role of NEF2 in an early step of the repair process. NEF2 does not, however, possess an enzymatic activity, and its function in the damage-specific incision reaction has not yet been defined. Here we use a DNA mobility shift assay to demonstrate that NEF2 binds specifically to UV-damaged DNA. Elimination of cyclobutane pyrimidine dimers from the UV-damaged DNA by enzymatic photoreactivation has little effect on the affinity of NEF2 for the DNA, suggesting that NEF2 recognizes the 6-(1, 2)-dihydro-2-oxo-4-pyrimidinyl)-5-methyl-2,4-(1H,3H)-pyrimidinedione photoproducts in the damaged DNA. These results highlight the intricacy of the DNA damage-demarcation reaction during nucleotide excision repair in eukaryotes.

EVIDENCE FOR BASE EXCISION REPAIR PROCESSING OF DNA INTERSTRAND CROSSLINKS

PubMed Central

Kothandapani, Anbarasi; Patrick, Steve M

2013-01-01

Many bifunctional alkylating agents and anticancer drugs exert their cytotoxicity by producing cross links between the two complementary strands of DNA, termed interstrand crosslinks (ICLs). This blocks the strand separating processes during DNA replication and transcription, which can lead to cell cycle arrest and apoptosis. Cells use multiple DNA repair systems to eliminate the ICLs. Concerted action of repair proteins involved in Nucleotide Excision Repair and Homologous Recombination pathways are suggested to play a key role in the ICL repair. However, recent studies indicate a possible role for Base Excision Repair (BER) in mediating the cytotoxicity of ICL inducing agents in mammalian cells. Elucidating the mechanism of BER mediated modulation of ICL repair would help in understanding the recognition and removal of ICLs and aid in the development of potential therapeutic agents. In this review, the influence of BER proteins on ICL DNA repair and the possible mechanisms of action are discussed. PMID:23219605

Improving efficiency and reliability of environmental DNA analysis for silver carp

USGS Publications Warehouse

Amberg, Jon J.; McCalla, S. Grace; Monroe, Emy; Lance, Richard; Baerwaldt, Kelly; Gaikowski, Mark P.

2015-01-01

Natural resource agencies have established surveillance programs which use environmental DNA (eDNA) for the early detection of bighead carp Hypophthalmichthys nobilis and silver carp Hypophthalmichthys molitrix before they establish populations within the Great Lakes. This molecular monitoring technique must be highly accurate and precise for confident interpretation and also efficient, both in detection threshold and cost. Therefore, we compared two DNA extraction techniques and compared a new quantitative PCR (qPCR) assay with the conventional PCR (cPCR) assay used by monitoring programs. Both the qPCR and cPCR assays were able to amplify the DNA of silver carp present in environmental samples taken from locations where mixed populations of bigheaded carps existed. However, the qPCR assay had substantially fewer PCR positive samples which were subsequently determined not to contain DNA of bigheaded carps than the cPCR assay. Additionally, the qPCR assay was able to amplify the DNA of bigheaded carps even in the presence of inhibitors that blocked amplification with cPCR. Also, the selection of an appropriate DNA extraction method can significantly alter the efficiency of eDNA surveillance programs by lowering detection limits and by decreasing costs associated with sample processing. The results reported herein are presently being incorporated into eDNA surveillance programs to decrease the costs, increase DNA yield and increase the confidence that assays are amplifying the target DNA. These results are critical to enhancing our ability to accurately and confidently interpret the results reported from monitoring programs using eDNA for early detection of invasive species.

Schistosomiasis elimination strategies and potential role of a vaccine in achieving global health goals.

PubMed

Mo, Annie X; Agosti, Jan M; Walson, Judd L; Hall, B Fenton; Gordon, Lance

2014-01-01

In March 2013, the National Institute of Allergy and Infectious Diseases and the Bill and Melinda Gates Foundation co-sponsored a meeting entitled "Schistosomiasis Elimination Strategy and Potential Role of a Vaccine in Achieving Global Health Goals" to discuss the potential role of schistosomiasis vaccines and other tools in the context of schistosomiasis control and elimination strategies. It was concluded that although schistosomiasis elimination in some focal areas may be achievable through current mass drug administration programs, global control and elimination will face several significant scientific and operational challenges, and will require an integrated approach with other, additional interventions. These challenges include vector (snail) control; environmental modification; water, sanitation, and hygiene; and other future innovative tools such as vaccines. Defining a clear product development plan that reflects a vaccine strategy as complementary to the existing control programs to combat different forms of schistosomiasis will be important to develop a vaccine effectively.

Potentiometric Aptasensing of Vibrio alginolyticus Based on DNA Nanostructure-Modified Magnetic Beads

PubMed Central

Zhao, Guangtao; Ding, Jiawang; Yu, Han; Yin, Tanji; Qin, Wei

2016-01-01

A potentiometric aptasensing assay that couples the DNA nanostructure-modified magnetic beads with a solid-contact polycation-sensitive membrane electrode for the detection of Vibrio alginolyticus is herein described. The DNA nanostructure-modified magnetic beads are used for amplification of the potential response and elimination of the interfering effect from a complex sample matrix. The solid-contact polycation-sensitive membrane electrode using protamine as an indicator is employed to chronopotentiometrically detect the change in the charge or DNA concentration on the magnetic beads, which is induced by the interaction between Vibrio alginolyticus and the aptamer on the DNA nanostructures. The present potentiometric aptasensing method shows a linear range of 10–100 CFU mL−1 with a detection limit of 10 CFU mL−1, and a good specificity for the detection of Vibrio alginolyticus. This proposed strategy can be used for the detection of other microorganisms by changing the aptamers in the DNA nanostructures. PMID:27918423

Multisubunit DNA-Dependent RNA Polymerases from Vaccinia Virus and Other Nucleocytoplasmic Large-DNA Viruses: Impressions from the Age of Structure.

PubMed

Mirzakhanyan, Yeva; Gershon, Paul D

2017-09-01

The past 17 years have been marked by a revolution in our understanding of cellular multisubunit DNA-dependent RNA polymerases (MSDDRPs) at the structural level. A parallel development over the past 15 years has been the emerging story of the giant viruses, which encode MSDDRPs. Here we link the two in an attempt to understand the specialization of multisubunit RNA polymerases in the domain of life encompassing the large nucleocytoplasmic DNA viruses (NCLDV), a superclade that includes the giant viruses and the biochemically well-characterized poxvirus vaccinia virus. The first half of this review surveys the recently determined structural biology of cellular RNA polymerases for a microbiology readership. The second half discusses a reannotation of MSDDRP subunits from NCLDV families and the apparent specialization of these enzymes by virus family and by subunit with regard to subunit or domain loss, subunit dissociability, endogenous control of polymerase arrest, and the elimination/customization of regulatory interactions that would confer higher-order cellular control. Some themes are apparent in linking subunit function to structure in the viral world: as with cellular RNA polymerases I and III and unlike cellular RNA polymerase II, the viral enzymes seem to opt for speed and processivity and seem to have eliminated domains associated with higher-order regulation. The adoption/loss of viral RNA polymerase proofreading functions may have played a part in matching intrinsic mutability to genome size. Copyright © 2017 American Society for Microbiology.

Environmental Health Assessment for Pyrotechnic Perchlorate Elimination/Mitigation Program for M118/M119 Simulators

DTIC Science & Technology

2009-09-11

evaluated. When toxicity data were not available, criteria were modeled using QSAR approaches. 3 Toxicology Report No. 87-XE-074Z-09C g. Potential...Environmental Health Assessment for Work Unit PYRO 06-08, Pyrotechnic Perchlorate Elimination/Mitigation Program for M118/M119 Simulators, Toxicology ...Directorate of Toxicology , Health Effects Research Program. He may be contacted at DSN (312) 584-7159 or commercial (410) 436-7159. FOR THE COMMANDER

Prospects for elimination of soil-transmitted helminths.

PubMed

Ásbjörnsdóttir, Kristjana H; Means, Arianna R; Werkman, Marleen; Walson, Judd L

2017-10-01

Soil-transmitted helminths (STH) are endemic in 120 countries and are associated with substantial morbidity and loss of economic productivity. Although current WHO guidelines focus on morbidity control through mass drug administration (MDA), there is global interest in whether a strategy targeting disease elimination might be feasible in some settings. This review summarizes the prospects for switching from control to an elimination strategy. STH control efforts have reduced the intensity of infections in targeted populations with associated reductions in morbidity. However, adults are not frequently targeted and remain important reservoirs for reinfection of treated children. Recent modeling suggests that transmission interruption may be possible through expanded community-wide delivery of MDA, the feasibility of which has been demonstrated by other programs. However, these models suggest that high levels of coverage and compliance must be achieved. Potential challenges include the risk of prematurely dismantling STH programs and the potential increased risk of antihelminthic resistance. Elimination of STH may offer an opportunity to eliminate substantial STH-related morbidity while reducing resource needs of neglected tropical disease programs. Evidence from large community trials is needed to determine the feasibility of interrupting the transmission of STH in some geographic settings.

Duck hepatitis B virus covalently closed circular DNA appears to survive hepatocyte mitosis in the growing liver

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

Reaiche-Miller, Georget Y.; Thorpe, Michael; Low, Huey Chi

Nucleos(t)ide analogues that inhibit hepatitis B virus (HBV) DNA replication are typically used as monotherapy for chronically infected patients. Treatment with a nucleos(t)ide analogue eliminates most HBV DNA replication intermediates and produces a gradual decline in levels of covalently closed circular DNA (cccDNA), the template for viral RNA synthesis. It remains uncertain if levels of cccDNA decline primarily through hepatocyte death, or if loss also occurs during hepatocyte mitosis. To determine if cccDNA survives mitosis, growing ducklings infected with duck hepatitis B virus (DHBV) were treated with the nucleoside analogue, Entecavir. Viremia was suppressed at least 10{sup 5}-fold, during amore » period when average liver mass increased 23-fold. Analysis of the data suggested that if cccDNA synthesis was completely inhibited, at least 49% of cccDNA survived hepatocyte mitosis. However, there was a large duck-to-duck variation in cccDNA levels, suggesting that low level cccDNA synthesis may contribute to this apparent survival through mitosis. - Highlights: • The hepatitis B virus nuclear template is covalently closed circular DNA (cccDNA). • cccDNA was studied during liver growth in duck hepatitis B virus infected ducks. • Virus DNA replication and new cccDNA synthesis were inhibited with Entecavir. • At least 49% of cccDNA appeared to survive hepatocyte mitosis. • Low level virus DNA synthesis may contribute to survival of cccDNA through mitosis.« less

[Plan to improve malaria control towards its elimination in Mesoamerica].

PubMed

Rodríguez, Mario Henry; Betanzos-Reyes, Angel Francisco

2011-01-01

To develop a plan to strengthen the control of malaria towards its elimination. In 2009, under the coordination of the National Public HealthInstitute ofMexico, atransdisciplinary equipment of technical and operative experts was conformed to carry out a situational analysis of malaria and control programs and for the selection of effective practices of intervention that would be incorporated to the plan, within the framework of an exercise in Theory of Change. Criteria for thestratificationof thelocalities, based ontheirtransmission characteristics were established. The structural and operative limitations of the control programs were identified. A plan of interventions was elaborated to improve the coverage of epidemiological surveillance, anti-malaria interventions and opportune diagnosis and treatment of cases. The plan delineates progressive phases of implementation: reorganization, intensification of interventions and evaluation of elimination feasibility. The adoption of a regional strategic plan will provide guidance and administrative elements to conform a system that coordinates the activities of the national control programs and facilitate the elimination of malaria in the region.

Cost-effectiveness of programs to eliminate disparities in elderly vaccination rates in the United States

PubMed Central

2014-01-01

Background There are disparities in influenza and pneumococcal vaccination rates among elderly minority groups and little guidance as to which intervention or combination of interventions to eliminate these disparities is likely to be most cost-effective. Here, we evaluate the cost-effectiveness of four hypothetical vaccination programs designed to eliminate disparities in elderly vaccination rates and differing in the number of interventions. Methods We developed a Markov model in which we assumed a healthcare system perspective, 10-year vaccination program and lifetime time horizon. The cohort was the combined African-American and Hispanic 65 year-old birth cohort in the United States in 2009. We evaluated five different vaccination strategies: no vaccination program and four vaccination programs that varied from “low intensity” to “very high intensity” based on the number of interventions deployed in each program, their cumulative cost and their cumulative impact on elderly minority influenza and pneumococcal vaccination rates. Results The very high intensity vaccination program ($24,479/quality-adjusted life year; QALY) was preferred at willingness-to-pay-thresholds of $50,000 and $100,000/QALY and prevented 37,178 influenza cases, 342 influenza deaths, 1,158 invasive pneumococcal disease (IPD) cases and 174 IPD deaths over the birth cohort’s lifetime. In one-way sensitivity analyses, the very high intensity program only became cost-prohibitive (>$100,000/QALY) at less likely values for the influenza vaccination rates achieved in year 10 of the high intensity (>73.5%) or very high intensity (<76.8%) vaccination programs. Conclusions A practice-based vaccination program designed to eliminate disparities in elderly minority vaccination rates and including four interventions would be cost-effective. PMID:25023889

Cost-effectiveness of programs to eliminate disparities in elderly vaccination rates in the United States.

PubMed

Michaelidis, Constantinos I; Zimmerman, Richard K; Nowalk, Mary Patricia; Smith, Kenneth J

2014-07-15

There are disparities in influenza and pneumococcal vaccination rates among elderly minority groups and little guidance as to which intervention or combination of interventions to eliminate these disparities is likely to be most cost-effective. Here, we evaluate the cost-effectiveness of four hypothetical vaccination programs designed to eliminate disparities in elderly vaccination rates and differing in the number of interventions. We developed a Markov model in which we assumed a healthcare system perspective, 10-year vaccination program and lifetime time horizon. The cohort was the combined African-American and Hispanic 65 year-old birth cohort in the United States in 2009. We evaluated five different vaccination strategies: no vaccination program and four vaccination programs that varied from "low intensity" to "very high intensity" based on the number of interventions deployed in each program, their cumulative cost and their cumulative impact on elderly minority influenza and pneumococcal vaccination rates. The very high intensity vaccination program ($24,479/quality-adjusted life year; QALY) was preferred at willingness-to-pay-thresholds of $50,000 and $100,000/QALY and prevented 37,178 influenza cases, 342 influenza deaths, 1,158 invasive pneumococcal disease (IPD) cases and 174 IPD deaths over the birth cohort's lifetime. In one-way sensitivity analyses, the very high intensity program only became cost-prohibitive (>$100,000/QALY) at less likely values for the influenza vaccination rates achieved in year 10 of the high intensity (>73.5%) or very high intensity (<76.8%) vaccination programs. A practice-based vaccination program designed to eliminate disparities in elderly minority vaccination rates and including four interventions would be cost-effective.

Evaluating Ethanol-based Sample Preservation to Facilitate Use of DNA Barcoding in Routine Freshwater Biomonitoring Programs Using Benthic Macroinvertebrates

EPA Science Inventory

Molecular methods, such as DNA barcoding, have the potential in enhance biomonitoring programs worldwide. Altering routinely used sample preservation methods to protect DNA from degradation may pose a potential impediment to application of DNA barcoding and metagenomics for biom...

Different effects of eubacterial and eukaryotic DNA topoisomerase II inhibitors on chloroplasts ofEuglena gracilis

NASA Astrophysics Data System (ADS)

Krajčovič, Juraj; Ebringer, Libor

1990-03-01

Inhibitors of eubacterial and eukaryotic DNA topoisomerases type II exhibited different effects on chloroplasts of the flagellateEuglena gracilis. Antibacterial agents (cinoxacin, nalidixic and oxolinic acids, ciprofloxacin, enoxacin, norfloxacin and ofloxacin) from the group of quinolones and coumarins (coumermycin A1, clorobiocin and novobiocin) — all inhibitors of prokaryotic DNA topoisomerase II — were very potent eliminators of chloroplasts fromE. gracilis. In contrast, antitumor drugs (adriamycin, etoposide, teniposide and mitoxantrone) — antagonists of the eukaryotic counterpart — did not affect these semiautonomous photosynthetic organelles. These findings point out again the close evolutionary relationships between eubacteria and chloroplasts and are in agreement with the hypothesis of an endosymbiotic origin of chloroplasts.

Rapid communications: antiperspirant induced DNA damage in canine cells by comet assay.

PubMed

Yiu, Gloria

2004-01-01

Abstract Millions of people around the world use antiperspirants to decrease or eliminate body odors. Most antiperspirants contain aluminum zirconium or another form of aluminum as its active ingredient. The present investigation applied Comet assay to detect if Secret Platinum for women, Old Spice for men, or Crystal Natural produced DNA damage in Madin-Darby canine kidney cells (MDCKII). This study has shown that antiperspirants cause DNA damage on a single-cell level. Additionally, our data showed us that in general, Secret Platinum for women and Old Spice for men, produced equivalent damage. Crystal Natural, marketed as being safer or less damaging, induced the most extensive damage of all three antiperspirants tested.

Product analysis illuminates the final steps of IES deletion in Tetrahymena thermophila

PubMed Central

Saveliev, Sergei V.; Cox, Michael M.

2001-01-01

DNA sequences (IES elements) eliminated from the developing macronucleus in the ciliate Tetrahymena thermophila are released as linear fragments, which have now been detected and isolated. A PCR-mediated examination of fragment end structures reveals three types of strand scission events, reflecting three steps in the deletion process. New evidence is provided for two steps proposed previously: an initiating double-stranded cleavage, and strand transfer to create a branched deletion intermediate. The fragment ends provide evidence for a previously uncharacterized third step: the branched DNA strand is cleaved at one of several defined sites located within 15–16 nucleotides of the IES boundary, liberating the deleted DNA in a linear form. PMID:11406601

Product analysis illuminates the final steps of IES deletion in Tetrahymena thermophila.

PubMed

Saveliev, S V; Cox, M M

2001-06-15

DNA sequences (IES elements) eliminated from the developing macronucleus in the ciliate Tetrahymena thermophila are released as linear fragments, which have now been detected and isolated. A PCR-mediated examination of fragment end structures reveals three types of strand scission events, reflecting three steps in the deletion process. New evidence is provided for two steps proposed previously: an initiating double-stranded cleavage, and strand transfer to create a branched deletion intermediate. The fragment ends provide evidence for a previously uncharacterized third step: the branched DNA strand is cleaved at one of several defined sites located within 15-16 nucleotides of the IES boundary, liberating the deleted DNA in a linear form.

Rapid detection of Wuchereria bancrofti and Brugia malayi in mosquito vectors (Diptera: Culicidae) using a real-time fluorescence resonance energy transfer multiplex PCR and melting curve analysis.

PubMed

Intapan, Pewpan M; Thanchomnang, Tongjit; Lulitanond, Viraphong; Maleewong, Wanchai

2009-01-01

We developed a single-step real-time fluorescence resonance energy transfer (FRET) multiplex polymerase chain reaction (PCR) merged with melting curve analysis for the detection of Wuchereria bancrofti and Brugia malayi DNA in blood-fed mosquitoes. Real-time FRET multiplex PCR is based on fluorescence melting curve analysis of a hybrid of amplicons generated from two families of repeated DNA elements: the 188 bp SspI repeated sequence, specific to W. bancrofti, and the 153-bp HhaI repeated sequence, specific to the genus Brugia and two pairs of specific fluorophore-labeled probes. Both W. bancrofti and B. malayi can be differentially detected in infected vectors by this process through their different fluorescence channel and melting temperatures. The assay could distinguish both human filarial DNAs in infected vectors from the DNAs of Dirofilaria immitis- and Plasmodium falciparum-infected human red blood cells and noninfected mosquitoes and human leukocytes. The technique showed 100% sensitivity and specificity and offers a rapid and reliable procedure for differentially identifying lymphatic filariasis. The introduced real-time FRET multiplex PCR can reduce labor time and reagent costs and is not prone to carry over contamination. The test can be used to screen mosquito vectors in endemic areas and therefore should be a useful diagnostic tool for the evaluation of infection rate of the mosquito populations and for xenomonitoring in the community after eradication programs such as the Global Program to Eliminate Lymphatic Filariasis.

Construction of a System for the Strawberry Nursery Production towards Elimination of Latent Infection of Anthracnose Fungi by a Combination of PCR and Microtube Hybridization

PubMed Central

Furuta, Kazuyoshi; Nagashima, Saki; Inukai, Tsuyoshi; Masuta, Chikara

2017-01-01

One of the major problems in strawberry production is difficulty in diagnosis of anthracnose caused by Colletotrichum acutatum or Glomerella cingulata in latent infection stage. We here developed a diagnostic tool for the latent infection consisting of initial culturing of fungi, DNA extraction, synthesis of PCR-amplified probes and microtube hybridization (MTH) using a macroarray. The initial culturing step is convenient to lure the fungi out of the plant tissues, and to extract PCR-inhibitor-free DNA directly from fungal hyphae. For specific detection of the fungi, PCR primers were designed to amplify the fungal MAT1-2 gene. The subsequent MTH step using the PCR products as probes can replace the laborious electrophoresis step providing us sequence information and high-throughput screening. Using this method, we have conducted a survey for a few thousands nursery plants every year for three consecutive years, and finally succeeded in eliminating latent infection in the third year of challenge. PMID:28167891

Intracellular Copper Does Not Catalyze the Formation of Oxidative DNA Damage in Escherichia coli▿

PubMed Central

Macomber, Lee; Rensing, Christopher; Imlay, James A.

2007-01-01

Because copper catalyzes the conversion of H2O2 to hydroxyl radicals in vitro, it has been proposed that oxidative DNA damage may be an important component of copper toxicity. Elimination of the copper export genes, copA, cueO, and cusCFBA, rendered Escherichia coli sensitive to growth inhibition by copper and provided forcing circumstances in which this hypothesis could be tested. When the cells were grown in medium supplemented with copper, the intracellular copper content increased 20-fold. However, the copper-loaded mutants were actually less sensitive to killing by H2O2 than cells grown without copper supplementation. The kinetics of cell death showed that excessive intracellular copper eliminated iron-mediated oxidative killing without contributing a copper-mediated component. Measurements of mutagenesis and quantitative PCR analysis confirmed that copper decreased the rate at which H2O2 damaged DNA. Electron paramagnetic resonance (EPR) spin trapping showed that the copper-dependent H2O2 resistance was not caused by inhibition of the Fenton reaction, for copper-supplemented cells exhibited substantial hydroxyl radical formation. However, copper EPR spectroscopy suggested that the majority of H2O2-oxidizable copper is located in the periplasm; therefore, most of the copper-mediated hydroxyl radical formation occurs in this compartment and away from the DNA. Indeed, while E. coli responds to H2O2 stress by inducing iron sequestration proteins, H2O2-stressed cells do not induce proteins that control copper levels. These observations do not explain how copper suppresses iron-mediated damage. However, it is clear that copper does not catalyze significant oxidative DNA damage in vivo; therefore, copper toxicity must occur by a different mechanism. PMID:17189367

Low-density lipoprotein peptide-combined DNA nanocomplex as an efficient anticancer drug delivery vehicle.

PubMed

Zhang, Nan; Tao, Jun; Hua, Haiying; Sun, Pengchao; Zhao, Yongxing

2015-08-01

DNA is a type of potential biomaterials for drug delivery due to its nanoscale geometry, loading capacity of therapeutics, biocompatibility, and biodegradability. Unfortunately, DNA is easily degraded by DNases in the body circulation and has low intracellular uptake. In the present study, we selected three cationic polymers polyethylenimine (PEI), hexadecyl trimethyl ammonium bromide (CTAB), and low-density lipoprotein (LDL) receptor targeted peptide (RLT), to modify DNA and improve the issues. A potent anti-tumor anthracycline-doxorubicin (DOX) was intercalated into DNA non-covalently and the DOX/DNA was then combined with PEI, CTAB, and RLT, respectively. Compact nanocomplexes were formed by electrostatic interaction and could potentially protect DNA from DNases. More importantly, RLT had the potential to enhance intracellular uptake by LDL receptor mediated endocytosis. In a series of in vitro experiments, RLT complexed DNA enhanced intracellular delivery of DOX, increased tumor cell death and intracellular ROS production, and reduced intracellular elimination of DOX. All results suggested that the easily prepared and targeted RLT/DNA nanocomplexes had great potential to be developed into a formulation for doxorubicin with enhanced anti-tumor activity. Copyright © 2015 Elsevier B.V. All rights reserved.

Tribulations of the Last Mile: Sides from a Regional Program.

PubMed

Del Rio Vilas, Victor J; Freire de Carvalho, Mary J; Vigilato, Marco A N; Rocha, Felipe; Vokaty, Alexandra; Pompei, Julio A; Molina Flores, Baldomero; Fenelon, Natael; Cosivi, Ottorino

2017-01-01

In Latin American and Caribbean (LAC) countries, the number of cases of dog-mediated human rabies is at its lowest since the onset of the Regional Program for Rabies Elimination in 1983, a commitment from LAC countries to eliminate dog-mediated rabies coordinated by the Pan American Health Organization. Despite minor setbacks, the decline in the number of human cases has been constant since 1983. While many LAC countries have significantly reduced rabies to a level where it is no longer significant public health concern, elimination has proven elusive and pockets of the disease remain across the region. In the 33-year period since 1983, the region has set and committed to four dates for elimination (1990, 2000, 2012, and 2015). In this paper, we ponder on the multiple causes behind the elusive goal of rabies elimination, such as blanket regional goals oblivious to the large heterogeneity in national rabies capacities. Looking ahead to the elimination of dog-mediated rabies in the region, now established for 2022, we also review the many challenges and questions that the region faces in the last mile of the epidemic. Given the advanced position of the Americas in the race toward elimination, our considerations could provide valuable knowledge to other regions pursuing elimination goals.

Progress toward elimination of onchocerciasis in the Americas.

PubMed

Sauerbrey, Mauricio; Rakers, Lindsay J; Richards, Frank O

2018-03-01

The Onchocerciasis Elimination Program for the Americas (OEPA) is a regional initiative and international partnership that has made considerable progress toward its goal since it was launched in 1993. Its strategy is based on mass drug administration of ivermectin (Mectizan, donated by MSD, also known as Merck & Co., Inc., Kenilworth, NJ, USA), twice or four times per year, with at least 85% coverage of eligible populations. From 1989 to 2016, 11 741 276 ivermectin treatments have been given in the Americas, eliminating transmission in 11 of 13 foci. The OEPA's success has had a great influence on programs in Africa, especially Sudan and Uganda, which moved from a control to an elimination strategy in 2006 and 2007, respectively. The successes in the Americas have also greatly influenced WHO guidelines for onchocerciasis transmission elimination. With four of the six originally endemic American countries now WHO verified as having eliminated onchocerciasis transmission, and 95% of ivermectin treatments in the region halted, the regional focus is now on the remaining active transmission zone, called the Yanomami Area, on the border between Venezuela and Brazil. Both countries have difficult political climates that hinder the elimination task in this remote and relatively neglected region. As with other elimination efforts, 'the final inch' is often the most difficult task of all.

Tribulations of the Last Mile: Sides from a Regional Program

PubMed Central

Del Rio Vilas, Victor J.; Freire de Carvalho, Mary J.; Vigilato, Marco A. N.; Rocha, Felipe; Vokaty, Alexandra; Pompei, Julio A.; Molina Flores, Baldomero; Fenelon, Natael; Cosivi, Ottorino

2017-01-01

In Latin American and Caribbean (LAC) countries, the number of cases of dog-mediated human rabies is at its lowest since the onset of the Regional Program for Rabies Elimination in 1983, a commitment from LAC countries to eliminate dog-mediated rabies coordinated by the Pan American Health Organization. Despite minor setbacks, the decline in the number of human cases has been constant since 1983. While many LAC countries have significantly reduced rabies to a level where it is no longer significant public health concern, elimination has proven elusive and pockets of the disease remain across the region. In the 33-year period since 1983, the region has set and committed to four dates for elimination (1990, 2000, 2012, and 2015). In this paper, we ponder on the multiple causes behind the elusive goal of rabies elimination, such as blanket regional goals oblivious to the large heterogeneity in national rabies capacities. Looking ahead to the elimination of dog-mediated rabies in the region, now established for 2022, we also review the many challenges and questions that the region faces in the last mile of the epidemic. Given the advanced position of the Americas in the race toward elimination, our considerations could provide valuable knowledge to other regions pursuing elimination goals. PMID:28197407

Additional mitochondrial DNA influences the interactions between the nuclear and mitochondrial genomes in a bovine embryo model of nuclear transfer.

PubMed

Srirattana, Kanokwan; St John, Justin C

2018-05-08

We generated cattle embryos using mitochondrial supplementation and somatic cell nuclear transfer (SCNT), named miNT, to determine how additional mitochondrial DNA (mtDNA) modulates the nuclear genome. To eliminate any confounding effects from somatic cell mtDNA in intraspecies SCNT, donor cell mtDNA was depleted prior to embryo production. Additional oocyte mtDNA did not affect embryo development rates but increased mtDNA copy number in blastocyst stage embryos. Moreover, miNT-derived blastocysts had different gene expression profiles when compared with SCNT-derived blastocysts. Additional mtDNA increased expression levels of genes involved in oxidative phosphorylation, cell cycle and DNA repair. Supplementing the embryo culture media with a histone deacetylase inhibitor, Trichostatin A (TSA), had no beneficial effects on the development of miNT-derived embryos, unlike SCNT-derived embryos. When compared with SCNT-derived blastocysts cultured in the presence of TSA, additional mtDNA alone had beneficial effects as the activity of glycolysis may increase and embryonic cell death may decrease. However, these beneficial effects were not found with additional mtDNA and TSA together, suggesting that additional mtDNA alone enhances reprogramming. In conclusion, additional mtDNA increased mtDNA copy number and expression levels of genes involved in energy production and embryo development in blastocyst stage embryos emphasising the importance of nuclear-mitochondrial interactions.

Treatment of PCR products with exonuclease I and heat-labile alkaline phosphatase improves the visibility of combined bisulfite restriction analysis

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

Watanabe, Kousuke; Emoto, Noriko; Sunohara, Mitsuhiro

2010-08-27

Research highlights: {yields} Incubating PCR products at a high temperature causes smears in gel electrophoresis. {yields} Smears interfere with the interpretation of methylation analysis using COBRA. {yields} Treatment with exonuclease I and heat-labile alkaline phosphatase eliminates smears. {yields} The elimination of smears improves the visibility of COBRA. -- Abstract: DNA methylation plays a vital role in the regulation of gene expression. Abnormal promoter hypermethylation is an important mechanism of inactivating tumor suppressor genes in human cancers. Combined bisulfite restriction analysis (COBRA) is a widely used method for identifying the DNA methylation of specific CpG sites. Here, we report that exonucleasemore » I and heat-labile alkaline phosphatase can be used for PCR purification for COBRA, improving the visibility of gel electrophoresis after restriction digestion. This improvement is observed when restriction digestion is performed at a high temperature, such as 60 {sup o}C or 65 {sup o}C, with BstUI and TaqI, respectively. This simple method can be applied instead of DNA purification using spin columns or phenol/chloroform extraction. It can also be applied to other situations when PCR products are digested by thermophile-derived restriction enzymes, such as PCR restriction fragment length polymorphism (RFLP) analysis.« less

Psoralen-induced DNA adducts are substrates for the base excision repair pathway in human cells

PubMed Central

Couvé-Privat, Sophie; Macé, Gaëtane; Saparbaev, Murat K.

2007-01-01

Interstrand cross-link (ICL) is a covalent modification of both strands of DNA, which prevents DNA strand separation during transcription and replication. Upon photoactivation 8-methoxypsoralen (8-MOP+UVA) alkylates both strands of DNA duplex at the 5,6-double bond of thymidines, generating monoadducts (MAs) and ICLs. It was thought that bulky DNA lesions such as MAs are eliminated only in the nucleotide excision repair pathway. Instead, non-bulky DNA lesions are substrates for DNA glycosylases and AP endonucleases which initiate the base excision repair (BER) pathway. Here we examined whether BER might be involved in the removal of psoralen–DNA photoadducts. The results show that in human cells DNA glycosylase NEIL1 excises the MAs in duplex DNA, subsequently the apurinic/apyrimidinic endonuclease 1, APE1, removes the 3′-phosphate residue at single-strand break generated by NEIL1. The apparent kinetic parameters suggest that NEIL1 excises MAs with high efficiency. Consistent with these results HeLa cells lacking APE1 and/or NEIL1 become hypersensitive to 8-MOP+UVA exposure. Furthermore, we demonstrate that bacterial homologues of NEIL1, the Fpg and Nei proteins, also excise MAs. New substrate specificity of the Fpg/Nei protein family provides an alternative repair pathway for ICLs and bulky DNA damage. PMID:17715144

Application of liquid chromatography/electrospray ionization ion trap tandem mass spectrometry for the evaluation of global nucleic acids: methylation in garden cress under exposure to CuO nanoparticles.

PubMed

Alcazar Magana, Armando; Wrobel, Kazimierz; Corrales Escobosa, Alma Rosa; Wrobel, Katarzyna

2016-01-15

A full understanding of the biological impact of nanomaterials demands analytical procedures suitable for the detection/quantification of epigenetic changes that occur in the exposed organisms. Here, the effect of CuO nanoparticles (NPs) on global methylation of nucleic acids in Lepidium sativum was evaluated by liquid chromatography/ion trap mass spectrometry. Enhanced selectivity toward cytosine-containing nucleosides was achieved by using their proton-bound dimers formed in positive electrospray ionization (ESI(+)) as precursor ions for multiple reaction monitoring (MRM) quantification based on one or two ion transitions. Plants were exposed to CuO NPs (0-1000 mg L(-1)); nucleic acid extracts were washed with bathocuproine disulfate; nucleosides were separated on a Luna C18 column coupled via ESI(+) to an AmaZon SL mass spectrometer (Bruker Daltonics). Cytidine, 2´-deoxycytidine, 5-methylcytidine, 5-methyl-2´-deoxycytidine and 5-hydroxymethyl-2´-deoxycytidine were quantified by MRM based on MS(3) ([2M+H](+)/[M+H](+)/[M+H-132](+) or [M+H-116](+)) and MS(2) ([2M+H](+)/[M+H](+) ). Bathocuproine disulfate, added as Cu(I) complexing agent, allowed for elimination of [2M+Cu](+) adducts from the mass spectra. Poorer instrumental detection limits were obtained for MS(3) (20-120 fmol) as compared to MS(2) (9.0-41 fmol); however, two ion transitions helped to eliminate matrix effects in plant extracts. The procedure was tested by analyzing salmon sperm DNA (Sigma) and applied for the evaluation of DNA and RNA methylation in plants; in the absence of NPs, 13.03% and 0.92% methylated cytosines were found in DNA and RNA, respectively; for NPs concentration >50 mg L(-1), DNA hypomethylation was observed with respect to unexposed plants. RNA methylation did not present significant changes upon plant exposure; 5-hydroxymethyl-2´-deoxycytidine was not detected in any sample. The MRM quantification proposed here of cytosine-containing nucleosides using their proton-bound homo-dimers as precursor ions proved its utility for the assessment of global methylation of DNA and RNA in plants under stress imposed by CuO NPs. Detection of copper adducts with cytosine-containing ions, and their elimination by washing extracts with Cu(I) chelator, calls for further investigation. Copyright © 2015 John Wiley & Sons, Ltd.

78 FR 77191 - Self-Regulatory Organizations; Chicago Board Options Exchange, Incorporated; Notice of Filing and...

Federal Register 2010, 2011, 2012, 2013, 2014

2013-12-20

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34 CFR Appendix A to Part 106 - Guidelines for Eliminating Discrimination and Denial of Services on the Basis of Race, Color...

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2013-07-01

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Code of Federal Regulations, 2012 CFR

2012-07-01

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MinGenome: An In Silico Top-Down Approach for the Synthesis of Minimized Genomes.

PubMed

Wang, Lin; Maranas, Costas D

2018-02-16

Genome minimized strains offer advantages as production chassis by reducing transcriptional cost, eliminating competing functions and limiting unwanted regulatory interactions. Existing approaches for identifying stretches of DNA to remove are largely ad hoc based on information on presumably dispensable regions through experimentally determined nonessential genes and comparative genomics. Here we introduce a versatile genome reduction algorithm MinGenome that implements a mixed-integer linear programming (MILP) algorithm to identify in size descending order all dispensable contiguous sequences without affecting the organism's growth or other desirable traits. Known essential genes or genes that cause significant fitness or performance loss can be flagged and their deletion can be prohibited. MinGenome also preserves needed transcription factors and promoter regions ensuring that retained genes will be properly transcribed while also avoiding the simultaneous deletion of synthetic lethal pairs. The potential benefit of removing even larger contiguous stretches of DNA if only one or two essential genes (to be reinserted elsewhere) are within the deleted sequence is explored. We applied the algorithm to design a minimized E. coli strain and found that we were able to recapitulate the long deletions identified in previous experimental studies and discover alternative combinations of deletions that have not yet been explored in vivo.

The Role of Research in Viral Disease Eradication and Elimination Programs: Lessons for Malaria Eradication

PubMed Central

Breman, Joel G.; de Quadros, Ciro A.; Dowdle, Walter R.; Foege, William H.; Henderson, Donald A.; John, T. Jacob; Levine, Myron M.

2011-01-01

By examining the role research has played in eradication or regional elimination initiatives for three viral diseases—smallpox, poliomyelitis, and measles—we derive nine cross-cutting lessons applicable to malaria eradication. In these initiatives, some types of research commenced as the programs began and proceeded in parallel. Basic laboratory, clinical, and field research all contributed notably to progress made in the viral programs. For each program, vaccine was the lynchpin intervention, but as the programs progressed, research was required to improve vaccine formulations, delivery methods, and immunization schedules. Surveillance was fundamental to all three programs, whilst polio eradication also required improved diagnostic methods to identify asymptomatic infections. Molecular characterization of pathogen isolates strengthened surveillance and allowed insights into the geographic source of infections and their spread. Anthropologic, sociologic, and behavioural research were needed to address cultural and religious beliefs to expand community acceptance. The last phases of elimination and eradication became increasingly difficult, as a nil incidence was approached. Any eradication initiative for malaria must incorporate flexible research agendas that can adapt to changing epidemiologic contingencies and allow planning for posteradication scenarios. PMID:21311582

32 CFR 291.6 - Procedures.

Code of Federal Regulations, 2010 CFR

2010-07-01

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32 CFR 291.6 - Procedures.

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2011-07-01

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32 CFR 291.6 - Procedures.

Code of Federal Regulations, 2014 CFR

2014-07-01

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32 CFR 291.6 - Procedures.

Code of Federal Regulations, 2012 CFR

2012-07-01

... PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM § 291.6 Procedures. (a) If HQ, DNA... handcarry the request to PAO. TDNM and AFRRI personnel will forward all FOIA requests to HQ, DNA, Attn: PAO. FCDNA will adhere to paragraph 6d and FCDNA Supplement to DNA Instruction 5400.7C. 2 2 Copies can be...

32 CFR 291.6 - Procedures.

Code of Federal Regulations, 2013 CFR

2013-07-01

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77 FR 60489 - Self-Regulatory Organizations; Chicago Board Options Exchange, Incorporated; Notice of Filing and...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-10-03

... Effectiveness of a Proposed Rule To Eliminate Position and Exercise Limits for Physically- Settled SPY Options... Proposed Rule Change CBOE proposes to amend its rules to eliminate position and exercise limits for... eliminate position and exercise limits for physically-settled SPY options pursuant to a pilot program.\\5...

A force-based protein biochip

NASA Astrophysics Data System (ADS)

Blank, K.; Mai, T.; Gilbert, I.; Schiffmann, S.; Rankl, J.; Zivin, R.; Tackney, C.; Nicolaus, T.; Spinnler, K.; Oesterhelt, F.; Benoit, M.; Clausen-Schaumann, H.; Gaub, H. E.

2003-09-01

A parallel assay for the quantification of single-molecule binding forces was developed based on differential unbinding force measurements where ligand-receptor interactions are compared with the unzipping forces of DNA hybrids. Using the DNA zippers as molecular force sensors, the efficient discrimination between specific and nonspecific interactions was demonstrated for small molecules binding to specific receptors, as well as for protein-protein interactions on protein arrays. Finally, an antibody sandwich assay with different capture antibodies on one chip surface and with the detection antibodies linked to a congruent surface via the DNA zippers was used to capture and quantify a recombinant hepatitis C antigen from solution. In this case, the DNA zippers enable not only discrimination between specific and nonspecific binding, but also allow for the local application of detection antibodies, thereby eliminating false-positive results caused by cross-reactive antibodies and nonspecific binding.

The Brain Physics: Multi Laser Beam Interaction with the Brain Topions (the Brain Neurocenters)

NASA Astrophysics Data System (ADS)

Stefan, V. Alexander

2015-03-01

A novel method for the treatment of the neurological diseases is proposed. The multiple-energy laser photons (the blue scanning photons and ultraviolet focusing photons) interact with the specific DNA molecules within the topion (such as Parkinson's and Alzheimer's brain topion) via the matching of laser frequency with the oscillation eigen-frequency of a particular molecule within the DNA. In this way, the corrupt molecules (the structure of molecules) can be manipulated so as to treat (eliminate) the neurological disease. Supported by Nikola Tesla Labs, Stefan University.

Diagnostic devices for isothermal nucleic acid amplification.

PubMed

Chang, Chia-Chen; Chen, Chien-Cheng; Wei, Shih-Chung; Lu, Hui-Hsin; Liang, Yang-Hung; Lin, Chii-Wann

2012-01-01

Since the development of the polymerase chain reaction (PCR) technique, genomic information has been retrievable from lesser amounts of DNA than previously possible. PCR-based amplifications require high-precision instruments to perform temperature cycling reactions; further, they are cumbersome for routine clinical use. However, the use of isothermal approaches can eliminate many complications associated with thermocycling. The application of diagnostic devices for isothermal DNA amplification has recently been studied extensively. In this paper, we describe the basic concepts of several isothermal amplification approaches and review recent progress in diagnostic device development.

Diagnostic Devices for Isothermal Nucleic Acid Amplification

PubMed Central

Chang, Chia-Chen; Chen, Chien-Cheng; Wei, Shih-Chung; Lu, Hui-Hsin; Liang, Yang-Hung; Lin, Chii-Wann

2012-01-01

Since the development of the polymerase chain reaction (PCR) technique, genomic information has been retrievable from lesser amounts of DNA than previously possible. PCR-based amplifications require high-precision instruments to perform temperature cycling reactions; further, they are cumbersome for routine clinical use. However, the use of isothermal approaches can eliminate many complications associated with thermocycling. The application of diagnostic devices for isothermal DNA amplification has recently been studied extensively. In this paper, we describe the basic concepts of several isothermal amplification approaches and review recent progress in diagnostic device development. PMID:22969402

Paternal leakage of mitochondrial DNA in experimental crosses of populations of the potato cyst nematode Globodera pallida.

PubMed

Hoolahan, Angelique H; Blok, Vivian C; Gibson, Tracey; Dowton, Mark

2011-12-01

Animal mtDNA is typically assumed to be maternally inherited. Paternal mtDNA has been shown to be excluded from entering the egg or eliminated post-fertilization in several animals. However, in the contact zones of hybridizing species and populations, the reproductive barriers between hybridizing organisms may not be as efficient at preventing paternal mtDNA inheritance, resulting in paternal leakage. We assessed paternal mtDNA leakage in experimental crosses of populations of a cyst-forming nematode, Globodera pallida. A UK population, Lindley, was crossed with two South American populations, P5A and P4A. Hybridization of these populations was supported by evidence of nuclear DNA from both the maternal and paternal populations in the progeny. To assess paternal mtDNA leakage, a ~3.4 kb non-coding mtDNA region was analyzed in the parental populations and in the progeny. Paternal mtDNA was evident in the progeny of both crosses involving populations P5A and P4A. Further, paternal mtDNA replaced the maternal mtDNA in 22 and 40 % of the hybrid cysts from these crosses, respectively. These results indicate that under appropriate conditions, paternal leakage occurs in the mtDNA of parasitic nematodes, and supports the hypothesis that hybrid zones facilitate paternal leakage. Thus, assumptions of strictly maternal mtDNA inheritance may be frequently violated, particularly when divergent populations interbreed.

40 CFR 35.310 - Purpose.

Code of Federal Regulations, 2010 CFR

2010-07-01

... establish and operate compliance monitoring programs to prevent or eliminate unreasonable risks to health or... which the Administrator is unable or not likely to take action for their prevention or elimination. (c...

Computer program for the IBM personal computer which searches for approximate matches to short oligonucleotide sequences in long target DNA sequences.

PubMed Central

Myers, E W; Mount, D W

1986-01-01

We describe a program which may be used to find approximate matches to a short predefined DNA sequence in a larger target DNA sequence. The program predicts the usefulness of specific DNA probes and sequencing primers and finds nearly identical sequences that might represent the same regulatory signal. The program is written in the C programming language and will run on virtually any computer system with a C compiler, such as the IBM/PC and other computers running under the MS/DOS and UNIX operating systems. The program has been integrated into an existing software package for the IBM personal computer (see article by Mount and Conrad, this volume). Some examples of its use are given. PMID:3753785

Interplay between Ku and Replication Protein A in the Restriction of Exo1-mediated DNA Break End Resection*

PubMed Central

Krasner, Danielle S.; Daley, James M.; Sung, Patrick; Niu, Hengyao

2015-01-01

DNA double-strand breaks can be eliminated via non-homologous end joining or homologous recombination. Non-homologous end joining is initiated by the association of Ku with DNA ends. In contrast, homologous recombination entails nucleolytic resection of the 5′-strands, forming 3′-ssDNA tails that become coated with replication protein A (RPA). Ku restricts end access by the resection nuclease Exo1. It is unclear how partial resection might affect Ku engagement and Exo1 restriction. Here, we addressed these questions in a reconstituted system with yeast proteins. With blunt-ended DNA, Ku protected against Exo1 in a manner that required its DNA end-binding activity. Despite binding poorly to ssDNA, Ku could nonetheless engage a 5′-recessed DNA end with a 40-nucleotide (nt) ssDNA overhang, where it localized to the ssDNA-dsDNA junction and efficiently blocked resection by Exo1. Interestingly, RPA could exclude Ku from a partially resected structure with a 22-nt ssDNA tail and thus restored processing by Exo1. However, at a 40-nt tail, Ku remained stably associated at the ssDNA-dsDNA junction, and RPA simultaneously engaged the ssDNA region. We discuss a model in which the dynamic equilibrium between Ku and RPA binding to a partially resected DNA end influences the timing and efficiency of the resection process. PMID:26067273

Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication

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

Haruta, Mayumi; Shimada, Midori, E-mail: midorism@med.nagoya-cu.ac.jp; Nishiyama, Atsuya

The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program.more » Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells. - Highlights: • DNMT1 depletion results in an abnormal DNA replication program. • Aberrant DNA replication is independent of the DNA damage checkpoint in DNMT1cKO. • DNMT1 catalytic activity and RFT domain are required for proper DNA replication. • DNMT1 catalytic activity and RFT domain are required for cell proliferation.« less

Molecular simulations of polycation-DNA binding exploring the effect of peptide chemistry and sequence in nuclear localization sequence based polycations.

PubMed

Elder, Robert M; Jayaraman, Arthi

2013-10-10

Gene therapy relies on the delivery of DNA into cells, and polycations are one class of vectors enabling efficient DNA delivery. Nuclear localization sequences (NLS), cationic oligopeptides that target molecules for nuclear entry, can be incorporated into polycations to improve their gene delivery efficiency. We use simulations to study the effect of peptide chemistry and sequence on the DNA-binding behavior of NLS-grafted polycations by systematically mutating the residues in the grafts, which are based on the SV40 NLS (peptide sequence PKKKRKV). Replacing arginine (R) with lysine (K) reduces binding strength by eliminating arginine-DNA interactions, but placing R in a less hindered location (e.g., farther from the grafting point to the polycation backbone) has surprisingly little effect on polycation-DNA binding strength. Changing the positions of the hydrophobic proline (P) and valine (V) residues relative to the polycation backbone changes hydrophobic aggregation within the polycation and, consequently, changes the conformational entropy loss that occurs upon polycation-DNA binding. Since conformational entropy loss affects the free energy of binding, the positions of P and V in the grafts affect DNA binding affinity. The insight from this work guides synthesis of polycations with tailored DNA binding affinity and, in turn, efficient DNA delivery.

Detection of DNA damage in individual cells by flow cytometric analysis using anti-DNA monoclonal antibody

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

Frankfurt, O.S.

A new method for the measurement of DNA damage in individual cells treated with alkylating agents is described. The method is based on the binding of anti-DNA monoclonal antibody to DNA in situ. Binding of antibody was evaluated by flow cytometry with indirect immunofluorescence. No binding of antibody to DNA in non-treated HeLa S3 cells was detected. Treatment of cells with HN2 or L-phenylalanine mustard induced binding of antibody to DNA in situ. Binding of antibody was observed after treating cells with doses of drugs which reduced the surviving fraction below 20%. Intensity of binding increased in proportion to themore » drug dose. In HN2-treated cells a cell subset with the lowest antibody binding was observed among cells in G1 phase. Binding of antibody to DNA in HN2-treated cells was eliminated by single-strand (ss) specific S1 nuclease. In competition assay, antibody was inhibited by thermally denatured DNA, but not by native double-stranded (ds) DNA, RNA, nucleosides and deoxyribohomopolymers. Immunoreactivity of cells with the monoclonal antibody F7-26 may be a useful probe for the assessment of cell damage induced by alkylating agents, especially in heterogeneous cell populations.« less

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

PubMed

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

2008-02-01

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

Comparison of methods of DNA extraction for real-time PCR in a model of pleural tuberculosis.

PubMed

Santos, Ana; Cremades, Rosa; Rodríguez, Juan Carlos; García-Pachón, Eduardo; Ruiz, Montserrat; Royo, Gloria

2010-01-01

Molecular methods have been reported to have different sensitivities in the diagnosis of pleural tuberculosis and this may in part be caused by the use of different methods of DNA extraction. Our study compares nine DNA extraction systems in an experimental model of pleural tuberculosis. An inoculum of Mycobacterium tuberculosis was added to 23 pleural liquid samples with different characteristics. DNA was subsequently extracted using nine different methods (seven manual and two automatic) for analysis with real-time PCR. Only two methods were able to detect the presence of M. tuberculosis DNA in all the samples: extraction using columns (Qiagen) and automated extraction with the TNAI system (Roche). The automatic method is more expensive, but requires less time. Almost all the false negatives were because of the difficulty involved in extracting M. tuberculosis DNA, as in general, all the methods studied are capable of eliminating inhibitory substances that block the amplification reaction. The method of M. tuberculosis DNA extraction used affects the results of the diagnosis of pleural tuberculosis by molecular methods. DNA extraction systems that have been shown to be effective in pleural liquid should be used.

Schistosomiasis Elimination Strategies and Potential Role of a Vaccine in Achieving Global Health Goals

PubMed Central

Mo, Annie X.; Agosti, Jan M.; Walson, Judd L.; Hall, B. Fenton; Gordon, Lance

2014-01-01

In March 2013, the National Institute of Allergy and Infectious Diseases and the Bill and Melinda Gates Foundation co-sponsored a meeting entitled “Schistosomiasis Elimination Strategy and Potential Role of a Vaccine in Achieving Global Health Goals” to discuss the potential role of schistosomiasis vaccines and other tools in the context of schistosomiasis control and elimination strategies. It was concluded that although schistosomiasis elimination in some focal areas may be achievable through current mass drug administration programs, global control and elimination will face several significant scientific and operational challenges, and will require an integrated approach with other, additional interventions. These challenges include vector (snail) control; environmental modification; water, sanitation, and hygiene; and other future innovative tools such as vaccines. Defining a clear product development plan that reflects a vaccine strategy as complementary to the existing control programs to combat different forms of schistosomiasis will be important to develop a vaccine effectively. PMID:24402703

Bacterial DNA-induced NK cell IFN-gamma production is dependent on macrophage secretion of IL-12.

PubMed

Chace, J H; Hooker, N A; Mildenstein, K L; Krieg, A M; Cowdery, J S

1997-08-01

Bacterial DNA (bDNA) activates B cells and macrophages and can augment inflammatory responses by inducing release of proinflammatory cytokines. We found that bDNA stimulation of mouse spleen cells induced NK cell IFN-gamma production that was dependent upon the presence of unmethylated CpG motifs, and oligonucleotides with internal CpG motifs could also induce splenocytes to secrete IFN-gamma. The bDNA-induced IFN-gamma response was strictly macrophages dependent. While splenocytes from SCID mice secreted IFN-gamma in response to bDNA, depletion of macrophages eliminated this response. Additionally, purified NK cells did not respond to bDNA; however, addition of macrophages restored the NK cell IFN-gamma response. Coculture of NK cells with preactivated macrophages further increased bDNA-induced NK cell IFN-gamma production. Anti-IL-12 or IL-10 inhibited bDNA-induced IFN-gamma response. Treatment of purified macrophages with bDNA resulted in IL-12 secretion accompanied by an increase in IL-12 p40 mRNA level. Although isolated NK cells did not make IFN-gamma in response to bDNA, NK cells costimulated with IL-12 gained the ability to respond to bDNA. These experiments show that bDNA induces macrophage IL-12 production which, in turn, stimulates NK cell IFN-gamma production. Macrophage-derived IL-12 renders NK cells responsive to bDNA permitting an even greater IFN-gamma response to bDNA.

Schistosomiasis Prevalence and Intensity of Infection in Latin America and the Caribbean Countries, 1942-2014: A Systematic Review in the Context of a Regional Elimination Goal

PubMed Central

2016-01-01

Background In 2012 the World Health Assembly adopted resolution WHA65.21 on elimination of schistosomiasis, calling for increased investment in schistosomiasis control and support for countries to initiate elimination programs. This study aims to analyze prevalence and intensity of Schistosoma mansoni infection in children in Latin America and the Caribbean countries and territories (LAC), at the second administrative level or lower. Methodology A systematic review of schistosomiasis prevalence and intensity of infection was conducted by searching at PubMed, LILACS and EMBASE. Experts on the topic were informally consulted and institutional web pages were reviewed (PAHO/WHO, Ministries of Health). Only SCH infection among children was registered because it can be a ‘proxi-indicator’ of recent transmission by the time the study is conducted. Principal Findings One hundred thirty two full-text articles met the inclusion criteria and provided 1,242 prevalence and 199 intensity of infection data points. Most of them were from Brazil (69.7%). Only Brazil published studies after 2001, showing several 'hot spots' with high prevalence. Brazil, Venezuela, Suriname and Saint Lucia need to update the epidemiological status of schistosomiasis to re-design their national programs and target the elimination of Schistosoma mansoni transmission by 2020. In Antigua and Barbuda, Dominican Republic, Guadeloupe, Martinique, Montserrat and Puerto Rico schistosomiasis transmission may be interrupted. However the compilation of an elimination dossier and follow-up surveys, per WHO recommendations, are needed to verify that status. Hence, the burden of subtle SCH chronic infection may be still present and even high in countries that may have eliminated transmission. Heterogeneity in the methodologies used for monitoring and evaluating the progress of the schistosomiasis programs was found, making cross-national and chronological comparisons difficult. Conclusions There is a need for updating the schistosomiasis status in the historically endemic countries and territories in LAC to address the required public health interventions for control and elimination programs or to verify the elimination of transmission of Schistosoma mansoni. Improved reporting and standardization of the monitoring and evaluation methodologies used are recommended, while using available WHO guidelines. Meeting a regional elimination goal will require additional and improved epidemiological data by age group and sex. PMID:27007193

Schistosomiasis Prevalence and Intensity of Infection in Latin America and the Caribbean Countries, 1942-2014: A Systematic Review in the Context of a Regional Elimination Goal.

PubMed

Zoni, Ana Clara; Catalá, Laura; Ault, Steven K

2016-03-01

In 2012 the World Health Assembly adopted resolution WHA65.21 on elimination of schistosomiasis, calling for increased investment in schistosomiasis control and support for countries to initiate elimination programs. This study aims to analyze prevalence and intensity of Schistosoma mansoni infection in children in Latin America and the Caribbean countries and territories (LAC), at the second administrative level or lower. A systematic review of schistosomiasis prevalence and intensity of infection was conducted by searching at PubMed, LILACS and EMBASE. Experts on the topic were informally consulted and institutional web pages were reviewed (PAHO/WHO, Ministries of Health). Only SCH infection among children was registered because it can be a 'proxi-indicator' of recent transmission by the time the study is conducted. One hundred thirty two full-text articles met the inclusion criteria and provided 1,242 prevalence and 199 intensity of infection data points. Most of them were from Brazil (69.7%). Only Brazil published studies after 2001, showing several 'hot spots' with high prevalence. Brazil, Venezuela, Suriname and Saint Lucia need to update the epidemiological status of schistosomiasis to re-design their national programs and target the elimination of Schistosoma mansoni transmission by 2020. In Antigua and Barbuda, Dominican Republic, Guadeloupe, Martinique, Montserrat and Puerto Rico schistosomiasis transmission may be interrupted. However the compilation of an elimination dossier and follow-up surveys, per WHO recommendations, are needed to verify that status. Hence, the burden of subtle SCH chronic infection may be still present and even high in countries that may have eliminated transmission. Heterogeneity in the methodologies used for monitoring and evaluating the progress of the schistosomiasis programs was found, making cross-national and chronological comparisons difficult. There is a need for updating the schistosomiasis status in the historically endemic countries and territories in LAC to address the required public health interventions for control and elimination programs or to verify the elimination of transmission of Schistosoma mansoni. Improved reporting and standardization of the monitoring and evaluation methodologies used are recommended, while using available WHO guidelines. Meeting a regional elimination goal will require additional and improved epidemiological data by age group and sex.

77 FR 62300 - Self-Regulatory Organizations; International Securities Exchange, LLC; Notice of Filing and...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-10-12

... Rule Change To Adopt a Pilot Program To Eliminate Position and Exercise Limits in SPY Options October 5... eliminate position and exercise limits for physically-settled options on the SPDR S&P ETF Trust (``SPY... Material .01 to ISE Rule 412 and Supplementary Material .01 of ISE Rule 414 to eliminate position and...

Eliminating Childhood Lead Poisoning: A Federal Strategy Targeting Lead Paint Hazards.

ERIC Educational Resources Information Center

2000

Noting that lead poisoning is a preventable disease, this report details a coordinated federal program to eliminate childhood lead poisoning in the United States. The report describes how lead poisoning harms children, how pervasive lead poisoning is, and how lead paint hazards in housing could be eliminated in 10 years. Following information on…

Impact of long-term treatment of onchocerciasis with ivermectin in Ecuador: potential for elimination of infection.

PubMed

Vieira, Juan Carlos; Cooper, Philip J; Lovato, Raquel; Mancero, Tamara; Rivera, Jorge; Proaño, Roberto; López, Andrea A; Guderian, Ronald H; Guzmán, José Rumbea

2007-05-23

Onchocerciasis is a leading cause of blindness worldwide, hence elimination of the infection is an important health priority. Community-based treatment programs with ivermectin form the basis of control programs for the disease in Latin America. The long-term administration of ivermectin could eliminate Onchocerca volvulus infection from endemic areas in Latin America. A strategy of annual to twice-annual treatments with ivermectin has been used for onchocerciasis in endemic communities in Ecuador for up to 14 years. The impact of ivermectin treatment on ocular morbidity, and O. volvulus infection and transmission was monitored in seven sentinel communities. Over the period 1990-2003, high rates of treatment coverage of the eligible population were maintained in endemic communities (mean 85.2% per treatment round). Ivermectin reduced the prevalence of anterior segment disease of the eye to 0% in sentinel communities and had a major impact on the prevalence and transmission of infection, with possible elimination of infection in some foci. The distribution of ivermectin in endemic communities in Ecuador might have eliminated ocular morbidity and significant progress has been made towards elimination of the infection. A strategy of more frequent treatments with ivermectin may be required in communities where the infection persists to achieve the objective of elimination of the infection from Ecuador. The elimination of the infection from an endemic country in Latin America would be a major public health achievement and could stimulate the implementation of elimination strategies in other endemic countries.

Studying long 16S rDNA sequences with ultrafast-metagenomic sequence classification using exact alignments (Kraken).

PubMed

Valenzuela-González, Fabiola; Martínez-Porchas, Marcel; Villalpando-Canchola, Enrique; Vargas-Albores, Francisco

2016-03-01

Ultrafast-metagenomic sequence classification using exact alignments (Kraken) is a novel approach to classify 16S rDNA sequences. The classifier is based on mapping short sequences to the lowest ancestor and performing alignments to form subtrees with specific weights in each taxon node. This study aimed to evaluate the classification performance of Kraken with long 16S rDNA random environmental sequences produced by cloning and then Sanger sequenced. A total of 480 clones were isolated and expanded, and 264 of these clones formed contigs (1352 ± 153 bp). The same sequences were analyzed using the Ribosomal Database Project (RDP) classifier. Deeper classification performance was achieved by Kraken than by the RDP: 73% of the contigs were classified up to the species or variety levels, whereas 67% of these contigs were classified no further than the genus level by the RDP. The results also demonstrated that unassembled sequences analyzed by Kraken provide similar or inclusively deeper information. Moreover, sequences that did not form contigs, which are usually discarded by other programs, provided meaningful information when analyzed by Kraken. Finally, it appears that the assembly step for Sanger sequences can be eliminated when using Kraken. Kraken cumulates the information of both sequence senses, providing additional elements for the classification. In conclusion, the results demonstrate that Kraken is an excellent choice for use in the taxonomic assignment of sequences obtained by Sanger sequencing or based on third generation sequencing, of which the main goal is to generate larger sequences. Copyright © 2016 Elsevier B.V. All rights reserved.

DNA Helicase HIM-6/BLM Both Promotes MutSγ-Dependent Crossovers and Antagonizes MutSγ-Independent Interhomolog Associations During Caenorhabditis elegans Meiosis

PubMed Central

Schvarzstein, Mara; Pattabiraman, Divya; Libuda, Diana E.; Ramadugu, Ajit; Tam, Angela; Martinez-Perez, Enrique; Roelens, Baptiste; Zawadzki, Karl A.; Yokoo, Rayka; Rosu, Simona; Severson, Aaron F.; Meyer, Barbara J.; Nabeshima, Kentaro; Villeneuve, Anne M.

2014-01-01

Meiotic recombination is initiated by the programmed induction of double-strand DNA breaks (DSBs), lesions that pose a potential threat to the genome. A subset of the DSBs induced during meiotic prophase become designated to be repaired by a pathway that specifically yields interhomolog crossovers (COs), which mature into chiasmata that temporarily connect the homologs to ensure their proper segregation at meiosis I. The remaining DSBs must be repaired by other mechanisms to restore genomic integrity prior to the meiotic divisions. Here we show that HIM-6, the Caenorhabditis elegans ortholog of the RecQ family DNA helicase BLM, functions in both of these processes. We show that him-6 mutants are competent to load the MutSγ complex at multiple potential CO sites, to generate intermediates that fulfill the requirements of monitoring mechanisms that enable meiotic progression, and to accomplish and robustly regulate CO designation. However, recombination events at a subset of CO-designated sites fail to mature into COs and chiasmata, indicating a pro-CO role for HIM-6/BLM that manifests itself late in the CO pathway. Moreover, we find that in addition to promoting COs, HIM-6 plays a role in eliminating and/or preventing the formation of persistent MutSγ-independent associations between homologous chromosomes. We propose that HIM-6/BLM enforces biased outcomes of recombination events to ensure that both (a) CO-designated recombination intermediates are reliably resolved as COs and (b) other recombination intermediates reliably mature into noncrossovers in a timely manner. PMID:25053665

Magnetic bead purification of labeled DNA fragments forhigh-throughput capillary electrophoresis sequencing

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

Elkin, Christopher; Kapur, Hitesh; Smith, Troy

2001-09-15

We have developed an automated purification method for terminator sequencing products based on a magnetic bead technology. This 384-well protocol generates labeled DNA fragments that are essentially free of contaminates for less than $0.005 per reaction. In comparison to laborious ethanol precipitation protocols, this method increases the phred20 read length by forty bases with various DNA templates such as PCR fragments, Plasmids, Cosmids and RCA products. Our method eliminates centrifugation and is compatible with both the MegaBACE 1000 and ABIPrism 3700 capillary instruments. As of September 2001, this method has produced over 1.6 million samples with 93 percent averaging 620more » phred20 bases as part of Joint Genome Institutes Production Process.« less

Accelerated measles control in the Western Pacific region.

PubMed

McFarland, Jeffrey W; Mansoor, Osman David; Yang, Baoping

2003-05-15

By the 1990s, an immunization program in the western Pacific had dramatically reduced measles morbidity and mortality. Building on the region's successful elimination of polio, several countries and areas achieved or are close to measles elimination, thus showing the potential for global eradication. The diverse challenges for measles control in different parts of the region have produced lessons that will help with future control, including the need for surveillance of sufficient standard to guide and monitor progress. A group of experts recognized both the potential and the challenges of the measles immunization program and proposed regional elimination as the appropriate disease control target for the region. No date was recommended for its achievement. If progress continues at the present rate, the western Pacific region should soon be able to set a target date for measles elimination.

Maternal and neonatal tetanus elimination: from protecting women and newborns to protecting all.

PubMed

Khan, Rownak; Vandelaer, Jos; Yakubu, Ahmadu; Raza, Azhar Abid; Zulu, Flint

2015-01-01

A total of 35 of the 59 countries that had not eliminated maternal and neonatal tetanus (MNT) as a public health problem in 1999 have since achieved the MNT-elimination goal. Neonatal tetanus deaths have decreased globally from 200,000 in 2000 to 49,000 in 2013. This is the result of increased immunization coverage with tetanus toxoid-containing vaccines among pregnant women, improved access to skilled birth attendance during delivery, and targeted campaigns with these vaccines for women of reproductive age in high-risk areas. In the process, inequities have been reduced, private-public partnerships fostered, and innovations triggered. However, lack of funding, poor accessibility to some areas, suboptimal surveillance, and a perceived low priority for the disease are among the main obstacles. To ensure MNT elimination is sustained, countries must build and maintain strong routine programs that reach people with vaccination and with clean deliveries. This should also be an opportunity to shift programs into preventing tetanus among all people. Regular assessments, and where needed appropriate action, are key to prevent increases in MNT incidence over time, especially in areas that are at higher risk. The main objective of the paper is to provide a detailed update on the progress toward MNT elimination between 1999 and 2014. It elaborates on the challenges and opportunities, and discusses how MNT elimination can be sustained and to shift the program to protect wider populations against tetanus.

Pre-steady-state fluorescence analysis of damaged DNA transfer from human DNA glycosylases to AP endonuclease APE1.

PubMed

Kuznetsova, Alexandra A; Kuznetsov, Nikita A; Ishchenko, Alexander A; Saparbaev, Murat K; Fedorova, Olga S

2014-10-01

DNA glycosylases remove the modified, damaged or mismatched bases from the DNA by hydrolyzing the N-glycosidic bonds. Some enzymes can further catalyze the incision of a resulting abasic (apurinic/apyrimidinic, AP) site through β- or β,δ-elimination mechanisms. In most cases, the incision reaction of the AP-site is catalyzed by special enzymes called AP-endonucleases. Here, we report the kinetic analysis of the mechanisms of modified DNA transfer from some DNA glycosylases to the AP endonuclease, APE1. The modified DNA contained the tetrahydrofurane residue (F), the analogue of the AP-site. DNA glycosylases AAG, OGG1, NEIL1, MBD4(cat) and UNG from different structural superfamilies were used. We found that all DNA glycosylases may utilise direct protein-protein interactions in the transient ternary complex for the transfer of the AP-containing DNA strand to APE1. We hypothesize a fast "flip-flop" exchange mechanism of damaged and undamaged DNA strands within this complex for monofunctional DNA glycosylases like MBD4(cat), AAG and UNG. Bifunctional DNA glycosylase NEIL1 creates tightly specific complex with DNA containing F-site thereby efficiently competing with APE1. Whereas APE1 fast displaces other bifunctional DNA glycosylase OGG1 on F-site thereby induces its shifts to undamaged DNA regions. Kinetic analysis of the transfer of DNA between human DNA glycosylases and APE1 allows us to elucidate the critical step in the base excision repair pathway. Copyright © 2014 Elsevier B.V. All rights reserved.

32 CFR Appendix A to Part 291 - Freedom of Information Act Request (DNA Form 524)

Code of Federal Regulations, 2011 CFR

2011-07-01

... 32 National Defense 2 2011-07-01 2011-07-01 false Freedom of Information Act Request (DNA Form 524... OF DEFENSE (CONTINUED) FREEDOM OF INFORMATION ACT PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM Pt. 291, App. A Appendix A to Part 291—Freedom of Information Act Request (DNA Form...

32 CFR Appendix A to Part 291 - Freedom of Information Act Request (DNA Form 524)

Code of Federal Regulations, 2010 CFR

2010-07-01

... 32 National Defense 2 2010-07-01 2010-07-01 false Freedom of Information Act Request (DNA Form 524... OF DEFENSE (CONTINUED) FREEDOM OF INFORMATION ACT PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM Pt. 291, App. A Appendix A to Part 291—Freedom of Information Act Request (DNA Form...

32 CFR Appendix A to Part 291 - Freedom of Information Act Request (DNA Form 524)

Code of Federal Regulations, 2014 CFR

2014-07-01

... 32 National Defense 2 2014-07-01 2014-07-01 false Freedom of Information Act Request (DNA Form 524... OF DEFENSE (CONTINUED) FREEDOM OF INFORMATION ACT PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM Pt. 291, App. A Appendix A to Part 291—Freedom of Information Act Request (DNA Form...

32 CFR Appendix A to Part 291 - Freedom of Information Act Request (DNA Form 524)

Code of Federal Regulations, 2013 CFR

2013-07-01

... 32 National Defense 2 2013-07-01 2013-07-01 false Freedom of Information Act Request (DNA Form 524... OF DEFENSE (CONTINUED) FREEDOM OF INFORMATION ACT PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM Pt. 291, App. A Appendix A to Part 291—Freedom of Information Act Request (DNA Form...

32 CFR Appendix A to Part 291 - Freedom of Information Act Request (DNA Form 524)

Code of Federal Regulations, 2012 CFR

2012-07-01

... 32 National Defense 2 2012-07-01 2012-07-01 false Freedom of Information Act Request (DNA Form 524... OF DEFENSE (CONTINUED) FREEDOM OF INFORMATION ACT PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM Pt. 291, App. A Appendix A to Part 291—Freedom of Information Act Request (DNA Form...

A successful traffic relief program

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

Dimino, R.A.; Bezkorovainy, G.; Campbell, B.

This article reports that in August 1986, under the direction of Mayor Raymond Flynn, the City of Boston initiated Phase I of a Traffic Relief Program (TRP). The program was an interagency effort of the Boston Transportation Department and the Boston Police Department, to provide increased enforcement of the city's traffic and parking regulations on congested roadways in downtown Boston. The TRP is a reaffirmation of the city's philosophy that major arterials' primary function is the movement of traffic during periods of heavy traffic flow. There were six objectives: to reduce vehicular travel time along travel corridors; to increase street/intersectionmore » capacity; to eliminate vehicular blockage at intersections; to eliminate double parking; to eliminate pedestrian/vehicular conflicts at intersections and thus reduce the potential number of accidents; and to provide clear regulatory and street name signage.« less

PubMed Central

Di Mauro, S.

2010-01-01

In this brief review, I have highlighted recent advances in several areas of mitochondrial medicine, including mtDNA-related diseases, mendelian mitochondrial encephalomyopathies, and therapy. The pathogenic mechanisms of mtDNA mutations, especially those affecting mitochondrial protein synthesis, are still largely unknown. The pathogenicity of homoplasmic mtDNA mutations has become evident but has also called attention to modifying nuclear genes, yet another example of impaired intergenomic signaling. The functional significance of the homoplasmic changes associated with mitochondrial haplogroups has been confirmed. Among the mendelian disorders, a new form of “indirect hit” has been described, in which the ultimate pathogenesis is toxic damage to the respiratory chain. Three therapeutic strategies look promising: (i) allogeneic hematopoietic stem cell transplantation in MNGIE (mitochondrial neurogastrointestinal encephalomyopathy); (ii) bezafibrate, an activator of PGC-1α, has proven effective in animal models of mitochondrial myopathy; and (iii) pronucleus transfer into a normal oocyte is effective in eliminating maternal transmission of mtDNA, thus preventing the appearance of mtDNA-related disorders. PMID:21314015

SLAP deficiency decreases dsDNA autoantibody production

PubMed Central

Peterson, Lisa K.; Pennington, Luke F.; Shaw, Laura A.; Brown, Meredith; Treacy, Eric C.; Friend, Samantha F.; Hatlevik, Øyvind; Rubtsova, Kira; Rubtsov, Anatoly V.; Dragone, Leonard L.

2014-01-01

Src-like adaptor protein (SLAP) adapts c-Cbl, an E3 ubiquitin ligase, to activated components of the BCR signaling complex regulating BCR levels and signaling in developing B cells. Based on this function, we asked whether SLAP deficiency could decrease the threshold for tolerance and eliminate development of autoreactive B cells in two models of autoantibody production. First, we sensitized mice with a dsDNA mimetope that causes an anti-dsDNA response. Despite equivalent production of anti-peptide antibodies compared to BALB/c controls, SLAP−/− mice did not produce anti-dsDNA. Second, we used the 56R tolerance model. SLAP−/− 56R mice had decreased levels of dsDNA-reactive antibodies compared to 56R mice due to skewed light chain usage. Thus, SLAP is a critical regulator of B-cell development and function and its deficiency leads to decreased autoreactive B cells that are otherwise maintained by inefficient receptor editing or failed negative selection. PMID:24440645

SLAP deficiency decreases dsDNA autoantibody production.

PubMed

Peterson, Lisa K; Pennington, Luke F; Shaw, Laura A; Brown, Meredith; Treacy, Eric C; Friend, Samantha F; Hatlevik, Øyvind; Rubtsova, Kira; Rubtsov, Anatoly V; Dragone, Leonard L

2014-02-01

Src-like adaptor protein (SLAP) adapts c-Cbl, an E3 ubiquitin ligase, to activated components of the BCR signaling complex regulating BCR levels and signaling in developing B cells. Based on this function, we asked whether SLAP deficiency could decrease the threshold for tolerance and eliminate development of autoreactive B cells in two models of autoantibody production. First, we sensitized mice with a dsDNA mimetope that causes an anti-dsDNA response. Despite equivalent production of anti-peptide antibodies compared to BALB/c controls, SLAP(-/-) mice did not produce anti-dsDNA. Second, we used the 56R tolerance model. SLAP(-/-) 56R mice had decreased levels of dsDNA-reactive antibodies compared to 56R mice due to skewed light chain usage. Thus, SLAP is a critical regulator of B-cell development and function and its deficiency leads to decreased autoreactive B cells that are otherwise maintained by inefficient receptor editing or failed negative selection. Copyright © 2013 Elsevier Inc. All rights reserved.

The effects of simulated hypogravity on murine bone marrow cells

NASA Technical Reports Server (NTRS)

Lawless, Desales

1989-01-01

Mouse bone marrow cells grown in complete medium at unit gravity were compared with a similar population cultured in conditions that mimic some aspects of microgravity. After the cells adjusted to the conditions that simulated microgravity, they proliferated as fetal or oncogenic populations; their numbers doubled in twelve hour periods. Differentiated subpopulations were depleted from the heterogeneous mixture with time and the undifferentiated hematopoietic stem cells increased in numbers. The cells in the control groups in unit gravity and those in the bioreactors in conditions of microgravity were monitored under a number of parameters. Each were phenotyped as to cell surface antigens using a panel of monoclonal antibodies and flow cytometry. Other parameters compared included: pH, glucose uptake, oxygen consumption and carbon-dioxide production. Nuclear DNA was monitored by flow cytometry. Functional responses were studied by mitogenic stimulation by various lectins. The importance of these findings should have relevance to the space program. Cells should behave predictably in zero gravity; specific populations can be eliminated from diverse populations and other populations isolated. The availability of stem cell populations will enhance both bone marrow and gene transplant programs. Stem cells will permit developmental biologists study the paths of hematopoiesis.

Spurring enrollment in Medicare savings programs through a substitute for the asset test focused on investment income.

PubMed

Dorn, Stan; Shang, Baoping

2012-02-01

Fewer than one-third of eligible Medicare beneficiaries enroll in Medicare savings programs, which pay premiums and, in some cases, eliminate out-of-pocket cost sharing for poor and near-poor enrollees. Many beneficiaries don't participate in savings programs because they must complete a cumbersome application process, including a burdensome asset test. We demonstrate that a streamlined alternative to the asset test-allowing seniors to qualify for Medicare savings programs by providing evidence of limited assets or showing a lack of investment income-would permit 78 percent of currently eligible seniors to bypass the asset test entirely. This simplified approach would increase the number of beneficiaries who qualify for Medicare savings programs from the current 3.6 million seniors to 4.6 million. Such an alternative would keep benefits targeted to people with low assets, eliminate costly administrative expenses and obstacles to enrollment associated with the asset test, and avoid the much larger influx of seniors that would occur if the asset test were eliminated entirely.

AtPDCD5 Plays a Role in Programmed Cell Death after UV-B Exposure in Arabidopsis1[OPEN

PubMed Central

Falcone Ferreyra, María Lorena; D’Andrea, Lucio; AbdElgawad, Hamada

2016-01-01

DNA damage responses have evolved to sense and react to DNA damage; the induction of DNA repair mechanisms can lead to genomic restoration or, if the damaged DNA cannot be adequately repaired, to the execution of a cell death program. In this work, we investigated the role of an Arabidopsis (Arabidopsis thaliana) protein, AtPDCD5, which is highly similar to the human PDCD5 protein; it is induced by ultraviolet (UV)-B radiation and participates in programmed cell death in the UV-B DNA damage response. Transgenic plants expressing AtPDCD5 fused to GREEN FLUORESCENT PROTEIN indicate that AtPDCD5 is localized both in the nucleus and the cytosol. By use of pdcd5 mutants, we here demonstrate that these plants have an altered antioxidant metabolism and accumulate higher levels of DNA damage after UV-B exposure, similar to levels in ham1ham2 RNA interference transgenic lines with decreased expression of acetyltransferases from the MYST family. By coimmunoprecipitation and pull-down assays, we provide evidence that AtPDCD5 interacts with HAM proteins, suggesting that both proteins participate in the same pathway of DNA damage responses. Plants overexpressing AtPDCD5 show less DNA damage but more cell death in root tips upon UV-B exposure. Finally, we here show that AtPDCD5 also participates in age-induced programmed cell death. Together, the data presented here demonstrate that AtPDCD5 plays an important role during DNA damage responses induced by UV-B radiation in Arabidopsis and also participates in programmed cell death programs. PMID:26884483

A Role for the Host DNA Damage Response in Hepatitis B Virus cccDNA Formation—and Beyond?

PubMed Central

Schreiner, Sabrina; Nassal, Michael

2017-01-01

Chronic hepatitis B virus (HBV) infection puts more than 250 million people at a greatly increased risk to develop end-stage liver disease. Like all hepadnaviruses, HBV replicates via protein-primed reverse transcription of a pregenomic (pg) RNA, yielding an unusually structured, viral polymerase-linked relaxed-circular (RC) DNA as genome in infectious particles. Upon infection, RC-DNA is converted into nuclear covalently closed circular (ccc) DNA. Associating with cellular proteins into an episomal minichromosome, cccDNA acts as template for new viral RNAs, ensuring formation of progeny virions. Hence, cccDNA represents the viral persistence reservoir that is not directly targeted by current anti-HBV therapeutics. Eliminating cccDNA will thus be at the heart of a cure for chronic hepatitis B. The low production of HBV cccDNA in most experimental models and the associated problems in reliable cccDNA quantitation have long hampered a deeper understanding of cccDNA molecular biology. Recent advancements including cccDNA-dependent cell culture systems have begun to identify select host DNA repair enzymes that HBV usurps for RC-DNA to cccDNA conversion. While this list is bound to grow, it may represent just one facet of a broader interaction with the cellular DNA damage response (DDR), a network of pathways that sense and repair aberrant DNA structures and in the process profoundly affect the cell cycle, up to inducing cell death if repair fails. Given the divergent interactions between other viruses and the DDR it will be intriguing to see how HBV copes with this multipronged host system. PMID:28531167

Application of virtual screening and molecular dynamics for the analysis of selectivity of inhibitors of HU proteins targeted to the DNA-recognition site

NASA Astrophysics Data System (ADS)

Talyzina, A. A.; Agapova, Yu. K.; Podshivalov, D. D.; Timofeev, V. I.; Sidorov-Biryukov, D. D.; Rakitina, T. V.

2017-11-01

DNA-Binding HU proteins are essential for the maintenance of genomic DNA supercoiling and compaction in prokaryotic cells and are promising pharmacological targets for the design of new antibacterial agents. The virtual screening for low-molecular-weight compounds capable of specifically interacting with the DNA-recognition loop of the HU protein from the mycoplasma Spiroplasma melliferum was performed. The ability of the initially selected ligands to form stable complexes with the protein target was assessed by molecular dynamics simulation. One compound, which forms an unstable complex, was eliminated by means of a combination of computational methods, resulting in a decrease in the number of compounds that will pass to the experimental test phase. This approach can be used to solve a wide range of problems related to the search for and validation of low-molecular-weight inhibitors specific for a particular protein target.

High mobility organic field-effect transistor based on water-soluble deoxyribonucleic acid via spray coating

NASA Astrophysics Data System (ADS)

Shi, Wei; Han, Shijiao; Huang, Wei; Yu, Junsheng

2015-01-01

High mobility organic field-effect transistors (OFETs) by inserting water-soluble deoxyribonucleic acid (DNA) buffer layer between electrodes and pentacene film through spray coating process were fabricated. Compared with the OFETs incorporated with DNA in the conventional organic solvents of ethanol and methanol: water mixture, the water-soluble DNA based OFET exhibited an over four folds enhancement of field-effect mobility from 0.035 to 0.153 cm2/Vs. By characterizing the surface morphology and the crystalline structure of pentacene active layer through atomic force microscope and X-ray diffraction, it was found that the adoption of water solvent in DNA solution, which played a key role in enhancing the field-effect mobility, was ascribed to both the elimination of the irreversible organic solvent-induced bulk-like phase transition of pentacene film and the diminution of a majority of charge trapping at interfaces in OFETs.

What’s the Damage? The Impact of Pathogens on Pathways that Maintain Host Genome Integrity

PubMed Central

Weitzman, Matthew D.; Weitzman, Jonathan B.

2014-01-01

Maintaining genome integrity and transmission of intact genomes is critical for cellular, organismal, and species survival. Cells can detect damaged DNA, activate checkpoints, and either enable DNA repair or trigger apoptosis to eliminate the damaged cell. Aberrations in these mechanisms lead to somatic mutations and genetic instability, which are hallmarks of cancer. Considering the long history of host-microbe coevolution, an impact of microbial infection on host genome integrity is not unexpected, and emerging links between microbial infections and oncogenesis further reinforce this idea. In this review, we compare strategies employed by viruses, bacteria, and parasites to alter, subvert, or otherwise manipulate host DNA damage and repair pathways. We highlight how microbes contribute to tumorigenesis by directly inducing DNA damage, inactivating checkpoint controls, or manipulating repair processes. We also discuss indirect effects resulting from inflammatory responses, changes in cellular metabolism, nuclear architecture, and epigenome integrity, and the associated evolutionary tradeoffs. PMID:24629335

Automation and validation of DNA-banking systems.

PubMed

Thornton, Melissa; Gladwin, Amanda; Payne, Robin; Moore, Rachael; Cresswell, Carl; McKechnie, Douglas; Kelly, Steve; March, Ruth

2005-10-15

DNA banking is one of the central capabilities on which modern genetic research rests. The DNA-banking system plays an essential role in the flow of genetic data from patients and genetics researchers to the application of genetic research in the clinic. Until relatively recently, large collections of DNA samples were not common in human genetics. Now, collections of hundreds of thousands of samples are common in academic institutions and private companies. Automation of DNA banking can dramatically increase throughput, eliminate manual errors and improve the productivity of genetics research. An increased emphasis on pharmacogenetics and personalized medicine has highlighted the need for genetics laboratories to operate within the principles of a recognized quality system such as good laboratory practice (GLP). Automated systems are suitable for such laboratories but require a level of validation that might be unfamiliar to many genetics researchers. In this article, we use the AstraZeneca automated DNA archive and reformatting system (DART) as a case study of how such a system can be successfully developed and validated within the principles of GLP.

Costs of eliminating malaria and the impact of the global fund in 34 countries.

PubMed

Zelman, Brittany; Kiszewski, Anthony; Cotter, Chris; Liu, Jenny

2014-01-01

International financing for malaria increased more than 18-fold between 2000 and 2011; the largest source came from The Global Fund to Fight AIDS, Tuberculosis and Malaria (Global Fund). Countries have made substantial progress, but achieving elimination requires sustained finances to interrupt transmission and prevent reintroduction. Since 2011, global financing for malaria has declined, fueling concerns that further progress will be impeded, especially for current malaria-eliminating countries that may face resurgent malaria if programs are disrupted. This study aims to 1) assess past total and Global Fund funding to the 34 current malaria-eliminating countries, and 2) estimate their future funding needs to achieve malaria elimination and prevent reintroduction through 2030. Historical funding is assessed against trends in country-level malaria annual parasite incidences (APIs) and income per capita. Following Kizewski et al. (2007), program costs to eliminate malaria and prevent reintroduction through 2030 are estimated using a deterministic model. The cost parameters are tailored to a package of interventions aimed at malaria elimination and prevention of reintroduction. The majority of Global Fund-supported countries experiencing increases in total funding from 2005 to 2010 coincided with reductions in malaria APIs and also overall GNI per capita average annual growth. The total amount of projected funding needed for the current malaria-eliminating countries to achieve elimination and prevent reintroduction through 2030 is approximately US$8.5 billion, or about $1.84 per person at risk per year (PPY) (ranging from $2.51 PPY in 2014 to $1.43 PPY in 2030). Although external donor funding, particularly from the Global Fund, has been key for many malaria-eliminating countries, sustained and sufficient financing is critical for furthering global malaria elimination. Projected cost estimates for elimination provide policymakers with an indication of the level of financial resources that should be mobilized to achieve malaria elimination goals.

Spreadsheet-based program for alignment of overlapping DNA sequences.

PubMed

Anbazhagan, R; Gabrielson, E

1999-06-01

Molecular biology laboratories frequently face the challenge of aligning small overlapping DNA sequences derived from a long DNA segment. Here, we present a short program that can be used to adapt Excel spreadsheets as a tool for aligning DNA sequences, regardless of their orientation. The program runs on any Windows or Macintosh operating system computer with Excel 97 or Excel 98. The program is available for use as an Excel file, which can be downloaded from the BioTechniques Web site. Upon execution, the program opens a specially designed customized workbook and is capable of identifying overlapping regions between two sequence fragments and displaying the sequence alignment. It also performs a number of specialized functions such as recognition of restriction enzyme cutting sites and CpG island mapping without costly specialized software.

Self-assembly programming of DNA polyominoes.

PubMed

Ong, Hui San; Syafiq-Rahim, Mohd; Kasim, Noor Hayaty Abu; Firdaus-Raih, Mohd; Ramlan, Effirul Ikhwan

2016-10-20

Fabrication of functional DNA nanostructures operating at a cellular level has been accomplished through molecular programming techniques such as DNA origami and single-stranded tiles (SST). During implementation, restrictive and constraint dependent designs are enforced to ensure conformity is attainable. We propose a concept of DNA polyominoes that promotes flexibility in molecular programming. The fabrication of complex structures is achieved through self-assembly of distinct heterogeneous shapes (i.e., self-organised optimisation among competing DNA basic shapes) with total flexibility during the design and assembly phases. In this study, the plausibility of the approach is validated using the formation of multiple 3×4 DNA network fabricated from five basic DNA shapes with distinct configurations (monomino, tromino and tetrominoes). Computational tools to aid the design of compatible DNA shapes and the structure assembly assessment are presented. The formations of the desired structures were validated using Atomic Force Microscopy (AFM) imagery. Five 3×4 DNA networks were successfully constructed using combinatorics of these five distinct DNA heterogeneous shapes. Our findings revealed that the construction of DNA supra-structures could be achieved using a more natural-like orchestration as compared to the rigid and restrictive conventional approaches adopted previously. Copyright © 2016 Elsevier B.V. All rights reserved.

32 CFR 291.5 - Responsibilities.

Code of Federal Regulations, 2011 CFR

2011-07-01

... INFORMATION ACT PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM § 291.5 Responsibilities. (a) The Director, DNA, as appellate authority, is responsible for reviewing and making the final... requests and has sole responsibility for withholding that information. (c) The DNA FOIA Officer, who is...

32 CFR 291.5 - Responsibilities.

Code of Federal Regulations, 2012 CFR

2012-07-01

... INFORMATION ACT PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM § 291.5 Responsibilities. (a) The Director, DNA, as appellate authority, is responsible for reviewing and making the final... requests and has sole responsibility for withholding that information. (c) The DNA FOIA Officer, who is...

32 CFR 291.5 - Responsibilities.

Code of Federal Regulations, 2013 CFR

2013-07-01

... INFORMATION ACT PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM § 291.5 Responsibilities. (a) The Director, DNA, as appellate authority, is responsible for reviewing and making the final... requests and has sole responsibility for withholding that information. (c) The DNA FOIA Officer, who is...

32 CFR 291.5 - Responsibilities.

Code of Federal Regulations, 2014 CFR

2014-07-01

... INFORMATION ACT PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM § 291.5 Responsibilities. (a) The Director, DNA, as appellate authority, is responsible for reviewing and making the final... requests and has sole responsibility for withholding that information. (c) The DNA FOIA Officer, who is...

The Estimated Health and Economic Benefits of Three Decades of Polio Elimination Efforts in India.

PubMed

Nandi, Arindam; Barter, Devra M; Prinja, Shankar; John, T Jacob

2016-08-07

In March 2014, India, the country with historically the highest burden of polio, was declared polio free, with no reported cases since January 2011. We estimate the health and economic benefits of polio elimination in India with the oral polio vaccine (OPV) during 1982-2012. Based on a pre-vaccine incidence rate, we estimate the counterfactual burden of polio in the hypothetical absence of the national polio elimination program in India. We attribute differences in outcomes between the actual (adjusted for under-reporting) and hypothetical counterfactual scenarios in our model to the national polio program. We measure health benefits as averted polio incidence, deaths, and disability adjusted life years (DALYs). We consider two methods to measure economic benefits: the value of statistical life approach, and equating one DALY to the Gross National Income (GNI) per capita. We estimate that the National Program against Polio averted 3.94 million (95% confidence interval [CI]: 3.89-3.99 million) paralytic polio cases, 393,918 polio deaths (95% CI: 388,897- 398,939), and 1.48 billion DALYs (95% CI: 1.46-1.50 billion). We also estimate that the program contributed to a $1.71 trillion (INR 76.91 trillion) gain (95% CI: $1.69-$1.73 trillion [INR 75.93-77.89 trillion]) in economic productivity between 1982 and 2012 in our base case analysis. Using the GNI and DALY method, the economic gain from the program is estimated to be $1.11 trillion (INR 50.13 trillion) (95% CI: $1.10-$1.13 trillion [INR 49.50-50.76 trillion]) over the same period. India accrued large health and economic benefits from investing in polio elimination efforts. Other programs to control/eliminate more vaccine-preventable diseases are likely to contribute to large health and economic benefits in India.

Conformational Dynamics of DNA Repair by Escherichia coli Endonuclease III*

PubMed Central

Kuznetsov, Nikita A.; Kladova, Olga A.; Kuznetsova, Alexandra A.; Ishchenko, Alexander A.; Saparbaev, Murat K.; Zharkov, Dmitry O.; Fedorova, Olga S.

2015-01-01

Escherichia coli endonuclease III (Endo III or Nth) is a DNA glycosylase with a broad substrate specificity for oxidized or reduced pyrimidine bases. Endo III possesses two types of activities: N-glycosylase (hydrolysis of the N-glycosidic bond) and AP lyase (elimination of the 3′-phosphate of the AP-site). We report a pre-steady-state kinetic analysis of structural rearrangements of the DNA substrates and uncleavable ligands during their interaction with Endo III. Oligonucleotide duplexes containing 5,6-dihydrouracil, a natural abasic site, its tetrahydrofuran analog, and undamaged duplexes carried fluorescent DNA base analogs 2-aminopurine and 1,3-diaza-2-oxophenoxazine as environment-sensitive reporter groups. The results suggest that Endo III induces several fast sequential conformational changes in DNA during binding, lesion recognition, and adjustment to a catalytically competent conformation. A comparison of two fluorophores allowed us to distinguish between the events occurring in the damaged and undamaged DNA strand. Combining our data with the available structures of Endo III, we conclude that this glycosylase uses a multistep mechanism of damage recognition, which likely involves Gln41 and Leu81 as DNA lesion sensors. PMID:25869130

INTERACTION OF BENZO(A)PYRENE DIOL EPOXIDE WITH SVAO MINICHROMOSOMES

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

Gamper, Howard B.; Yokota, Hisao A.; Bartholomew, James C.

SV40 minichromosomes were reacted with (+)7{beta},8{alpha}-dihydroxy-9{alpha},10{alpha}-epoxy- 7,8,9,10-tetrahydrobenzo[a]pyrene (BaP diol epoxide). Low levels of modification (< 5 DNA adducts/minichromosome) did not detectably alter the structure of the minichromosomes but high levels (> 200 DNA adducts/minichromosome) led to extensive fragmentation. Relative to naked SV40 DNA BaP diol epoxide induced alkylation and strand scission of minichromosomal DNA was reduced or enhanced by factors of 1.5 and 2.0, respectively. The reduction in covalent binding was attributed to the presence of histones, which competed with DNA for the hydrocarbon and reduced the probability of BaP diol epoxide intercalation by tightening the helix. The enhancement ofmore » strand scission was probably due to the catalytic effect of histones on the rate of S-elimination at apurinic sites, although an altered adduct profile or the presence of a repair endonuclease were not excluded. Staphylococcal nuclease digestion indicated that BaP dial epoxide randomly alkylated the minichromosomal DNA. This is in contrast to studies with cellular chromatin where internucleosomal DNA was preferentially modified. Differences in the minichromosomal protein complement were responsible for this altered susceptibility.« less

Systems biology approach identifies the kinase Csnk1a1 as a regulator of the DNA damage response in embryonic stem cells.

PubMed

Carreras Puigvert, Jordi; von Stechow, Louise; Siddappa, Ramakrishnaiah; Pines, Alex; Bahjat, Mahnoush; Haazen, Lizette C J M; Olsen, Jesper V; Vrieling, Harry; Meerman, John H N; Mullenders, Leon H F; van de Water, Bob; Danen, Erik H J

2013-01-22

In pluripotent stem cells, DNA damage triggers loss of pluripotency and apoptosis as a safeguard to exclude damaged DNA from the lineage. An intricate DNA damage response (DDR) signaling network ensures that the response is proportional to the severity of the damage. We combined an RNA interference screen targeting all kinases, phosphatases, and transcription factors with global transcriptomics and phosphoproteomics to map the DDR in mouse embryonic stem cells treated with the DNA cross-linker cisplatin. Networks derived from canonical pathways shared in all three data sets were implicated in DNA damage repair, cell cycle and survival, and differentiation. Experimental probing of these networks identified a mode of DNA damage-induced Wnt signaling that limited apoptosis. Silencing or deleting the p53 gene demonstrated that genotoxic stress elicited Wnt signaling in a p53-independent manner. Instead, this response occurred through reduced abundance of Csnk1a1 (CK1α), a kinase that inhibits β-catenin. Together, our findings reveal a balance between p53-mediated elimination of stem cells (through loss of pluripotency and apoptosis) and Wnt signaling that attenuates this response to tune the outcome of the DDR.

Chromosome demise in the wake of ligase-deficient replication.

PubMed

Kouzminova, Elena A; Kuzminov, Andrei

2012-06-01

Bacterial DNA ligases, NAD⁺-dependent enzymes, are distinct from eukaryotic ATP-dependent ligases, representing promising targets for broad-spectrum antimicrobials. Yet, the chromosomal consequences of ligase-deficient DNA replication, during which Okazaki fragments accumulate, are still unclear. Using ligA251(Ts), the strongest ligase mutant of Escherichia coli, we studied ligase-deficient DNA replication by genetic and physical approaches. Here we show that replication without ligase kills after a short resistance period. We found that double-strand break repair via RecA, RecBCD, RuvABC and RecG explains the transient resistance, whereas irreparable chromosomal fragmentation explains subsequent cell death. Remarkably, death is mostly prevented by elimination of linear DNA degradation activity of ExoV, suggesting that non-allelic double-strand breaks behind replication forks precipitate DNA degradation that enlarge them into allelic double-strand gaps. Marker frequency profiling of synchronized replication reveals stalling of ligase-deficient forks with subsequent degradation of the DNA synthesized without ligase. The mechanism that converts unsealed nicks behind replication forks first into repairable double-strand breaks and then into irreparable double-strand gaps may be behind lethality of any DNA damaging treatment. © 2012 Blackwell Publishing Ltd.

DNA context represents transcription regulation of the gene in mouse embryonic stem cells

NASA Astrophysics Data System (ADS)

Ha, Misook; Hong, Soondo

2016-04-01

Understanding gene regulatory information in DNA remains a significant challenge in biomedical research. This study presents a computational approach to infer gene regulatory programs from primary DNA sequences. Using DNA around transcription start sites as attributes, our model predicts gene regulation in the gene. We find that H3K27ac around TSS is an informative descriptor of the transcription program in mouse embryonic stem cells. We build a computational model inferring the cell-type-specific H3K27ac signatures in the DNA around TSS. A comparison of embryonic stem cell and liver cell-specific H3K27ac signatures in DNA shows that the H3K27ac signatures in DNA around TSS efficiently distinguish the cell-type specific H3K27ac peaks and the gene regulation. The arrangement of the H3K27ac signatures inferred from the DNA represents the transcription regulation of the gene in mESC. We show that the DNA around transcription start sites is associated with the gene regulatory program by specific interaction with H3K27ac.

DNA context represents transcription regulation of the gene in mouse embryonic stem cells.

PubMed

Ha, Misook; Hong, Soondo

2016-04-14

Understanding gene regulatory information in DNA remains a significant challenge in biomedical research. This study presents a computational approach to infer gene regulatory programs from primary DNA sequences. Using DNA around transcription start sites as attributes, our model predicts gene regulation in the gene. We find that H3K27ac around TSS is an informative descriptor of the transcription program in mouse embryonic stem cells. We build a computational model inferring the cell-type-specific H3K27ac signatures in the DNA around TSS. A comparison of embryonic stem cell and liver cell-specific H3K27ac signatures in DNA shows that the H3K27ac signatures in DNA around TSS efficiently distinguish the cell-type specific H3K27ac peaks and the gene regulation. The arrangement of the H3K27ac signatures inferred from the DNA represents the transcription regulation of the gene in mESC. We show that the DNA around transcription start sites is associated with the gene regulatory program by specific interaction with H3K27ac.

Are Alternative Strategies Required to Accelerate the Global Elimination of Lymphatic Filariasis? Insights From Mathematical Models

PubMed Central

Stolk, Wilma A; Prada, Joaquin M; Smith, Morgan E; Kontoroupis, Periklis; de Vos, Anneke S; Touloupou, Panayiota; Irvine, Michael A; Brown, Paul; Subramanian, Swaminathan; Kloek, Marielle; Michael, E; Hollingsworth, T Deirdre; de Vlas, Sake J

2018-01-01

Abstract Background With the 2020 target year for elimination of lymphatic filariasis (LF) approaching, there is an urgent need to assess how long mass drug administration (MDA) programs with annual ivermectin + albendazole (IA) or diethylcarbamazine + albendazole (DA) would still have to be continued, and how elimination can be accelerated. We addressed this using mathematical modeling. Methods We used 3 structurally different mathematical models for LF transmission (EPIFIL, LYMFASIM, TRANSFIL) to simulate trends in microfilariae (mf) prevalence for a range of endemic settings, both for the current annual MDA strategy and alternative strategies, assessing the required duration to bring mf prevalence below the critical threshold of 1%. Results Three annual MDA rounds with IA or DA and good coverage (≥65%) are sufficient to reach the threshold in settings that are currently at mf prevalence <4%, but the required duration increases with increasing mf prevalence. Switching to biannual MDA or employing triple-drug therapy (ivermectin, diethylcarbamazine, and albendazole [IDA]) could reduce program duration by about one-third. Optimization of coverage reduces the time to elimination and is particularly important for settings with a history of poorly implemented MDA (low coverage, high systematic noncompliance). Conclusions Modeling suggests that, in several settings, current annual MDA strategies will be insufficient to achieve the 2020 LF elimination targets, and programs could consider policy adjustment to accelerate, guided by recent monitoring and evaluation data. Biannual treatment and IDA hold promise in reducing program duration, provided that coverage is good, but their efficacy remains to be confirmed by more extensive field studies. PMID:29860286

Are Alternative Strategies Required to Accelerate the Global Elimination of Lymphatic Filariasis? Insights From Mathematical Models.

PubMed

Stolk, Wilma A; Prada, Joaquin M; Smith, Morgan E; Kontoroupis, Periklis; de Vos, Anneke S; Touloupou, Panayiota; Irvine, Michael A; Brown, Paul; Subramanian, Swaminathan; Kloek, Marielle; Michael, E; Hollingsworth, T Deirdre; de Vlas, Sake J

2018-06-01

With the 2020 target year for elimination of lymphatic filariasis (LF) approaching, there is an urgent need to assess how long mass drug administration (MDA) programs with annual ivermectin + albendazole (IA) or diethylcarbamazine + albendazole (DA) would still have to be continued, and how elimination can be accelerated. We addressed this using mathematical modeling. We used 3 structurally different mathematical models for LF transmission (EPIFIL, LYMFASIM, TRANSFIL) to simulate trends in microfilariae (mf) prevalence for a range of endemic settings, both for the current annual MDA strategy and alternative strategies, assessing the required duration to bring mf prevalence below the critical threshold of 1%. Three annual MDA rounds with IA or DA and good coverage (≥65%) are sufficient to reach the threshold in settings that are currently at mf prevalence <4%, but the required duration increases with increasing mf prevalence. Switching to biannual MDA or employing triple-drug therapy (ivermectin, diethylcarbamazine, and albendazole [IDA]) could reduce program duration by about one-third. Optimization of coverage reduces the time to elimination and is particularly important for settings with a history of poorly implemented MDA (low coverage, high systematic noncompliance). Modeling suggests that, in several settings, current annual MDA strategies will be insufficient to achieve the 2020 LF elimination targets, and programs could consider policy adjustment to accelerate, guided by recent monitoring and evaluation data. Biannual treatment and IDA hold promise in reducing program duration, provided that coverage is good, but their efficacy remains to be confirmed by more extensive field studies.

Apoptosis-Like Death in Bacteria Induced by HAMLET, a Human Milk Lipid-Protein Complex

PubMed Central

Hakansson, Anders P.; Roche-Hakansson, Hazeline; Mossberg, Ann-Kristin; Svanborg, Catharina

2011-01-01

Background Apoptosis is the primary means for eliminating unwanted cells in multicellular organisms in order to preserve tissue homeostasis and function. It is characterized by distinct changes in the morphology of the dying cell that are orchestrated by a series of discrete biochemical events. Although there is evidence of primitive forms of programmed cell death also in prokaryotes, no information is available to suggest that prokaryotic death displays mechanistic similarities to the highly regulated programmed death of eukaryotic cells. In this study we compared the characteristics of tumor and bacterial cell death induced by HAMLET, a human milk complex of alpha-lactalbumin and oleic acid. Methodology/Principal Findings We show that HAMLET-treated bacteria undergo cell death with mechanistic and morphologic similarities to apoptotic death of tumor cells. In Jurkat cells and Streptococcus pneumoniae death was accompanied by apoptosis-like morphology such as cell shrinkage, DNA condensation, and DNA degradation into high molecular weight fragments of similar sizes, detected by field inverse gel electrophoresis. HAMLET was internalized into tumor cells and associated with mitochondria, causing a rapid depolarization of the mitochondrial membrane and bound to and induced depolarization of the pneumococcal membrane with similar kinetic and magnitude as in mitochondria. Membrane depolarization in both systems required calcium transport, and both tumor cells and bacteria were found to require serine protease activity (but not caspase activity) to execute cell death. Conclusions/Significance Our results suggest that many of the morphological changes and biochemical responses associated with apoptosis are present in prokaryotes. Identifying the mechanisms of bacterial cell death has the potential to reveal novel targets for future antimicrobial therapy and to further our understanding of core activation mechanisms of cell death in eukaryote cells. PMID:21423701

Apoptosis-like death in bacteria induced by HAMLET, a human milk lipid-protein complex.

PubMed

Hakansson, Anders P; Roche-Hakansson, Hazeline; Mossberg, Ann-Kristin; Svanborg, Catharina

2011-03-10

Apoptosis is the primary means for eliminating unwanted cells in multicellular organisms in order to preserve tissue homeostasis and function. It is characterized by distinct changes in the morphology of the dying cell that are orchestrated by a series of discrete biochemical events. Although there is evidence of primitive forms of programmed cell death also in prokaryotes, no information is available to suggest that prokaryotic death displays mechanistic similarities to the highly regulated programmed death of eukaryotic cells. In this study we compared the characteristics of tumor and bacterial cell death induced by HAMLET, a human milk complex of alpha-lactalbumin and oleic acid. We show that HAMLET-treated bacteria undergo cell death with mechanistic and morphologic similarities to apoptotic death of tumor cells. In Jurkat cells and Streptococcus pneumoniae death was accompanied by apoptosis-like morphology such as cell shrinkage, DNA condensation, and DNA degradation into high molecular weight fragments of similar sizes, detected by field inverse gel electrophoresis. HAMLET was internalized into tumor cells and associated with mitochondria, causing a rapid depolarization of the mitochondrial membrane and bound to and induced depolarization of the pneumococcal membrane with similar kinetic and magnitude as in mitochondria. Membrane depolarization in both systems required calcium transport, and both tumor cells and bacteria were found to require serine protease activity (but not caspase activity) to execute cell death. Our results suggest that many of the morphological changes and biochemical responses associated with apoptosis are present in prokaryotes. Identifying the mechanisms of bacterial cell death has the potential to reveal novel targets for future antimicrobial therapy and to further our understanding of core activation mechanisms of cell death in eukaryote cells.

Newborn screening for cystic fibrosis in Wisconsin: comparison of biochemical and molecular methods.

PubMed

Gregg, R G; Simantel, A; Farrell, P M; Koscik, R; Kosorok, M R; Laxova, A; Laessig, R; Hoffman, G; Hassemer, D; Mischler, E H; Splaingard, M

1997-06-01

To evaluate neonatal screening for cystic fibrosis (CF), including study of the screening procedures and characteristics of false-positive infants, over the past 10 years in Wisconsin. An important objective evolving from the original design has been to compare use of a single-tier immunoreactive trypsinogen (IRT) screening method with that of a two-tier method using IRT and analyses of samples for the most common cystic fibrosis transmembrane regulator (CFTR) (DeltaF508) mutation. We also examined the benefit of including up to 10 additional CFTR mutations in the screening protocol. From 1985 to 1994, using either the IRT or IRT/DNA protocol, 220 862 and 104 308 neonates, respectively, were screened for CF. For the IRT protocol, neonates with an IRT >/=180 ng/mL were considered positive, and the standard sweat chloride test was administered to determine CF status. For the IRT/DNA protocol, samples from the original dried-blood specimen on the Guthrie card of neonates with an IRT >/=110 ng/mL were tested for the presence of the DeltaF508 CFTR allele, and if the DNA test revealed one or two DeltaF508 alleles, a sweat test was obtained. Both screening procedures had very high specificity. The sensitivity tended to be higher with the IRT/DNA protocol, but the differences were not statistically significant. The positive predictive value of the IRT/DNA screening protocol was 15.2% compared with 6.4% if the same samples had been screened by the IRT method. Assessment of the false-positive IRT/DNA population revealed that the two-tier method eliminates the disproportionate number of infants with low Apgar scores and also the high prevalence of African-Americans identified previously in our study of newborns with high IRT levels. We found that 55% of DNA-positive CF infants were homozygous for DeltaF508 and 40% had one DeltaF508 allele. Adding analyses for 10 more CFTR mutations has only a small effect on the sensitivity but is likely to add significantly to the cost of screening. Advantages of the IRT/DNA protocol over IRT analysis include improved positive predictive value, reduction of false-positive infants, and more rapid diagnosis with elimination of recall specimens.

Impact of long-term treatment of onchocerciasis with ivermectin in Ecuador: potential for elimination of infection

PubMed Central

Vieira, Juan Carlos; Cooper, Philip J; Lovato, Raquel; Mancero, Tamara; Rivera, Jorge; Proaño, Roberto; López, Andrea A; Guderian, Ronald H; Guzmán, José Rumbea

2007-01-01

Background Onchocerciasis is a leading cause of blindness worldwide, hence elimination of the infection is an important health priority. Community-based treatment programs with ivermectin form the basis of control programs for the disease in Latin America. The long-term administration of ivermectin could eliminate Onchocerca volvulus infection from endemic areas in Latin America. Methods A strategy of annual to twice-annual treatments with ivermectin has been used for onchocerciasis in endemic communities in Ecuador for up to 14 years. The impact of ivermectin treatment on ocular morbidity, and O. volvulus infection and transmission was monitored in seven sentinel communities. Results Over the period 1990–2003, high rates of treatment coverage of the eligible population were maintained in endemic communities (mean 85.2% per treatment round). Ivermectin reduced the prevalence of anterior segment disease of the eye to 0% in sentinel communities and had a major impact on the prevalence and transmission of infection, with possible elimination of infection in some foci. Conclusion The distribution of ivermectin in endemic communities in Ecuador might have eliminated ocular morbidity and significant progress has been made towards elimination of the infection. A strategy of more frequent treatments with ivermectin may be required in communities where the infection persists to achieve the objective of elimination of the infection from Ecuador. The elimination of the infection from an endemic country in Latin America would be a major public health achievement and could stimulate the implementation of elimination strategies in other endemic countries. PMID:17521449

GENESUS: a two-step sequence design program for DNA nanostructure self-assembly.

PubMed

Tsutsumi, Takanobu; Asakawa, Takeshi; Kanegami, Akemi; Okada, Takao; Tahira, Tomoko; Hayashi, Kenshi

2014-01-01

DNA has been recognized as an ideal material for bottom-up construction of nanometer scale structures by self-assembly. The generation of sequences optimized for unique self-assembly (GENESUS) program reported here is a straightforward method for generating sets of strand sequences optimized for self-assembly of arbitrarily designed DNA nanostructures by a generate-candidates-and-choose-the-best strategy. A scalable procedure to prepare single-stranded DNA having arbitrary sequences is also presented. Strands for the assembly of various structures were designed and successfully constructed, validating both the program and the procedure.

To amend the DNA Analysis Backlog Elimination Act of 2000 to provide for Debbie Smith grants for auditing sexual assault evidence backlogs, and for other purposes.

THOMAS, 112th Congress

Sen. Cornyn, John [R-TX

2012-05-24

Senate - 01/02/2013 Message on House action received in Senate and at desk: House amendments to Senate bill. (All Actions) Tracker: This bill has the status Passed HouseHere are the steps for Status of Legislation:

Genome-wide association of a novel porcine stress-syndrome and isoflurane sensitivity to dystrophin

USDA-ARS?s Scientific Manuscript database

Losses of slaughter-weight pigs due to transport stress are an economic concern to pork producers. Historically, the HAL-1843 mutation in the ryanodine receptor 1 gene was considered responsible for most of the losses; however, DNA testing has effectively eliminated this mutation from commercial her...

32 CFR 291.9 - For official use only (FOUO).

Code of Federal Regulations, 2011 CFR

2011-07-01

...) FREEDOM OF INFORMATION ACT PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM § 291.9... authorized as an anemic form of classification to protect national security interests. See DNA Instruction... papers. Records, such as notes, working papers, and drafts retained as historical evidence of DNA actions...

32 CFR 291.7 - Administrative instruction.

Code of Federal Regulations, 2011 CFR

2011-07-01

... OF INFORMATION ACT PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM § 291.7... requester. The requester must provide a description of the desired record that enables DNA to locate it with..., both on the envelope and in the body of the letter. Persons appealing DNA denial letters should include...

32 CFR 291.4 - Policy.

Code of Federal Regulations, 2010 CFR

2010-07-01

... PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM § 291.4 Policy. (a) Compliance with the FOIA. DNA personnel are expected to comply with the FOIA and this part in both letter and spirit. This strict adherence is necessary to provide uniformity in the implementation of the DNA FOIA...

32 CFR 291.7 - Administrative instruction.

Code of Federal Regulations, 2010 CFR

2010-07-01

... OF INFORMATION ACT PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM § 291.7... requester. The requester must provide a description of the desired record that enables DNA to locate it with..., both on the envelope and in the body of the letter. Persons appealing DNA denial letters should include...

32 CFR 291.4 - Policy.

Code of Federal Regulations, 2011 CFR

2011-07-01

... PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM § 291.4 Policy. (a) Compliance with the FOIA. DNA personnel are expected to comply with the FOIA and this part in both letter and spirit. This strict adherence is necessary to provide uniformity in the implementation of the DNA FOIA...

32 CFR 291.7 - Administrative instruction.

Code of Federal Regulations, 2013 CFR

2013-07-01

... OF INFORMATION ACT PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM § 291.7... requester. The requester must provide a description of the desired record that enables DNA to locate it with..., both on the envelope and in the body of the letter. Persons appealing DNA denial letters should include...

32 CFR 291.4 - Policy.

Code of Federal Regulations, 2013 CFR

2013-07-01

... PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM § 291.4 Policy. (a) Compliance with the FOIA. DNA personnel are expected to comply with the FOIA and this part in both letter and spirit. This strict adherence is necessary to provide uniformity in the implementation of the DNA FOIA...

32 CFR 291.9 - For official use only (FOUO).

Code of Federal Regulations, 2013 CFR

2013-07-01

...) FREEDOM OF INFORMATION ACT PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM § 291.9... authorized as an anemic form of classification to protect national security interests. See DNA Instruction... papers. Records, such as notes, working papers, and drafts retained as historical evidence of DNA actions...

32 CFR 291.4 - Policy.

Code of Federal Regulations, 2012 CFR

2012-07-01

... PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM § 291.4 Policy. (a) Compliance with the FOIA. DNA personnel are expected to comply with the FOIA and this part in both letter and spirit. This strict adherence is necessary to provide uniformity in the implementation of the DNA FOIA...

32 CFR 291.7 - Administrative instruction.

Code of Federal Regulations, 2014 CFR

2014-07-01

... OF INFORMATION ACT PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM § 291.7... requester. The requester must provide a description of the desired record that enables DNA to locate it with..., both on the envelope and in the body of the letter. Persons appealing DNA denial letters should include...

32 CFR 291.4 - Policy.

Code of Federal Regulations, 2014 CFR

2014-07-01

... PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM § 291.4 Policy. (a) Compliance with the FOIA. DNA personnel are expected to comply with the FOIA and this part in both letter and spirit. This strict adherence is necessary to provide uniformity in the implementation of the DNA FOIA...

32 CFR 291.7 - Administrative instruction.

Code of Federal Regulations, 2012 CFR

2012-07-01

... OF INFORMATION ACT PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM § 291.7... requester. The requester must provide a description of the desired record that enables DNA to locate it with..., both on the envelope and in the body of the letter. Persons appealing DNA denial letters should include...

Endogenous overexpression of an active phosphorylated form of DNA polymerase β under oxidative stress in Trypanosoma cruzi.

PubMed

Rojas, Diego A; Urbina, Fabiola; Moreira-Ramos, Sandra; Castillo, Christian; Kemmerling, Ulrike; Lapier, Michel; Maya, Juan Diego; Solari, Aldo; Maldonado, Edio

2018-02-01

Trypanosoma cruzi is exposed during its life to exogenous and endogenous oxidative stress, leading to damage of several macromolecules such as DNA. There are many DNA repair pathways in the nucleus and mitochondria (kinetoplast), where specific protein complexes detect and eliminate damage to DNA. One group of these proteins is the DNA polymerases. In particular, Tc DNA polymerase β participates in kinetoplast DNA replication and repair. However, the mechanisms which control its expression under oxidative stress are still unknown. Here we describe the effect of oxidative stress on the expression and function of Tc DNA polymerase β To this end parasite cells (epimastigotes and trypomastigotes) were exposed to peroxide during short periods of time. Tc DNA polymerase β which was associated physically with kinetoplast DNA, showed increased protein levels in response to peroxide damage in both parasite forms analyzed. Two forms of DNA polymerase β were identified and overexpressed after peroxide treatment. One of them was phosphorylated and active in DNA synthesis after renaturation on polyacrylamide electrophoresis gel. This phosphorylated form showed 3-4-fold increase in both parasite forms. Our findings indicate that these increments in protein levels are not under transcriptional control because the level of Tc DNA polymerase β mRNA is maintained or slightly decreased during the exposure to oxidative stress. We propose a mechanism where a DNA repair pathway activates a cascade leading to the increment of expression and phosphorylation of Tc DNA polymerase β in response to oxidative damage, which is discussed in the context of what is known in other trypanosomes which lack transcriptional control.

Optimum sensitivity derivatives of objective functions in nonlinear programming

NASA Technical Reports Server (NTRS)

Barthelemy, J.-F. M.; Sobieszczanski-Sobieski, J.

1983-01-01

The feasibility of eliminating second derivatives from the input of optimum sensitivity analyses of optimization problems is demonstrated. This elimination restricts the sensitivity analysis to the first-order sensitivity derivatives of the objective function. It is also shown that when a complete first-order sensitivity analysis is performed, second-order sensitivity derivatives of the objective function are available at little additional cost. An expression is derived whose application to linear programming is presented.

Figures of Merit for Aeronautics Programs and Addition to NASA LARC Fire Station

NASA Technical Reports Server (NTRS)

Harper, Belinda M.

1995-01-01

This report accounts details of two research projects for the Langley Aerospace Research Summer Scholars (LARSS) program. The first project, with the Office of Mission Assurance, involved subjectively predicting the probable success of two aeronautics programs by means of a tool called a Figure of Merit. The figure of merit bases program success on the quality and reliability of the following factors: parts, complexity of research, quality programs, hazards elimination, and single point failures elimination. The second project, for the Office of Safety and Facilities Assurance, required planning, layouts, and source seeking for an addition to the fire house. Forecasted changes in facility layout necessitate this addition which will serve as housing for the fire fighters.

Evolution of rDNA in Nicotiana Allopolyploids: A Potential Link between rDNA Homogenization and Epigenetics

PubMed Central

Kovarik, Ales; Dadejova, Martina; Lim, Yoong K.; Chase, Mark W.; Clarkson, James J.; Knapp, Sandra; Leitch, Andrew R.

2008-01-01

Background The evolution and biology of rDNA have interested biologists for many years, in part, because of two intriguing processes: (1) nucleolar dominance and (2) sequence homogenization. We review patterns of evolution in rDNA in the angiosperm genus Nicotiana to determine consequences of allopolyploidy on these processes. Scope Allopolyploid species of Nicotiana are ideal for studying rDNA evolution because phylogenetic reconstruction of DNA sequences has revealed patterns of species divergence and their parents. From these studies we also know that polyploids formed over widely different timeframes (thousands to millions of years), enabling comparative and temporal studies of rDNA structure, activity and chromosomal distribution. In addition studies on synthetic polyploids enable the consequences of de novo polyploidy on rDNA activity to be determined. Conclusions We propose that rDNA epigenetic expression patterns established even in F1 hybrids have a material influence on the likely patterns of divergence of rDNA. It is the active rDNA units that are vulnerable to homogenization, which probably acts to reduce mutational load across the active array. Those rDNA units that are epigenetically silenced may be less vulnerable to sequence homogenization. Selection cannot act on these silenced genes, and they are likely to accumulate mutations and eventually be eliminated from the genome. It is likely that whole silenced arrays will be deleted in polyploids of 1 million years of age and older. PMID:18310159

Identification of Disubstituted Sulfonamide Compounds as Specific Inhibitors of Hepatitis B Virus Covalently Closed Circular DNA Formation

PubMed Central

Cai, Dawei; Mills, Courtney; Yu, Wenquan; Yan, Ran; Aldrich, Carol E.; Saputelli, Jeffry R.; Mason, William S.; Xu, Xiaodong; Guo, Ju-Tao; Block, Timothy M.

2012-01-01

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a central role in viral infection and persistence and is the basis for viral rebound after the cessation of therapy, as well as the elusiveness of a cure even after extended treatment. Therefore, there is an urgent need for the development of novel therapeutic agents that directly target cccDNA formation and maintenance. By employing an innovative cell-based cccDNA assay in which secreted HBV e antigen is a cccDNA-dependent surrogate, we screened an in-house small-molecule library consisting of 85,000 drug-like compounds. Two structurally related disubstituted sulfonamides (DSS), termed CCC-0975 and CCC-0346, emerged and were confirmed as inhibitors of cccDNA production, with low micromolar 50% effective concentrations (EC50s) in cell culture. Further mechanistic studies demonstrated that DSS compound treatment neither directly inhibited HBV DNA replication in cell culture nor reduced viral polymerase activity in the in vitro endogenous polymerase assay but synchronously reduced the levels of HBV cccDNA and its putative precursor, deproteinized relaxed circular DNA (DP-rcDNA). However, DSS compounds did not promote the intracellular decay of HBV DP-rcDNA and cccDNA, suggesting that the compounds interfere primarily with rcDNA conversion into cccDNA. In addition, we demonstrated that CCC-0975 was able to reduce cccDNA biosynthesis in duck HBV-infected primary duck hepatocytes. This is the first attempt, to our knowledge, to identify small molecules that target cccDNA formation, and DSS compounds thus potentially serve as proof-of-concept drug candidates for development into therapeutics to eliminate cccDNA from chronic HBV infection. PMID:22644022

JavaScript DNA translator: DNA-aligned protein translations.

PubMed

Perry, William L

2002-12-01

There are many instances in molecular biology when it is necessary to identify ORFs in a DNA sequence. While programs exist for displaying protein translations in multiple ORFs in alignment with a DNA sequence, they are often expensive, exist as add-ons to software that must be purchased, or are only compatible with a particular operating system. JavaScript DNA Translator is a shareware application written in JavaScript, a scripting language interpreted by the Netscape Communicator and Internet Explorer Web browsers, which makes it compatible with several different operating systems. While the program uses a familiar Web page interface, it requires no connection to the Internet since calculations are performed on the user's own computer. The program analyzes one or multiple DNA sequences and generates translations in up to six reading frames aligned to a DNA sequence, in addition to displaying translations as separate sequences in FASTA format. ORFs within a reading frame can also be displayed as separate sequences. Flexible formatting options are provided, including the ability to hide ORFs below a minimum size specified by the user. The program is available free of charge at the BioTechniques Software Library (www.Biotechniques.com).

40 CFR 122.46 - Duration of permits (applicable to State programs, see § 123.25).

Code of Federal Regulations, 2011 CFR

2011-07-01

... AGENCY (CONTINUED) WATER PROGRAMS EPA ADMINISTERED PERMIT PROGRAMS: THE NATIONAL POLLUTANT DISCHARGE ELIMINATION SYSTEM Permit Conditions § 122.46 Duration of permits (applicable to State programs, see § 123.25...

Role of the C-terminal residue of the DNA polymerase of bacteriophage T7.

PubMed

Kumar, J K; Tabor, S; Richardson, C C

2001-09-14

The crystal structure of the DNA polymerase encoded by gene 5 of bacteriophage T7, in a complex with its processivity factor, Escherichia coli thioredoxin, a primer-template, and an incoming deoxynucleoside triphosphate reveals a putative hydrogen bond between the C-terminal residue, histidine 704 of gene 5 protein, and an oxygen atom on the penultimate phosphate diester of the primer strand. Elimination of this electrostatic interaction by replacing His(704) with alanine renders the phage nonviable, and no DNA synthesis is observed in vivo. Polymerase activity of the genetically altered enzyme on primed M13 DNA is only 12% of the wild-type enzyme, and its processivity is drastically reduced. Kinetic parameters for binding a primer-template (K(D)(app)), nucleotide binding (K(m)), and k(off) for dissociation of the altered polymerase from a primer-template are not significantly different from that of wild-type T7 DNA polymerase. However, the decrease in polymerase activity is concomitant with increased hydrolytic activity, judging from the turnover of nucleoside triphosphate into the corresponding nucleoside monophosphate (percentage of turnover, 65%) during DNA synthesis. Biochemical data along with structural observations imply that the terminal amino acid residue of T7 DNA polymerase plays a critical role in partitioning DNA between the polymerase and exonuclease sites.

Programming Self-Assembly of DNA Origami Honeycomb Two-Dimensional Lattices and Plasmonic Metamaterials.

PubMed

Wang, Pengfei; Gaitanaros, Stavros; Lee, Seungwoo; Bathe, Mark; Shih, William M; Ke, Yonggang

2016-06-22

Scaffolded DNA origami has proven to be a versatile method for generating functional nanostructures with prescribed sub-100 nm shapes. Programming DNA-origami tiles to form large-scale 2D lattices that span hundreds of nanometers to the micrometer scale could provide an enabling platform for diverse applications ranging from metamaterials to surface-based biophysical assays. Toward this end, here we design a family of hexagonal DNA-origami tiles using computer-aided design and demonstrate successful self-assembly of micrometer-scale 2D honeycomb lattices and tubes by controlling their geometric and mechanical properties including their interconnecting strands. Our results offer insight into programmed self-assembly of low-defect supra-molecular DNA-origami 2D lattices and tubes. In addition, we demonstrate that these DNA-origami hexagon tiles and honeycomb lattices are versatile platforms for assembling optical metamaterials via programmable spatial arrangement of gold nanoparticles (AuNPs) into cluster and superlattice geometries.

Comprehensive splicing functional analysis of DNA variants of the BRCA2 gene by hybrid minigenes

PubMed Central

2012-01-01

Introduction The underlying pathogenic mechanism of a large fraction of DNA variants of disease-causing genes is the disruption of the splicing process. We aimed to investigate the effect on splicing of the BRCA2 variants c.8488-1G > A (exon 20) and c.9026_9030del (exon 23), as well as 41 BRCA2 variants reported in the Breast Cancer Information Core (BIC) mutation database. Methods DNA variants were analyzed with the splicing prediction programs NNSPLICE and Human Splicing Finder. Functional analyses of candidate variants were performed by lymphocyte RT-PCR and/or hybrid minigene assays. Forty-one BIC variants of exons 19, 20, 23 and 24 were bioinformatically selected and generated by PCR-mutagenesis of the wild type minigenes. Results Lymphocyte RT-PCR of c.8488-1G > A showed intron 19 retention and a 12-nucleotide deletion in exon 20, whereas c.9026_9030del did not show any splicing anomaly. Minigene analysis of c.8488-1G > A displayed the aforementioned aberrant isoforms but also exon 20 skipping. We further evaluated the splicing outcomes of 41 variants of four BRCA2 exons by minigene analysis. Eighteen variants presented splicing aberrations. Most variants (78.9%) disrupted the natural splice sites, whereas four altered putative enhancers/silencers and had a weak effect. Fluorescent RT-PCR of minigenes accurately detected 14 RNA isoforms generated by cryptic site usage, exon skipping and intron retention events. Fourteen variants showed total splicing disruptions and were predicted to truncate or eliminate essential domains of BRCA2. Conclusions A relevant proportion of BRCA2 variants are correlated with splicing disruptions, indicating that RNA analysis is a valuable tool to assess the pathogenicity of a particular DNA change. The minigene system is a straightforward and robust approach to detect variants with an impact on splicing and contributes to a better knowledge of this gene expression step. PMID:22632462

The Zinc-finger protein ASCIZ regulates B cell development via DYNLL1 and Bim

PubMed Central

Jurado, Sabine; Gleeson, Kimberly; O’Donnell, Kristy; Izon, David J.; Walkley, Carl R.; Strasser, Andreas; Tarlinton, David M.

2012-01-01

Developing B lymphocytes expressing defective or autoreactive pre-B or B cell receptors (BCRs) are eliminated by programmed cell death, but how the balance between death and survival signals is regulated to prevent immunodeficiency and autoimmunity remains incompletely understood. In this study, we show that absence of the essential ATM (ataxia telangiectasia mutated) substrate Chk2-interacting Zn2+-finger protein (ASCIZ; also known as ATMIN/ZNF822), a protein with dual functions in the DNA damage response and as a transcription factor, leads to progressive cell loss from the pre-B stage onwards and severely diminished splenic B cell numbers in mice. This lymphopenia cannot be suppressed by deletion of p53 or complementation with a prearranged BCR, indicating that it is not caused by impaired DNA damage responses or defective V(D)J recombination. Instead, ASCIZ-deficient B cell precursors contain highly reduced levels of DYNLL1 (dynein light chain 1; LC8), a recently identified transcriptional target of ASCIZ, and normal B cell development can be restored by ectopic Dynll1 expression. Remarkably, the B cell lymphopenia in the absence of ASCIZ can also be fully suppressed by deletion of the proapoptotic DYNLL1 target Bim. Our findings demonstrate a key role for ASCIZ in regulating the survival of developing B cells by activating DYNLL1 expression, which may then modulate Bim-dependent apoptosis. PMID:22891272

32 CFR 291.2 - Applicability.

Code of Federal Regulations, 2011 CFR

2011-07-01

... INFORMATION ACT PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM § 291.2 Applicability. This part applies to Headquarters, Defense Nuclear Agency (HQ, DNA), Field Command, Defense Nuclear...

32 CFR 291.2 - Applicability.

Code of Federal Regulations, 2010 CFR

2010-07-01

... INFORMATION ACT PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM § 291.2 Applicability. This part applies to Headquarters, Defense Nuclear Agency (HQ, DNA), Field Command, Defense Nuclear...

32 CFR 291.2 - Applicability.

Code of Federal Regulations, 2012 CFR

2012-07-01

... INFORMATION ACT PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM § 291.2 Applicability. This part applies to Headquarters, Defense Nuclear Agency (HQ, DNA), Field Command, Defense Nuclear...

32 CFR 291.2 - Applicability.

Code of Federal Regulations, 2013 CFR

2013-07-01

... INFORMATION ACT PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM § 291.2 Applicability. This part applies to Headquarters, Defense Nuclear Agency (HQ, DNA), Field Command, Defense Nuclear...

32 CFR 291.2 - Applicability.

Code of Federal Regulations, 2014 CFR

2014-07-01

... INFORMATION ACT PROGRAM DEFENSE NUCLEAR AGENCY (DNA) FREEDOM OF INFORMATION ACT PROGRAM § 291.2 Applicability. This part applies to Headquarters, Defense Nuclear Agency (HQ, DNA), Field Command, Defense Nuclear...

Does Tyrosyl DNA Phosphodiesterase-2 Play a Role in Hepatitis B Virus Genome Repair?

PubMed Central

Boregowda, Rajeev; Sohn, Ji A.; Ledesma, Felipe Cortes; Caldecott, Keith W.; Seeger, Christoph; Hu, Jianming

2015-01-01

Hepatitis B virus (HBV) replication and persistence are sustained by a nuclear episome, the covalently closed circular (CCC) DNA, which serves as the transcriptional template for all viral RNAs. CCC DNA is converted from a relaxed circular (RC) DNA in the virion early during infection as well as from RC DNA in intracellular progeny nucleocapsids via an intracellular amplification pathway. Current antiviral therapies suppress viral replication but cannot eliminate CCC DNA. Thus, persistence of CCC DNA remains an obstacle toward curing chronic HBV infection. Unfortunately, very little is known about how CCC DNA is formed. CCC DNA formation requires removal of the virally encoded reverse transcriptase (RT) protein from the 5’ end of the minus strand of RC DNA. Tyrosyl DNA phosphodiesterase-2 (Tdp2) was recently identified as the enzyme responsible for cleavage of tyrosyl-5’ DNA linkages formed between topoisomerase II and cellular DNA. Because the RT-DNA linkage is also a 5’ DNA-phosphotyrosyl bond, it has been hypothesized that Tdp2 might be one of several elusive host factors required for CCC DNA formation. Therefore, we examined the role of Tdp2 in RC DNA deproteination and CCC DNA formation. We demonstrated Tdp2 can cleave the tyrosyl-minus strand DNA linkage using authentic HBV RC DNA isolated from nucleocapsids and using RT covalently linked to short minus strand DNA produced in vitro. On the other hand, our results showed that Tdp2 gene knockout did not block CCC DNA formation during HBV infection of permissive human hepatoma cells and did not prevent intracellular amplification of duck hepatitis B virus CCC DNA. These results indicate that although Tdp2 can remove the RT covalently linked to the 5’ end of the HBV minus strand DNA in vitro, this protein might not be required for CCC DNA formation in vivo. PMID:26079492

Microsatellite DNA Analysis Revealed a Drastic Genetic Change of Plasmodium vivax Population in the Republic of Korea During 2002 and 2003

PubMed Central

Iwagami, Moritoshi; Hwang, Seung-Young; Kim, So-Hee; Park, So-Jung; Lee, Ga-Young; Matsumoto-Takahashi, Emilie Louise Akiko; Kho, Weon-Gyu; Kano, Shigeyuki

2013-01-01

Background Vivax malaria was successfully eliminated in the Republic of Korea (South Korea) in the late 1970s, but it was found to have re-emerged from 1993. In order to control malaria and evaluate the effectiveness of malaria controls, it is important to develop a spatiotemporal understanding of the genetic structure of the parasite population. Here, we estimated the population structure and temporal dynamics of the transmission of Plasmodium vivax in South Korea by analyzing microsatellite DNA markers of the parasite. Methodology/Principal Findings We analyzed 14 microsatellite DNA loci of the P. vivax genome from 163 South Korean isolates collected from 1994 to 2008. Allelic data were used to analyze linkage disequilibrium (LD), genetic differentiation and population structure, in order to make a detailed estimate of temporal change in the parasite population. The LD analysis showed a gradual decrease in LD levels, while the levels of genetic differentiation between successive years and analysis of the population structure based on the Bayesian approach suggested that a drastic genetic change occurred in the South Korean population during 2002 and 2003. Conclusions/Significance Although relapse and asymptomatic parasite carriage might influence the population structure to some extent, our results suggested the continual introduction of P. vivax into South Korea through other parasite population sources. One possible source, particularly during 2002 and 2003, is North Korea. Molecular epidemiology using microsatellite DNA of the P. vivax population is effective for assessing the population structure and temporal dynamics of parasite transmission; information that can assist in the elimination of vivax malaria in endemic areas. PMID:24205429

GUI to Facilitate Research on Biological Damage from Radiation

NASA Technical Reports Server (NTRS)

Cucinotta, Frances A.; Ponomarev, Artem Lvovich

2010-01-01

A graphical-user-interface (GUI) computer program has been developed to facilitate research on the damage caused by highly energetic particles and photons impinging on living organisms. The program brings together, into one computational workspace, computer codes that have been developed over the years, plus codes that will be developed during the foreseeable future, to address diverse aspects of radiation damage. These include codes that implement radiation-track models, codes for biophysical models of breakage of deoxyribonucleic acid (DNA) by radiation, pattern-recognition programs for extracting quantitative information from biological assays, and image-processing programs that aid visualization of DNA breaks. The radiation-track models are based on transport models of interactions of radiation with matter and solution of the Boltzmann transport equation by use of both theoretical and numerical models. The biophysical models of breakage of DNA by radiation include biopolymer coarse-grained and atomistic models of DNA, stochastic- process models of deposition of energy, and Markov-based probabilistic models of placement of double-strand breaks in DNA. The program is designed for use in the NT, 95, 98, 2000, ME, and XP variants of the Windows operating system.

Community Attitudes toward Mass Drug Administration for Control and Elimination of Neglected Tropical Diseases after the 2014 Outbreak of Ebola Virus Disease in Lofa County, Liberia

PubMed Central

Bogus, Joshua; Gankpala, Lincoln; Fischer, Kerstin; Krentel, Alison; Weil, Gary J.; Fischer, Peter U.; Kollie, Karsor; Bolay, Fatorma K.

2016-01-01

The recent outbreak of Ebola virus disease (EVD) interrupted mass drug administration (MDA) programs to control and eliminate neglected tropical diseases in Liberia. MDA programs treat entire communities with medication regardless of infection status to interrupt transmission and eliminate lymphatic filariasis and onchocerciasis. Following reports of hostilities toward health workers and fear that they might be spreading EVD, it was important to determine whether attitudes toward MDA might have changed after the outbreak. We surveyed 140 community leaders from 32 villages in Lofa County, Liberia, that had previously participated in MDA and are located in an area that was an early epicenter of the EVD outbreak. Survey respondents reported a high degree of community trust in the MDA program, and 97% thought their communities were ready to resume MDA. However, respondents predicted that fewer people would comply with MDA after the EVD epidemic than before. The survey also uncovered fears in the community that EVD and MDA might be linked. Respondents suggested that MDA programs emphasize to people that the medications are identical to those previously distributed and that MDA programs have nothing to do with EVD. PMID:26666700

SDM-Assist software to design site-directed mutagenesis primers introducing “silent” restriction sites

PubMed Central

2013-01-01

Background Over the past decades site-directed mutagenesis (SDM) has become an indispensable tool for biological structure-function studies. In principle, SDM uses modified primer pairs in a PCR reaction to introduce a mutation in a cDNA insert. DpnI digestion of the reaction mixture is used to eliminate template copies before amplification in E. coli; however, this process is inefficient resulting in un-mutated clones which can only be distinguished from mutant clones by sequencing. Results We have developed a program – ‘SDM-Assist’ which creates SDM primers adding a specific identifier: through additional silent mutations a restriction site is included or a previous one removed which allows for highly efficient identification of ‘mutated clones’ by a simple restriction digest. Conclusions The direct identification of SDM clones will save time and money for researchers. SDM-Assist also scores the primers based on factors such as Tm, GC content and secondary structure allowing for simplified selection of optimal primer pairs. PMID:23522286

Cellular chromosome DNA interferes with fluorescence quantitative real-time PCR detection of HBV DNA in culture medium.

PubMed

Pan, Xiao-Ben; Wei, Lai; Han, Jin-Chao; Gao, Yan

2008-01-01

Fluorescence quantitative real-time PCR (FQ-PCR) is a recently developed technique increasingly used for clinical diagnosis by detection of hepatitis B virus (HBV) DNA in serum. FQ-PCR is also used in scientific research for detection of HBV DNA in cell culture. Understanding potential FQ-PCR interference factors can improve the accuracy of HBV DNA quantification in cell culture medium. HBV positive serum was diluted with culture medium to produce three test groups with HBV DNA levels of 5 x 10(7) copies/ml (high), 5 x 10(5) copies/ml (medium), and 5 x 10(3) copies/ml (low). Chromosome DNA was extracted from HepG2 cells and then added to high, medium, and low group samples at final concentrations of 0, 12.5, 25, 50, and 100 microg/ml. The samples were quantified by FQ-PCR and data were evaluated using statistical software. No marked changes were seen in the quantitative curves for high level HBV DNA samples when the samples were supplemented with 0-100 microg/ml of chromosome DNA. Interference was observed in medium level samples when 50 and 100 microg/ml of chromosome DNA was added. Interference was also observed in low level HBV DNA samples when the concentration of added chromosome DNA was greater than 25 microg/ml. The interference was eliminated when samples were digested by DNase I prior to PCR detection. In Conclusions, the presence of cellular chromosome DNA can interfere with the detection of HBV DNA by FQ-PCR. Removal of cellular chromosome DNA from culture media prior to FQ-PCR is necessary for reliable HBV DNA quantitative detection. (c) 2007 Wiley-Liss, Inc.

Existing and emerging detection technologies for DNA (Deoxyribonucleic Acid) finger printing, sequencing, bio- and analytical chips: a multidisciplinary development unifying molecular biology, chemical and electronics engineering.

PubMed

Kumar Khanna, Vinod

2007-01-01

The current status and research trends of detection techniques for DNA-based analysis such as DNA finger printing, sequencing, biochips and allied fields are examined. An overview of main detectors is presented vis-à-vis these DNA operations. The biochip method is explained, the role of micro- and nanoelectronic technologies in biochip realization is highlighted, various optical and electrical detection principles employed in biochips are indicated, and the operational mechanisms of these detection devices are described. Although a diversity of biochips for diagnostic and therapeutic applications has been demonstrated in research laboratories worldwide, only some of these chips have entered the clinical market, and more chips are awaiting commercialization. The necessity of tagging is eliminated in refractive-index change based devices, but the basic flaw of indirect nature of most detection methodologies can only be overcome by generic and/or reagentless DNA sensors such as the conductance-based approach and the DNA-single electron transistor (DNA-SET) structure. Devices of the electrical detection-based category are expected to pave the pathway for the next-generation DNA chips. The review provides a comprehensive coverage of the detection technologies for DNA finger printing, sequencing and related techniques, encompassing a variety of methods from the primitive art to the state-of-the-art scenario as well as promising methods for the future.

40 CFR 122.64 - Termination of permits (applicable to State programs, see § 123.25).

Code of Federal Regulations, 2010 CFR

2010-07-01

... AGENCY (CONTINUED) WATER PROGRAMS EPA ADMINISTERED PERMIT PROGRAMS: THE NATIONAL POLLUTANT DISCHARGE ELIMINATION SYSTEM Transfer, Modification, Revocation and Reissuance, and Termination of Permits § 122.64...

40 CFR 122.43 - Establishing permit conditions (applicable to State programs, see § 123.25).

Code of Federal Regulations, 2011 CFR

2011-07-01

... PROTECTION AGENCY (CONTINUED) WATER PROGRAMS EPA ADMINISTERED PERMIT PROGRAMS: THE NATIONAL POLLUTANT DISCHARGE ELIMINATION SYSTEM Permit Conditions § 122.43 Establishing permit conditions (applicable to State...

40 CFR 122.61 - Transfer of permits (applicable to State programs, see § 123.25).

Code of Federal Regulations, 2010 CFR

2010-07-01

... AGENCY (CONTINUED) WATER PROGRAMS EPA ADMINISTERED PERMIT PROGRAMS: THE NATIONAL POLLUTANT DISCHARGE ELIMINATION SYSTEM Transfer, Modification, Revocation and Reissuance, and Termination of Permits § 122.61...

40 CFR 122.64 - Termination of permits (applicable to State programs, see § 123.25).

Code of Federal Regulations, 2011 CFR

2011-07-01

... AGENCY (CONTINUED) WATER PROGRAMS EPA ADMINISTERED PERMIT PROGRAMS: THE NATIONAL POLLUTANT DISCHARGE ELIMINATION SYSTEM Transfer, Modification, Revocation and Reissuance, and Termination of Permits § 122.64...

40 CFR 122.61 - Transfer of permits (applicable to State programs, see § 123.25).

Code of Federal Regulations, 2011 CFR

2011-07-01

... AGENCY (CONTINUED) WATER PROGRAMS EPA ADMINISTERED PERMIT PROGRAMS: THE NATIONAL POLLUTANT DISCHARGE ELIMINATION SYSTEM Transfer, Modification, Revocation and Reissuance, and Termination of Permits § 122.61...

40 CFR 122.43 - Establishing permit conditions (applicable to State programs, see § 123.25).

Code of Federal Regulations, 2010 CFR

2010-07-01

... PROTECTION AGENCY (CONTINUED) WATER PROGRAMS EPA ADMINISTERED PERMIT PROGRAMS: THE NATIONAL POLLUTANT DISCHARGE ELIMINATION SYSTEM Permit Conditions § 122.43 Establishing permit conditions (applicable to State...

Shuffle Optimizer: A Program to Optimize DNA Shuffling for Protein Engineering.

PubMed

Milligan, John N; Garry, Daniel J

2017-01-01

DNA shuffling is a powerful tool to develop libraries of variants for protein engineering. Here, we present a protocol to use our freely available and easy-to-use computer program, Shuffle Optimizer. Shuffle Optimizer is written in the Python computer language and increases the nucleotide homology between two pieces of DNA desired to be shuffled together without changing the amino acid sequence. In addition we also include sections on optimal primer design for DNA shuffling and library construction, a small-volume ultrasonicator method to create sheared DNA, and finally a method to reassemble the sheared fragments and recover and clone the library. The Shuffle Optimizer program and these protocols will be useful to anyone desiring to perform any of the nucleotide homology-dependent shuffling methods.

Designing DNA nanodevices for compatibility with the immune system of higher organisms

NASA Astrophysics Data System (ADS)

Surana, Sunaina; Shenoy, Avinash R.; Krishnan, Yamuna

2015-09-01

DNA is proving to be a powerful scaffold to construct molecularly precise designer DNA devices. Recent trends reveal their ever-increasing deployment within living systems as delivery devices that not only probe but also program and re-program a cell, or even whole organisms. Given that DNA is highly immunogenic, we outline the molecular, cellular and organismal response pathways that designer nucleic acid nanodevices are likely to elicit in living systems. We address safety issues applicable when such designer DNA nanodevices interact with the immune system. In light of this, we discuss possible molecular programming strategies that could be integrated with such designer nucleic acid scaffolds to either evade or stimulate the host response with a view to optimizing and widening their applications in higher organisms.

Escherichia coli mutants thermosensitive for deoxyribonucleic acid gyrase subunit A: effects on deoxyribonucleic acid replication, transcription, and bacteriophage growth.

PubMed

Kreuzer, K N; Cozzarelli, N R

1979-11-01

Temperature-sensitive nalA mutants of Escherichia coli have been used to investigate the structure and functions of deoxyribonucleic acid (DNA) gyrase. Extracts of one such mutant (nalA43) had thermosensitive DNA gyrase subunit A activity but normal gyrase subunit B activity, proving definitively that nalA is the structural gene for subunit A. Extracts of a second nalA (Ts) mutant (nalA45) had a 50-fold deficiency of gyrase subunit A activity. The residual DNA supertwisting was catalyzed by the mutant DNA gyrase rather than by a novel supertwisting enzyme. The nalA45(Ts) extract was also deficient in the nalidixic acid target, which is defined as the protein necessary to confer drug sensitivity to in vitro DNA replication directed by a nalidixic acid-resistant mutant extract. Thus, gyrase subunit A and the nalidixic acid target are one and the same protein, the nalA gene product. Shift of the nalA43(Ts) mutant to a nonpermissive temperature resulted in a precipitous decline in the rate of [(3)H]thymidine incorporation, demonstrating an obligatory role of the nalA gene product in DNA replication. The rates of incorporation of [(3)H]uridine pulses and continuously administered [(3)H]uracil were quickly reduced approximately twofold upon temperature shift of the nalA43(Ts) mutant, and therefore some but not all transcription requires the nalA gene product. The thermosensitive growth of bacteriophages phiX174 and T4 in the nalA43(Ts) host shows that these phages depend on the host nalA gene product. In contrast, the growth of phage T7 was strongly inhibited by nalidixic acid but essentially unaffected by the nalA43(Ts) mutation. The inhibition of T7 growth by nalidixic acid was, however, eliminated by temperature inactivation of the nal43 gene product. Therefore, nalidixic acid may block T7 growth by a corruption rather than a simple elimination of the nalidixic acid target. Possible mechanisms for such a corruption are considered, and their relevance to the puzzling dominance of drug sensitivity is discussed.

Rapid and sensitive detection of Citrus Bacterial Canker by loop-mediated isothermal amplification combined with simple visual evaluation methods

PubMed Central

2010-01-01

Background Citrus Bacterial Canker (CBC) is a major, highly contagious disease of citrus plants present in many countries in Asia, Africa and America, but not in the Mediterranean area. There are three types of Citrus Bacterial Canker, named A, B, and C that have different genotypes and posses variation in host range within citrus species. The causative agent for type A CBC is Xanthomonas citri subsp. citri, while Xanthomonas fuscans subsp. aurantifolii, strain B causes type B CBC and Xanthomonas fuscans subsp. aurantifolii strain C causes CBC type C. The early and accurate identification of those bacteria is essential for the protection of the citrus industry. Detection methods based on bacterial isolation, antibodies or polymerase chain reaction (PCR) have been developed previously; however, these approaches may be time consuming, laborious and, in the case of PCR, it requires expensive laboratory equipment. Loop-mediated isothermal amplification (LAMP), which is a novel isothermal DNA amplification technique, is sensitive, specific, fast and requires no specialized laboratory equipment. Results A loop-mediated isothermal amplification assay for the diagnosis of Citrus Bacterial Canker (CBC-LAMP) was developed and evaluated. DNA samples were obtained from infected plants or cultured bacteria. A typical ladder-like pattern on gel electrophoresis was observed in all positive samples in contrast to the negative controls. In addition, amplification products were detected by visual inspection using SYBRGreen and using a lateral flow dipstick, eliminating the need for gel electrophoresis. The sensitivity and specificity of the assay were evaluated in different conditions and using several sample sources which included purified DNA, bacterium culture and infected plant tissue. The sensitivity of the CBC-LAMP was 10 fg of pure Xcc DNA, 5 CFU in culture samples and 18 CFU in samples of infected plant tissue. No cross reaction was observed with DNA of other phytopathogenic bacteria. The assay was capable of detecting CBC-causing strains from several geographical origins and pathotypes. Conclusions The CBC-LAMP technique is a simple, fast, sensitive and specific method for the diagnosis of Citrus Bacterial Canker. This method can be useful in the phytosanitary programs of the citrus industry worldwide. PMID:20565886

The combination of kinetic and flow cytometric semen parameters as a tool to predict fertility in cryopreserved bull semen.

PubMed

Gliozzi, T M; Turri, F; Manes, S; Cassinelli, C; Pizzi, F

2017-11-01

Within recent years, there has been growing interest in the prediction of bull fertility through in vitro assessment of semen quality. A model for fertility prediction based on early evaluation of semen quality parameters, to exclude sires with potentially low fertility from breeding programs, would therefore be useful. The aim of the present study was to identify the most suitable parameters that would provide reliable prediction of fertility. Frozen semen from 18 Italian Holstein-Friesian proven bulls was analyzed using computer-assisted semen analysis (CASA) (motility and kinetic parameters) and flow cytometry (FCM) (viability, acrosomal integrity, mitochondrial function, lipid peroxidation, plasma membrane stability and DNA integrity). Bulls were divided into two groups (low and high fertility) based on the estimated relative conception rate (ERCR). Significant differences were found between fertility groups for total motility, active cells, straightness, linearity, viability and percentage of DNA fragmented sperm. Correlations were observed between ERCR and some kinetic parameters, and membrane instability and some DNA integrity indicators. In order to define a model with high relation between semen quality parameters and ERCR, backward stepwise multiple regression analysis was applied. Thus, we obtained a prediction model that explained almost half (R 2=0.47, P<0.05) of the variation in the conception rate and included nine variables: five kinetic parameters measured by CASA (total motility, active cells, beat cross frequency, curvilinear velocity and amplitude of lateral head displacement) and four parameters related to DNA integrity evaluated by FCM (degree of chromatin structure abnormality Alpha-T, extent of chromatin structure abnormality (Alpha-T standard deviation), percentage of DNA fragmented sperm and percentage of sperm with high green fluorescence representative of immature cells). A significant relationship (R 2=0.84, P<0.05) was observed between real and predicted fertility. Once the accuracy of fertility prediction has been confirmed, the model developed in the present study could be used by artificial insemination centers for bull selection or for elimination of poor fertility ejaculates.

DNA helicase HIM-6/BLM both promotes MutSγ-dependent crossovers and antagonizes MutSγ-independent interhomolog associations during caenorhabditis elegans meiosis.

PubMed

Schvarzstein, Mara; Pattabiraman, Divya; Libuda, Diana E; Ramadugu, Ajit; Tam, Angela; Martinez-Perez, Enrique; Roelens, Baptiste; Zawadzki, Karl A; Yokoo, Rayka; Rosu, Simona; Severson, Aaron F; Meyer, Barbara J; Nabeshima, Kentaro; Villeneuve, Anne M

2014-09-01

Meiotic recombination is initiated by the programmed induction of double-strand DNA breaks (DSBs), lesions that pose a potential threat to the genome. A subset of the DSBs induced during meiotic prophase become designated to be repaired by a pathway that specifically yields interhomolog crossovers (COs), which mature into chiasmata that temporarily connect the homologs to ensure their proper segregation at meiosis I. The remaining DSBs must be repaired by other mechanisms to restore genomic integrity prior to the meiotic divisions. Here we show that HIM-6, the Caenorhabditis elegans ortholog of the RecQ family DNA helicase BLM, functions in both of these processes. We show that him-6 mutants are competent to load the MutSγ complex at multiple potential CO sites, to generate intermediates that fulfill the requirements of monitoring mechanisms that enable meiotic progression, and to accomplish and robustly regulate CO designation. However, recombination events at a subset of CO-designated sites fail to mature into COs and chiasmata, indicating a pro-CO role for HIM-6/BLM that manifests itself late in the CO pathway. Moreover, we find that in addition to promoting COs, HIM-6 plays a role in eliminating and/or preventing the formation of persistent MutSγ-independent associations between homologous chromosomes. We propose that HIM-6/BLM enforces biased outcomes of recombination events to ensure that both (a) CO-designated recombination intermediates are reliably resolved as COs and (b) other recombination intermediates reliably mature into noncrossovers in a timely manner. Copyright © 2014 by the Genetics Society of America.

Association Between Preovulatory Concentrations of Estradiol and Expression of Uterine Milk Protein Precursor, Inhibin Beta A, Period 1, Proenkephalin, and Receptors for Oxytocin, Progesterone, and Estradiol

USDA-ARS?s Scientific Manuscript database

Eliminating the preovulatory surge of estradiol decreased uterine weight, uterine protein, RNA to DNA ratio, rate of protein synthesis, and embryo survival following embryo transfer in sheep. Furthermore, cows that did not exhibit standing estrus (decreased preovulatory concentrations of estradiol) ...

Transmission models and management of lymphatic filariasis elimination.

PubMed

Michael, Edwin; Gambhir, Manoj

2010-01-01

The planning and evaluation of parasitic control programmes are complicated by the many interacting population dynamic and programmatic factors that determine infection trends under different control options. A key need is quantification about the status of the parasite system state at any one given timepoint and the dynamic change brought upon that state as an intervention program proceeds. Here, we focus on the control and elimination of the vector-borne disease, lymphatic filariasis, to show how mathematical models of parasite transmission can provide a quantitative framework for aiding the design of parasite elimination and monitoring programs by their ability to support (1) conducting rational analysis and definition of endpoints for different programmatic aims or objectives, including transmission endpoints for disease elimination, (2) undertaking strategic analysis to aid the optimal design of intervention programs to meet set endpoints under different endemic settings and (3) providing support for performing informed evaluations of ongoing programs, including aiding the formation of timely adaptive management strategies to correct for any observed deficiencies in program effectiveness. The results also highlight how the use of a model-based framework will be critical to addressing the impacts of ecological complexities, heterogeneities and uncertainties on effective parasite management and thereby guiding the development of strategies to resolve and overcome such real-world complexities. In particular, we underscore how this approach can provide a link between ecological science and policy by revealing novel tools and measures to appraise and enhance the biological controllability or eradicability of parasitic diseases. We conclude by emphasizing an urgent need to develop and apply flexible adaptive management frameworks informed by mathematical models that are based on learning and reducing uncertainty using monitoring data, apply phased or sequential decision-making to address extant uncertainty and focus on developing ecologically resilient management strategies, in ongoing efforts to control or eliminate filariasis and other parasitic diseases in resource-poor communities.

Citings on the Educational Horizon

ERIC Educational Resources Information Center

Mathies, Lorraine

1975-01-01

Article reviewed programs designed to help blind persons take advantage of information not in print form, programs which offer information on careers, programs designed to foster private sponsorship of basic research expeditions, and programs that eliminate social bias in educational materials. (Author/RK)

40 CFR 125.120 - Scope and purpose.

Code of Federal Regulations, 2010 CFR

2010-07-01

....120 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) WATER PROGRAMS CRITERIA AND STANDARDS FOR THE NATIONAL POLLUTANT DISCHARGE ELIMINATION SYSTEM Ocean Discharge Criteria § 125.120 Scope... Elimination System (NPDES) permits for the discharge of pollutants from a point source into the territorial...

40 CFR 125.120 - Scope and purpose.

Code of Federal Regulations, 2011 CFR

2011-07-01

....120 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) WATER PROGRAMS CRITERIA AND STANDARDS FOR THE NATIONAL POLLUTANT DISCHARGE ELIMINATION SYSTEM Ocean Discharge Criteria § 125.120 Scope... Elimination System (NPDES) permits for the discharge of pollutants from a point source into the territorial...

Sustaining visceral leishmaniasis elimination in Bangladesh – Could a policy brief help?

PubMed Central

Ahmed, Be-Nazir; Olliaro, Piero

2017-01-01

Bangladesh has made significant progress towards elimination of visceral leishmaniasis, and is on track to achieve its target of less than one case per 10,000 inhabitants in each subdistrict in 2017. As the incidence of disease falls, it is likely that the political capital and financial resources dedicated towards the elimination of visceral leishmaniasis may decrease, raising the prospect of disease resurgence. Policy memos may play a crucial role during the transition of the elimination plan from the ‘attack’ to the ‘consolidation’ and ‘maintenance’ phases, highlighting key stakeholders and areas where ongoing investment is crucial. An example of a policy brief is outlined in this paper. The background to the current elimination efforts is highlighted, with emphasis on remaining uncertainties including the impact of disease reservoirs and sustainable surveillance strategies. A stakeholder map is provided outlining the current and projected future activities of key bodies. Identification of key stakeholders subsequently frames the discussion of three key policy recommendations in the Bangladeshi context for the transition to the consolidation and maintenance phases of the elimination program. Recommendations include determining optimal vector control and surveillance strategies, shifting the emphasis towards horizontal integration of disease programs, and prioritising remaining research questions with a focus on operational and technical capacity. Achieving elimination is as much a political as a scientific question. Integrating the discussion of key stakeholders with policy priorities and the research agenda provides a novel insight into potential pathways forwards in the elimination of visceral leishmaniasis in Bangladesh and in the rest of the Indian subcontinent. PMID:29232385

Sustaining visceral leishmaniasis elimination in Bangladesh - Could a policy brief help?

PubMed

Fitzpatrick, Alyssa; Al-Kobaisi, Noor Saad M S; Beitman Maya, Jessica; Ren Chung, Yu; Duhan, Satyender; Elbegdorj, Erdene; Jain, Sushant; Kuhn, Edward; Nastase, Alexandra; Ahmed, Be-Nazir; Olliaro, Piero

2017-12-01

Bangladesh has made significant progress towards elimination of visceral leishmaniasis, and is on track to achieve its target of less than one case per 10,000 inhabitants in each subdistrict in 2017. As the incidence of disease falls, it is likely that the political capital and financial resources dedicated towards the elimination of visceral leishmaniasis may decrease, raising the prospect of disease resurgence. Policy memos may play a crucial role during the transition of the elimination plan from the 'attack' to the 'consolidation' and 'maintenance' phases, highlighting key stakeholders and areas where ongoing investment is crucial. An example of a policy brief is outlined in this paper. The background to the current elimination efforts is highlighted, with emphasis on remaining uncertainties including the impact of disease reservoirs and sustainable surveillance strategies. A stakeholder map is provided outlining the current and projected future activities of key bodies. Identification of key stakeholders subsequently frames the discussion of three key policy recommendations in the Bangladeshi context for the transition to the consolidation and maintenance phases of the elimination program. Recommendations include determining optimal vector control and surveillance strategies, shifting the emphasis towards horizontal integration of disease programs, and prioritising remaining research questions with a focus on operational and technical capacity. Achieving elimination is as much a political as a scientific question. Integrating the discussion of key stakeholders with policy priorities and the research agenda provides a novel insight into potential pathways forwards in the elimination of visceral leishmaniasis in Bangladesh and in the rest of the Indian subcontinent.

Requirement for XLF/Cernunnos in alignment-based gap filling by DNA polymerases lambda and mu for nonhomologous end joining in human whole-cell extracts.

PubMed

Akopiants, Konstantin; Zhou, Rui-Zhe; Mohapatra, Susovan; Valerie, Kristoffer; Lees-Miller, Susan P; Lee, Kyung-Jong; Chen, David J; Revy, Patrick; de Villartay, Jean-Pierre; Povirk, Lawrence F

2009-07-01

XLF/Cernunnos is a core protein of the nonhomologous end-joining pathway of DNA double-strand break repair. To better define the role of Cernunnos in end joining, whole-cell extracts were prepared from Cernunnos-deficient human cells. These extracts effected little joining of DNA ends with cohesive 5' or 3' overhangs, and no joining at all of partially complementary 3' overhangs that required gap filling prior to ligation. Assays in which gap-filled but unligated intermediates were trapped using dideoxynucleotides revealed that there was no gap filling on aligned DSB ends in the Cernunnos-deficient extracts. Recombinant Cernunnos protein restored gap filling and end joining of partially complementary overhangs, and stimulated joining of cohesive ends more than twentyfold. XLF-dependent gap filling was nearly eliminated by immunodepletion of DNA polymerase lambda, but was restored by addition of either polymerase lambda or polymerase mu. Thus, Cernunnos is essential for gap filling by either polymerase during nonhomologous end joining, suggesting that it plays a major role in aligning the two DNA ends in the repair complex.

Xeroderma pigmentosum variants have a slow recovery of DNA synthesis after irradiation with ultraviolet light

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

Cleaver, J.E.; Thomas, G.H.; Park, S.D.

1979-01-01

Human cells (normal and xeroderma pigmentosum variant) irradiated with ultraviolet light and pulse-labelled with (/sup 3/H)thymidine underwent transient decline and recovery of molecular weights of newly synthesized DNA and rates of (/sup 3/H)thymidine incorporation. The ability to synthesize normal-sized DNA recovered more rapidly in both cell types than thymidine incorporation. During recovery cells steadily increased in their ability to replicate normal-sized DNA on damaged templates. The molecular weight versus time curves fitted exponential functions with similar rate constants in normal and heterozygous xeroderma pigmentosum cells, but with a slower rate in two xeroderma pigmentosum variant cell lines. Caffeine added duringmore » the post-irradiation period eliminated the recovery of molecular weights in xeroderma pigmentosum variant but not in normal cells. The recovery of the ability to synthesize normal-sized DNA represents a combination of a number of cellular regulatory processes, some of which are constitutive, and one of which is altered in the xeroderma pigmentosum variant such that recovery becomes slow and caffeine sensitive.« less

A detailed experimental study of a DNA computer with two endonucleases.

PubMed

Sakowski, Sebastian; Krasiński, Tadeusz; Sarnik, Joanna; Blasiak, Janusz; Waldmajer, Jacek; Poplawski, Tomasz

2017-07-14

Great advances in biotechnology have allowed the construction of a computer from DNA. One of the proposed solutions is a biomolecular finite automaton, a simple two-state DNA computer without memory, which was presented by Ehud Shapiro's group at the Weizmann Institute of Science. The main problem with this computer, in which biomolecules carry out logical operations, is its complexity - increasing the number of states of biomolecular automata. In this study, we constructed (in laboratory conditions) a six-state DNA computer that uses two endonucleases (e.g. AcuI and BbvI) and a ligase. We have presented a detailed experimental verification of its feasibility. We described the effect of the number of states, the length of input data, and the nondeterminism on the computing process. We also tested different automata (with three, four, and six states) running on various accepted input words of different lengths such as ab, aab, aaab, ababa, and of an unaccepted word ba. Moreover, this article presents the reaction optimization and the methods of eliminating certain biochemical problems occurring in the implementation of a biomolecular DNA automaton based on two endonucleases.

Effect of caffeine on radiation-induced apoptosis in TK6 cells

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

Zhen, W.; Vaughan, A.T.M.

1995-02-01

Apoptosis has been measured in cells of the human TK6 lymphoblastoid cell line by recording the release of endonuclease-digested DNA from affected cells using flow cytometry. In asynchronously dividing cells, DNA degradation characteristic of apoptosis was first seen 12 h after irradiation as a defined DNA fluorescent peak of sub-G{sub 1}-phase content, reaching a maximum of 30-50% of the population by 24-72 h. Treating cells with 2 mM caffeine either before or up to 3 h after irradiation eliminated the degradation of DNA entirely. In addition, the percentage of cells in which apoptosis could be detected microscopically decreased from 62.4more » {+-} 0.95% to 16.7 {+-} 1.5% 72 h after caffeine treatment. Delaying caffeine treatment for 12 h after irradiation reduced DNA degradation by approximately 50% compared to cells receiving radiation alone. DNA degradation induced by serum deprivation was unaffected by caffeine treatment. These data support the contention that irradiation of TK6 cells produces a long-lived cellular signal which triggers apoptosis. Apoptosis produced by serum deprivation does not operate through the same pathway. 36 refs., 5 figs.« less

AIDing Chromatin and Transcription-Coupled Orchestration of Immunoglobulin Class-Switch Recombination

PubMed Central

Vaidyanathan, Bharat; Yen, Wei-Feng; Pucella, Joseph N.; Chaudhuri, Jayanta

2014-01-01

Secondary diversification of the antibody repertoire upon antigenic challenge, in the form of immunoglobulin heavy chain (IgH) class-switch recombination (CSR) endows mature, naïve B cells in peripheral lymphoid organs with a limitless ability to mount an optimal humoral immune response, thus expediting pathogen elimination. CSR replaces the default constant (CH) region exons (Cμ) of IgH with any of the downstream CH exons (Cγ, Cε, or Cα), thereby altering effector functions of the antibody molecule. This process depends on, and is orchestrated by, activation-induced deaminase (AID), a DNA cytidine deaminase that acts on single-stranded DNA exposed during transcription of switch (S) region sequences at the IgH locus. DNA lesions thus generated are processed by components of several general DNA repair pathways to drive CSR. Given that AID can instigate DNA lesions and genomic instability, stringent checks are imposed that constrain and restrict its mutagenic potential. In this review, we will discuss how AID expression and substrate specificity and activity is rigorously enforced at the transcriptional, post-transcriptional, post-translational, and epigenetic levels, and how the DNA-damage response is choreographed with precision to permit targeted activity while limiting bystander catastrophe. PMID:24734031

In vivo induction of interferon gamma expression in grey horses with metastatic melanoma resulting from direct injection of plasmid DNA coding for equine interleukin 12.

PubMed

Müller, J-M V; Wissemann, J; Meli, M L; Dasen, G; Lutz, H; Heinzerling, L; Feige, K

2011-11-01

Whole blood pharmacokinetics of intratumourally injected naked plasmid DNA coding for equine Interleukin 12 (IL-12) was assessed as a means of in vivo gene transfer in the treatment of melanoma in grey horses. The expression of induced interferon gamma (IFN-g) was evaluated in order to determine the pharmacodynamic properties of in vivo gene transduction. Seven grey horses bearing melanoma were injected intratumourally with 250 µg naked plasmid DNA coding for IL-12. Peripheral blood and biopsies from the injection site were taken at 13 time points until day 14 post injection (p.i.). Samples were analysed using quantitative real-time PCR. Plasmid DNA was quantified in blood samples and mRNA expression for IFN-g in tissue samples. Plasmid DNA showed fast elimination kinetics with more than 99 % of the plasmid disappearing within 36 hours. IFN-g expression increased quickly after IL-12 plasmid injection, but varied between individual horses. Intratumoural injection of plasmid DNA is a feasible method for inducing transgene expression in vivo. Biological activity of the transgene IL-12 was confirmed by measuring expression of IFN-g.

Optical selection and collection of DNA fragments

DOEpatents

Roslaniec, Mary C.; Martin, John C.; Jett, James H.; Cram, L. Scott

1998-01-01

Optical selection and collection of DNA fragments. The present invention includes the optical selection and collection of large (>.mu.g) quantities of clonable, chromosome-specific DNA from a sample of chromosomes. Chromosome selection is based on selective, irreversible photoinactivation of unwanted chromosomal DNA. Although more general procedures may be envisioned, the invention is demonstrated by processing chromosomes in a conventional flow cytometry apparatus, but where no droplets are generated. All chromosomes in the sample are first stained with at least one fluorescent analytic dye and bonded to a photochemically active species which can render chromosomal DNA unclonable if activated. After passing through analyzing light beam(s), unwanted chromosomes are irradiated using light which is absorbed by the photochemically active species, thereby causing photoinactivation. As desired chromosomes pass this photoinactivation point, the inactivating light source is deflected by an optical modulator; hence, desired chromosomes are not photoinactivated and remain clonable. The selection and photoinactivation processes take place on a microsecond timescale. By eliminating droplet formation, chromosome selection rates 50 times greater than those possible with conventional chromosome sorters may be obtained. Thus, usable quantities of clonable DNA from any source thereof may be collected.

First Polish DNA "manhunt"--an application of Y-chromosome STRs.

PubMed

Dettlaff-Kakol, A; Pawlowski, R

2002-10-01

This study presents the application of Y-chromosomal STR polymorphisms to male identification in the case of a serial rapist and woman murderer in Poland. Since August 1996 a rapist from Swinoujscie (northwest Poland) committed at least 14 rapes. In the year 2000 he brutally raped 8 young girls and murdered a 22-year-old girl. DNA profiles obtained from semen stains left at the scenes of crime gave information that one and the same man had committed all the rapes. The Y-chromosome haplotype (9 loci) obtained was used for the elimination process of 421 suspects. One man was found who had an identical DNA profile in all Y-chromosome STR loci analysed and possessed common alleles in 9 out of 10 autosomal loci, strongly suggesting that the real rapist and the typed man were closely related males. Analysis of reference DNA obtained from the man's brother revealed an identical DNA STR profile to that identified at the crime scenes. To the best of our knowledge this is the first case in Poland and probably in Eastern Europe where DNA typing of a large population was used to identify the offender.

Electrostatic coupling between DNA and its counterions modulates the observed translational diffusion coefficients.

PubMed

Stellwagen, Earle; Stellwagen, Nancy C

2015-09-01

Free solution capillary electrophoresis (CE) is a useful technique for measuring the translational diffusion coefficients of charged analytes. The measurements are relatively fast if the polarity of the electric field is reversed to drive the analyte back and forth past the detection window during each run. We have tested the validity of the resulting diffusion coefficients using double-stranded DNA molecules ranging in size from 20 to 960 base pairs as the model system. The diffusion coefficients of small DNAs are equal to values in the literature measured by other techniques. However, the diffusion coefficients of DNA molecules larger than ∼30 base pairs are anomalously high and deviate increasingly from the literature values with increasing DNA molar mass. The anomalously high diffusion coefficients are due to electrostatic coupling between the DNA and its counterions. As a result, the measured diffusion coefficients vary with the diffusion coefficient of the counterion, as well as with cation concentration and electric field strength. These effects can be reduced or eliminated by measuring apparent diffusion coefficients of the DNA at several different electric field strengths and extrapolating the results to zero electric field.

Model studies on the detectability of genetically modified feeds in milk.

PubMed

Poms, R E; Hochsteiner, W; Luger, K; Glössl, J; Foissy, H

2003-02-01

Detecting the use of genetically modified feeds in milk has become important, because the voluntary labeling of milk and dairy products as "GMO free" or as "organically grown" prohibits the employment of genetically modified organisms (GMOs). The aim of this work was to investigate whether a DNA transfer from foodstuffs like soya and maize was analytically detectable in cow's milk after digestion and transportation via the bloodstream of dairy cows and, thus, whether milk could report for the employment of transgene feeds. Blood, milk, urine, and feces of dairy cows were examined, and foreign DNA was detected by polymerase chain reaction by specifically amplifying a 226-bp fragment of the maize invertase gene and a 118-bp fragment of the soya lectin gene. An intravenous application of purified plant DNA showed a fast elimination of marker DNA in blood or its reduction below the detection limit. With feeding experiments, it could be demonstrated that a specific DNA transfer from feeds into milk was not detectable. Therefore, foreign DNA in milk cannot serve as an indicator for the employment of transgene feeds unless milk is directly contaminated with feed components or airborne feed particles.

DNase I and Proteinase K eliminate DNA from injured or dead bacteria but not from living bacteria in microbial reference systems and natural drinking water biofilms for subsequent molecular biology analyses.

PubMed

Villarreal, Jessica Varela; Jungfer, Christina; Obst, Ursula; Schwartz, Thomas

2013-09-01

Molecular techniques, such as polymerase chain reaction (PCR) and quantitative PCR (qPCR), are very sensitive, but may detect total DNA present in a sample, including extracellular DNA (eDNA) and DNA coming from live and dead cells. DNase I is an endonuclease that non-specifically cleaves single- and double-stranded DNA. This enzyme was tested in this study to analyze its capacity of digesting DNA coming from dead cells with damaged cell membranes, leaving DNA from living cells with intact cell membranes available for DNA-based methods. For this purpose, an optimized DNase I/Proteinase K (DNase/PK) protocol was developed. Intact Staphylococcus aureus cells, heat-killed Pseudomonas aeruginosa cells, free genomic DNA of Salmonella enterica, and a mixture of these targets were treated according to the developed DNase/PK protocol. In parallel, these samples were treated with propidium monoazide (PMA) as an already described assay for live-dead discrimination. Quantitative PCR and PCR-DGGE of the eubacterial 16S rDNA fragment were used to test the ability of the DNase/PK and PMA treatments to distinguish DNA coming from cells with intact cell membranes in the presence of DNA from dead cells and free genomic DNA. The methods were applied to three months old autochthonous drinking water biofilms from a pilot facility built at a German waterworks. Shifts in the DNA patterns observed after DGGE analysis demonstrated the applicability of DNase/PK as well as of the PMA treatment for natural biofilm investigation. However, the DNase/PK treatment demonstrated some practical advantages in comparison with the PMA treatment for live/dead discrimination of bacterial targets in drinking water systems. © 2013 Elsevier B.V. All rights reserved.

Endogenous overexpression of an active phosphorylated form of DNA polymerase β under oxidative stress in Trypanosoma cruzi

PubMed Central

Moreira-Ramos, Sandra; Castillo, Christian; Kemmerling, Ulrike; Lapier, Michel; Maya, Juan Diego; Solari, Aldo

2018-01-01

Trypanosoma cruzi is exposed during its life to exogenous and endogenous oxidative stress, leading to damage of several macromolecules such as DNA. There are many DNA repair pathways in the nucleus and mitochondria (kinetoplast), where specific protein complexes detect and eliminate damage to DNA. One group of these proteins is the DNA polymerases. In particular, Tc DNA polymerase β participates in kinetoplast DNA replication and repair. However, the mechanisms which control its expression under oxidative stress are still unknown. Here we describe the effect of oxidative stress on the expression and function of Tc DNA polymerase β To this end parasite cells (epimastigotes and trypomastigotes) were exposed to peroxide during short periods of time. Tc DNA polymerase β which was associated physically with kinetoplast DNA, showed increased protein levels in response to peroxide damage in both parasite forms analyzed. Two forms of DNA polymerase β were identified and overexpressed after peroxide treatment. One of them was phosphorylated and active in DNA synthesis after renaturation on polyacrylamide electrophoresis gel. This phosphorylated form showed 3-4-fold increase in both parasite forms. Our findings indicate that these increments in protein levels are not under transcriptional control because the level of Tc DNA polymerase β mRNA is maintained or slightly decreased during the exposure to oxidative stress. We propose a mechanism where a DNA repair pathway activates a cascade leading to the increment of expression and phosphorylation of Tc DNA polymerase β in response to oxidative damage, which is discussed in the context of what is known in other trypanosomes which lack transcriptional control. PMID:29432450

Tribal Waste Management Program

EPA Pesticide Factsheets

The EPA’s Tribal Waste Management Program encourages environmentally sound waste management practices that promote resource conservation through recycling, recovery, reduction, clean up, and elimination of waste.

Indoor household residual spraying program performance in Matabeleland South province, Zimbabwe: 2011 to 2012; a descriptive cross-sectional study.

PubMed

Chimberengwa, Pugie Tawanda; Masuka, Nyasha; Gombe, Notion Tafara; Tshimanga, Mufuta; Takundwa, Lucia; Bangure, Donewell

2015-01-01

Matabeleland South launched the malaria pre-elimination campaign in 2012 but provincial spraying coverage has failed to attain 95% target, with some districts still encountering malaria outbreaks. A study was conducted to evaluate program performance against achieving malaria pre-elimination. A descriptive cross sectional study was done in 5 districts carrying out IRS using the logical framework involving inputs, process, outputs and outcome evaluation. Health workers recruited into the study included direct program implementers, district and provincial program managers. An interviewer administered questionnaire, checklists, key informant interviewer guide and desk review of records were used to collect data. We enrolled 37 primary respondents and 5 key informants. Pre-elimination, Epidemic Preparedness and Response plans were absent in all districts. Shortages of inputs were reported by 97% of respondents, with districts receiving 80% of requested budget. Insecticides were procured centrally at national level. Spraying started late and districts failed to spray all targeted households by end of December. The province is using makeshift camps with inappropriate evaporation ponds where liquid DDT waste is not safely accounted for. The provincial IHRS coverage for 2011 was 84%. Challenges cited included; food shortages for spraymen, late delivery of inputs and poor state of IHRS equipment. The province has failed to achieve Malaria pre-elimination IRS coverage targets for 2011/12 season. Financial and logistical challenges led to delays in supply of program inputs, recruitment and training of sprayers. The Province should establish camping infrastructure with standard evaporation ponds to minimise contamination of the environment.

Curve fitting and modeling with splines using statistical variable selection techniques

NASA Technical Reports Server (NTRS)

Smith, P. L.

1982-01-01

The successful application of statistical variable selection techniques to fit splines is demonstrated. Major emphasis is given to knot selection, but order determination is also discussed. Two FORTRAN backward elimination programs, using the B-spline basis, were developed. The program for knot elimination is compared in detail with two other spline-fitting methods and several statistical software packages. An example is also given for the two-variable case using a tensor product basis, with a theoretical discussion of the difficulties of their use.

DNA extraction from benthic Cyanobacteria: comparative assessment and optimization.

PubMed

Gaget, V; Keulen, A; Lau, M; Monis, P; Brookes, J D

2017-01-01

Benthic Cyanobacteria produce toxic and odorous compounds similar to their planktonic counterparts, challenging the quality of drinking water supplies. The biofilm that benthic algae and other micro-organisms produce is a complex and protective matrix. Monitoring to determine the abundance and identification of Cyanobacteria, therefore, relies on molecular techniques, with the choice of DNA isolation technique critical. This study investigated which DNA extraction method is optimal for DNA recovery in order to guarantee the best DNA yield for PCR-based analysis of benthic Cyanobacteria. The conventional phenol-chloroform extraction method was compared with five commercial kits, with the addition of chemical and physical cell-lysis steps also trialled. The efficacy of the various methods was evaluated by measuring the quantity and quality of DNA by UV spectrophotometry and by quantitative PCR (qPCR) using Cyanobacteria-specific primers. The yield and quality of DNA retrieved with the commercial kits was significantly higher than that of DNA obtained with the phenol-chloroform protocol. Kits including a physical cell-lysis step, such as the MO BIO Power Soil and Biofilm kits, were the most efficient for DNA isolation from benthic Cyanobacteria. These commercial kits allow greater recovery and the elimination of dangerous chemicals for DNA extraction, making them the method of choice for the isolation of DNA from benthic mats. They also facilitate the extraction of DNA from benthic Cyanobacteria, which can help to improve the characterization of Cyanobacteria in environmental studies using qPCRs or population composition analysis using next-generation sequencing. © 2016 The Society for Applied Microbiology.

DNA confinement in nanochannels: physics and biological applications

NASA Astrophysics Data System (ADS)

Reisner, Walter; Pedersen, Jonas N.; Austin, Robert H.

2012-10-01

DNA is the central storage molecule of genetic information in the cell, and reading that information is a central problem in biology. While sequencing technology has made enormous advances over the past decade, there is growing interest in platforms that can readout genetic information directly from long single DNA molecules, with the ultimate goal of single-cell, single-genome analysis. Such a capability would obviate the need for ensemble averaging over heterogeneous cellular populations and eliminate uncertainties introduced by cloning and molecular amplification steps (thus enabling direct assessment of the genome in its native state). In this review, we will discuss how the information contained in genomic-length single DNA molecules can be accessed via physical confinement in nanochannels. Due to self-avoidance interactions, DNA molecules will stretch out when confined in nanochannels, creating a linear unscrolling of the genome along the channel for analysis. We will first review the fundamental physics of DNA nanochannel confinement—including the effect of varying ionic strength—and then discuss recent applications of these systems to genomic mapping. Apart from the intense biological interest in extracting linear sequence information from elongated DNA molecules, from a physics view these systems are fascinating as they enable probing of single-molecule conformation in environments with dimensions that intersect key physical length-scales in the 1 nm to 100 µm range.

Electrochemical evaluation of DNA methylation level based on the stoichiometric relationship between purine and pyrimidine bases.

PubMed

Wang, Po; Chen, Hanbin; Tian, Jiuying; Dai, Zong; Zou, Xiaoyong

2013-07-15

An efficient electrochemical approach for the evaluation of DNA methylation level was proposed according to the oxidation signal of DNA bases at an overoxidized polypyrrole (PPyox) directed multiwalled carbon nanotubes (MWNTs) film modified glassy carbon electrode (GCE). The PPyox/MWNTs/GCE exhibited remarkable electrocatalytic activities towards the oxidation of DNA bases due to the advantages of wide potential window, large effective surface area, and excellent antifouling property. As a result, all purine and pyrimidine bases of guanine (G), adenine (A), thymine (T), cytosine (C) and 5-methylcytosine (5-mC) exhibited well identified oxidation peaks at the PPyox/MWNTs/GCE. The direct potential resolution between 5-mC and C was obtained to be 180 mV, which was large enough for their signal recognition and accurate detection in mixture. In particular, the signal interference from T, a great challenge in exploring DNA methylation, was successfully eliminated by an innovative strategy, which was developed based on the stoichiometric relationship between purine and pyrimidine bases in DNA molecular structure. The proposed method was effectively applied to the rapid detection of DNA methylation status in real sample within 45 min with satisfactory results. Copyright © 2013 Elsevier B.V. All rights reserved.

The mitochondrial plasmid of the true slime mold Physarum polycephalum bypasses uniparental inheritance by promoting mitochondrial fusion.

PubMed

Sakurai, Rakusa; Nomura, Hideo; Moriyam, Yohsuke; Kawano, Shigeyuki

2004-08-01

Mitochondrial DNA (mtDNA) is inherited maternally in most eukaryotes. Linear mitochondrial plasmids in higher plants and fungi are also transmitted from the maternal parent to the progeny. However, mF, which is a mitochondrial linear plasmid of Physarum polycephalum, evades uniparental mitochondrial inheritance. We examined 36 myxamoebal strains of Physarum and isolated three novel mF+ strains (JE8, TU111, NG111) that harbored free mF plasmids. These strains were mated with the mF- strain KM88. Of the three mF- x mF+ crosses, only KM88 x JE8 displayed complete uniparental inheritance. However, in KM88 x TU111 and KM88 x NG111, the mtDNA of KM88 and mF of TU111 and NG111 were inherited by the plasmodia and showed recombination. For example, although the mtDNA of TU111 was eliminated, the mF of TU111 persisted and became inserted into the mtDNA of KM88, such that recombinant mtDNA represented 80% of the total mtDNA. The parental mitochondria fused to yield giant mitochondria with two or more mitochondrial nucleoids. The mF appears to exchange mitochondria from the recipient (paternal) to the donor (maternal) by promoting mitochondrial fusion.

Analysis of informational redundancy in the protein-assembling machinery

NASA Astrophysics Data System (ADS)

Berkovich, Simon

2004-03-01

Entropy analysis of the DNA structure does not reveal a significant departure from randomness indicating lack of informational redundancy. This signifies the absence of a hidden meaning in the genome text and supports the 'barcode' interpretation of DNA given in [1]. Lack of informational redundancy is a characteristic property of an identification label rather than of a message of instructions. Yet randomness of DNA has to induce non-random structures of the proteins. Protein synthesis is a two-step process: transcription into RNA with gene splicing and formation a structure of amino acids. Entropy estimations, performed by A. Djebbari, show typical values of redundancy of the biomolecules along these pathways: DNA gene 4proteins 15-40in gene expression, the RNA copy carries the same information as the original DNA template. Randomness is essentially eliminated only at the step of the protein creation by a degenerate code. According to [1], the significance of the substitution of U for T with a subsequent gene splicing is that these transformations result in a different pattern of RNA oscillations, so the vital DNA communications are protected against extraneous noise coming from the protein making activities. 1. S. Berkovich, "On the 'barcode' functionality of DNA, or the Phenomenon of Life in the Physical Universe", Dorrance Publishing Co., Pittsburgh, 2003

DNA confinement in nanochannels: physics and biological applications.

PubMed

Reisner, Walter; Pedersen, Jonas N; Austin, Robert H

2012-10-01

DNA is the central storage molecule of genetic information in the cell, and reading that information is a central problem in biology. While sequencing technology has made enormous advances over the past decade, there is growing interest in platforms that can readout genetic information directly from long single DNA molecules, with the ultimate goal of single-cell, single-genome analysis. Such a capability would obviate the need for ensemble averaging over heterogeneous cellular populations and eliminate uncertainties introduced by cloning and molecular amplification steps (thus enabling direct assessment of the genome in its native state). In this review, we will discuss how the information contained in genomic-length single DNA molecules can be accessed via physical confinement in nanochannels. Due to self-avoidance interactions, DNA molecules will stretch out when confined in nanochannels, creating a linear unscrolling of the genome along the channel for analysis. We will first review the fundamental physics of DNA nanochannel confinement--including the effect of varying ionic strength--and then discuss recent applications of these systems to genomic mapping. Apart from the intense biological interest in extracting linear sequence information from elongated DNA molecules, from a physics view these systems are fascinating as they enable probing of single-molecule conformation in environments with dimensions that intersect key physical length-scales in the 1 nm to 100 µm range.

Unique Helicase Determinants in the Essential Conjugative TraI Factor from Salmonella enterica Serovar Typhimurium Plasmid pCU1

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

McLaughlin, K. J.; Nash, R. P.; Redinbo, M. R.

The widespread development of multidrug-resistant bacteria is a major health emergency. Conjugative DNA plasmids, which harbor a wide range of antibiotic resistance genes, also encode the protein factors necessary to orchestrate the propagation of plasmid DNA between bacterial cells through conjugative transfer. Successful conjugative DNA transfer depends on key catalytic components to nick one strand of the duplex DNA plasmid and separate the DNA strands while cell-to-cell transfer occurs. The TraI protein from the conjugative Salmonella plasmid pCU1 fulfills these key catalytic roles, as it contains both single-stranded DNA-nicking relaxase and ATP-dependent helicase domains within a single, 1,078-residue polypeptide. Inmore » this work, we unraveled the helicase determinants of Salmonella pCU1 TraI through DNA binding, ATPase, and DNA strand separation assays. TraI binds DNA substrates with high affinity in a manner influenced by nucleic acid length and the presence of a DNA hairpin structure adjacent to the nick site. TraI selectively hydrolyzes ATP, and mutations in conserved helicase motifs eliminate ATPase activity. Surprisingly, the absence of a relatively short (144-residue) domain at the extreme C terminus of the protein severely diminishes ATP-dependent strand separation. Collectively, these data define the helicase motifs of the conjugative factor TraI from Salmonella pCU1 and reveal a previously uncharacterized C-terminal functional domain that uncouples ATP hydrolysis from strand separation activity.« less

Loss of maintenance DNA methylation results in abnormal DNA origin firing during DNA replication.

PubMed

Haruta, Mayumi; Shimada, Midori; Nishiyama, Atsuya; Johmura, Yoshikazu; Le Tallec, Benoît; Debatisse, Michelle; Nakanishi, Makoto

2016-01-22

The mammalian maintenance methyltransferase DNMT1 [DNA (cytosine-5-)-methyltransferase 1] mediates the inheritance of the DNA methylation pattern during replication. Previous studies have shown that depletion of DNMT1 causes a severe growth defect and apoptosis in differentiated cells. However, the detailed mechanisms behind this phenomenon remain poorly understood. Here we show that conditional ablation of Dnmt1 in murine embryonic fibroblasts (MEFs) resulted in an aberrant DNA replication program showing an accumulation of late-S phase replication and causing severely defective growth. Furthermore, we found that the catalytic activity and replication focus targeting sequence of DNMT1 are required for a proper DNA replication program. Taken together, our findings suggest that the maintenance of DNA methylation by DNMT1 plays a critical role in proper regulation of DNA replication in mammalian cells. Copyright © 2015 Elsevier Inc. All rights reserved.

​Plant centromeres​.

PubMed

Comai, Luca; Maheshwari, Shamoni; Marimuthu, Mohan P A

2017-04-01

Plant centromeres, which are determined epigenetically by centromeric histone 3 (CENH3) have revealed surprising structural diversity, ranging from the canonical monocentric seen in vertebrates, to polycentric, and holocentric. Normally stable, centromeres can change position over evolutionary times or upon genomic stress, such as when chromosomes are broken. At the DNA level, centromeres can be based on single copy DNA or more commonly on repeats. Rapid evolution of centromeric sequences and of CENH3 protein remains a mystery, as evidence of co-adaptation is lacking. Epigenetic differences between parents can trigger uniparental centromere failure and genome elimination, contributing to postzygotic hybridization barriers.​. Copyright © 2017 Elsevier Ltd. All rights reserved.

Hearing to Restore Eligibility in the Special Milk Program. Hearing before the Subcommittee on Elementary, Secondary, and Vocational Education of the Committee on Education and Labor. House of Representatives, Ninety-Eighth Congress, First Session on H.R. 904 to Amend the Child Nutrition Act of 1966 to Eliminate Certain Restrictions on the Eligibility of Schools to Participate in the Special Milk Program.

ERIC Educational Resources Information Center

Congress of the U.S., Washington, DC. House Committee on Education and Labor.

On March 23, 1983, testimony was heard concerning H.R. 904, a bill amending the Child Nutrition Act of 1966 to eliminate certain restrictions on the eligibility of schools to participate in the Special Milk Program. Included in this brief publication are statements focusing on (1) the "false economy" of not providing surplus milk to…

78 FR 5997 - Amendments to National Marine Sanctuary Regulations

Federal Register 2010, 2011, 2012, 2013, 2014

2013-01-28

...The National Oceanic and Atmospheric Administration (NOAA) proposes to amend the program regulations of the national marine sanctuaries. This rule would update and reorganize the existing regulations, eliminate redundancies across sanctuaries, eliminate outmoded regulations, adopt standard boundary descriptions, and consolidate general and permitting procedures.

76 FR 81488 - Agency Information Collection Activities; Proposed Collection; Comment Request; National...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-12-28

... Activities; Proposed Collection; Comment Request; National Pollutant Discharge Elimination System (NPDES... viruses. For additional information about EPA's public docket, visit the EPA Docket Center homepage at... Pollutant Discharge Elimination System (NPDES) Program (Renewal). ICR Number: EPA ICR No. 0229.20, OMB...

4 CFR 7.2 - Equal employment opportunity.

Code of Federal Regulations, 2011 CFR

2011-01-01

... eliminating minority and women underrepresentation. (c) Statutory rights and remedies. Nothing in this order... continuing programs for the recruitment of members of minorities and women for positions in GAO in a manner designed to eliminate underrepresentation of minorities and women in the various categories of employment...

4 CFR 7.2 - Equal employment opportunity.

Code of Federal Regulations, 2010 CFR

2010-01-01

... eliminating minority and women underrepresentation. (c) Statutory rights and remedies. Nothing in this order... continuing programs for the recruitment of members of minorities and women for positions in GAO in a manner designed to eliminate underrepresentation of minorities and women in the various categories of employment...

4 CFR 7.2 - Equal employment opportunity.

Code of Federal Regulations, 2014 CFR

2014-01-01

... eliminating minority and women underrepresentation. (c) Statutory rights and remedies. Nothing in this order... continuing programs for the recruitment of members of minorities and women for positions in GAO in a manner designed to eliminate underrepresentation of minorities and women in the various categories of employment...

4 CFR 7.2 - Equal employment opportunity.

Code of Federal Regulations, 2012 CFR

2012-01-01

... eliminating minority and women underrepresentation. (c) Statutory rights and remedies. Nothing in this order... continuing programs for the recruitment of members of minorities and women for positions in GAO in a manner designed to eliminate underrepresentation of minorities and women in the various categories of employment...

4 CFR 7.2 - Equal employment opportunity.

Code of Federal Regulations, 2013 CFR

2013-01-01

... eliminating minority and women underrepresentation. (c) Statutory rights and remedies. Nothing in this order... continuing programs for the recruitment of members of minorities and women for positions in GAO in a manner designed to eliminate underrepresentation of minorities and women in the various categories of employment...

Patient identification error among prostate needle core biopsy specimens--are we ready for a DNA time-out?

PubMed

Suba, Eric J; Pfeifer, John D; Raab, Stephen S

2007-10-01

Patient identification errors in surgical pathology often involve switches of prostate or breast needle core biopsy specimens among patients. We assessed strategies for decreasing the occurrence of these uncommon and yet potentially catastrophic events. Root cause analyses were performed following 3 cases of patient identification error involving prostate needle core biopsy specimens. Patient identification errors in surgical pathology result from slips and lapses of automatic human action that may occur at numerous steps during pre-laboratory, laboratory and post-laboratory work flow processes. Patient identification errors among prostate needle biopsies may be difficult to entirely prevent through the optimization of work flow processes. A DNA time-out, whereby DNA polymorphic microsatellite analysis is used to confirm patient identification before radiation therapy or radical surgery, may eliminate patient identification errors among needle biopsies.

High mobility organic field-effect transistor based on water-soluble deoxyribonucleic acid via spray coating

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

Shi, Wei; Han, Shijiao; Huang, Wei

High mobility organic field-effect transistors (OFETs) by inserting water-soluble deoxyribonucleic acid (DNA) buffer layer between electrodes and pentacene film through spray coating process were fabricated. Compared with the OFETs incorporated with DNA in the conventional organic solvents of ethanol and methanol: water mixture, the water-soluble DNA based OFET exhibited an over four folds enhancement of field-effect mobility from 0.035 to 0.153 cm{sup 2}/Vs. By characterizing the surface morphology and the crystalline structure of pentacene active layer through atomic force microscope and X-ray diffraction, it was found that the adoption of water solvent in DNA solution, which played a key role inmore » enhancing the field-effect mobility, was ascribed to both the elimination of the irreversible organic solvent-induced bulk-like phase transition of pentacene film and the diminution of a majority of charge trapping at interfaces in OFETs.« less

Autoclave method for rapid preparation of bacterial PCR-template DNA.

PubMed

Simmon, Keith E; Steadman, Dewey D; Durkin, Sarah; Baldwin, Amy; Jeffrey, Wade H; Sheridan, Peter; Horton, Rene; Shields, Malcolm S

2004-02-01

An autoclave method for preparing bacterial DNA for PCR template is presented, it eliminates the use of detergents, organic solvents, and mechanical cellular disruption approaches, thereby significantly reducing processing time and costs while increasing reproducibility. Bacteria are lysed by rapid heating and depressurization in an autoclave. The lysate, cleared by microcentrifugation, was either used directly in the PCR reaction, or concentrated by ultrafiltration. This approach was compared with seven established methods of DNA template preparation from four bacterial sources which included boiling Triton X-100 and SDS, bead beating, lysozyme/proteinase K, and CTAB lysis method components. Bacteria examined were Enterococcus and Escherichia coli, a natural marine bacterial community and an Antarctic cyanobacterial-mat. DNAs were tested for their suitability as PCR templates by repetitive element random amplified polymorphic DNA (RAPD) and denaturing gradient gel electrophoresis (DGGE) analysis. The autoclave method produced PCR amplifiable template comparable or superior to the other methods, with greater reproducibility, much shorter processing time, and at a significantly lower cost.

Correcting for Sample Contamination in Genotype Calling of DNA Sequence Data

PubMed Central

Flickinger, Matthew; Jun, Goo; Abecasis, Gonçalo R.; Boehnke, Michael; Kang, Hyun Min

2015-01-01

DNA sample contamination is a frequent problem in DNA sequencing studies and can result in genotyping errors and reduced power for association testing. We recently described methods to identify within-species DNA sample contamination based on sequencing read data, showed that our methods can reliably detect and estimate contamination levels as low as 1%, and suggested strategies to identify and remove contaminated samples from sequencing studies. Here we propose methods to model contamination during genotype calling as an alternative to removal of contaminated samples from further analyses. We compare our contamination-adjusted calls to calls that ignore contamination and to calls based on uncontaminated data. We demonstrate that, for moderate contamination levels (5%–20%), contamination-adjusted calls eliminate 48%–77% of the genotyping errors. For lower levels of contamination, our contamination correction methods produce genotypes nearly as accurate as those based on uncontaminated data. Our contamination correction methods are useful generally, but are particularly helpful for sample contamination levels from 2% to 20%. PMID:26235984

New LightCycler PCR for Rapid and Sensitive Quantification of Parvovirus B19 DNA Guides Therapeutic Decision-Making in Relapsing Infections

PubMed Central

Harder, Timm C.; Hufnagel, Markus; Zahn, Katrin; Beutel, Karin; Schmitt, Heinz-Josef; Ullmann, Uwe; Rautenberg, Peter

2001-01-01

Detection of parvovirus B19 DNA offers diagnostic advantages over serology, particularly in persistent infections of immunocompromised patients. A rapid, novel method of B19 DNA detection and quantification is introduced. This method, a quantitative PCR assay, is based on real-time glass capillary thermocycling (LightCycler [LC]) and fluorescence resonance energy transfer (FRET). The PCR assay allowed quantification over a dynamic range of over 7 logs and could quantify as little as 250 B19 genome equivalents (geq) per ml as calculated for plasmid DNA (i.e., theoretically ≥5 geq per assay). Interrater agreement analysis demonstrated equivalence of LC-FRET PCR and conventional nested PCR in the diagnosis of an active B19 infection (kappa coefficient = 0.83). The benefit of the new method was demonstrated in an immunocompromised child with a relapsing infection, who required an attenuation of the immunosuppressive therapy in addition to repeated doses of immunoglobulin to eliminate the virus. PMID:11724854

Detection of BRAF mutations from solid tumors using Tumorplex™ technology

PubMed Central

Yo, Jacob; Hay, Katie S.L.; Vinayagamoorthy, Dilanthi; Maryanski, Danielle; Carter, Mark; Wiegel, Joseph; Vinayagamoorthy, Thuraiayah

2015-01-01

Allele specific multiplex sequencing (Tumorplex™) is a new molecular platform for the detection of single base mutation in tumor biopsies with high sensitivity for clinical testing. Tumorplex™ is a novel modification of Sanger sequencing technology that generates both mutant and wild type nucleotide sequences simultaneously in the same electropherogram. The molecular weight of the two sequencing primers are different such that the two sequences generated are separated, thus eliminating possible suppression of mutant signal by the more abundant wild type signal. Tumorplex™ platform technology was tested using BRAF mutation V600E. These studies were performed with cloned BRAF mutations and genomic DNA extracted from tumor cells carrying 50% mutant allele. The lower limit of detection for BRAF V600E was found to be 20 genome equivalents (GE) using genomic DNA extracted from mutation specific cell lines. Sensitivity of the assay was tested by challenging the mutant allele with wild type allele at 20 GE, and was able to detect BRAF mutant signal at a GE ration of 20:1 × 107 (mutant to wild-type). This level of sensitivity can detect low abundance of clonal mutations in tumor biopsies and eliminate the need for cell enrichment. • Tumorplex™ is a single tube assay that permits the recognition of mutant allele without suppression by wildtype signal. • Tumorplex™ provides a high level of sensitivity. • Tumorplex™ can be used with small sample size with mixed population of cells carrying heterogeneous gDNA. PMID:26258049

Discerning Viable from Nonviable Yersinia pestis pgm- and Bacillus anthracis Sterne using Propidium Monoazide in the Presence of White Powders

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

Hess, Becky M.; Kaiser, Brooke LD; Sydor, Michael A.

ABSTRACT Aims To develop and optimize an assay to determine viability status of Bacillus anthracis Sterne and Yersinia pestis pgm- strains in the presence of white powders by coupling propidium monoazide (PMA) treatment with real-time PCR (qPCR) analysis. Methods and Results PMA selectively enters nonviable cells and binds DNA, thereby increasing qPCR assay cycle threshold (CT) values compared to untreated samples. Dye concentration, cell number and fitness, incubation time, inactivation methods, and assay buffer were optimized for B. anthracis Sterne and Y. pestis pgm-. Differences in CT values in nonviable cells compared to untreated samples were consistently > 9 formore » both B. anthracis Sterne vegetative cells and Y. pestis pgm- in the presence and absence of three different white powders. Our method eliminates the need for a DNA extraction step prior to detection by qPCR. Conclusions The developed assay enables simultaneous identification and viability assessment for B. anthracis Sterne and Y. pestis pgm- under laboratory conditions, even in the presence of white powders. Eliminating the DNA extraction step that is typically used reduces total assay time and labor requirements for sample analysis. Significance and Impact of the Study The method developed for simultaneous detection and viability assessment for B. anthracis and Y. pestis can be employed in forming decisions about the severity of a biothreat event or the safety of food. Keywords Bacillus anthracis, Yersinia pestis, Propidium Monoazide, qPCR, White Powders, Rapid Viability Detection« less

Undetectable Transcription of cap in a Clinical AAV Vector: Implications for Preformed Capsid in Immune Responses

PubMed Central

Hauck, Bernd; Murphy, Samuel L; Smith, Peter H; Qu, Guang; Liu, Xingge; Zelenaia, Olga; Mingozzi, Federico; Sommer, Jürg M; High, Katherine A; Wright, J. Fraser

2008-01-01

In a gene therapy clinical trial for hemophilia B, adeno-associated virus 2 (AAV2) capsid–specific CD8+ T cells were previously implicated in the elimination of vector-transduced hepatocytes, resulting in loss of human factor IX (hFIX) transgene expression. To test the hypothesis that expression of AAV2 cap DNA impurities in the AAV2-hFIX vector was the source of epitopes presented on transduced cells, transcription of cap was assessed by quantitative reverse transcription–PCR (Q-RT-PCR) following transduction of target cells with the vector used in the clinical trial. Transcriptional profiling was also performed for residual AmpR, and adenovirus E2A and E4. Although trace amounts of DNA impurities were present in the clinical vector, transcription of these sequences was not detected after transduction of human hepatocytes, nor in mice administered a dose 26-fold above the highest dose administered in the clinical study. Two methods used to minimize encapsidated DNA impurities in the clinical vector were: (i) a vector (cis) production plasmid with a backbone exceeding the packaging limit of AAV; and (ii) a vector purification step that achieved separation of the vector from vector-related impurities (e.g., empty capsids). In conclusion, residual cap expression was undetectable following transduction with AAV2-hFIX clinical vectors. Preformed capsid protein is implicated as the source of epitopes recognized by CD8+ T cells that eliminated vector-transduced cells in the clinical study. PMID:18941440

Evaluating perspectives for PRRS virus elimination from pig dense areas with a risk factor based herd index.

PubMed

Fahrion, A S; Beilage, E grosse; Nathues, H; Dürr, S; Doherr, M G

2014-06-01

Porcine reproductive and respiratory syndrome virus (PRRSV) is wide-spread in pig populations globally. In many regions of Europe with intensive pig production and high herd densities, the virus is endemic and can cause disease and production losses. This fuels discussion about the feasibility and sustainability of virus elimination from larger geographic regions. The implementation of a program aiming at virus elimination for areas with high pig density is unprecedented and its potential success is unknown. The objective of this work was to approach pig population data with a simple method that could support assessing the feasibility of a sustainable regional PRRSV elimination. Based on known risk factors such as pig herd structure and neighborhood conditions, an index characterizing individual herds' potential for endemic virus circulation and reinfection was designed. This index was subsequently used to compare data of all pig herds in two regions with different pig- and herd-densities in Lower Saxony (North-West Germany) where PRRSV is endemic. Distribution of the indexed herds was displayed using GIS. Clusters of high herd index densities forming potential risk hot spots were identified which could represent key target areas for surveillance and biosecurity measures under a control program aimed at virus elimination. In an additional step, for the study region with the higher pig density (2463 pigs/km(2) farmland), the potential distribution of PRRSV-free and non-free herds during the implementation of a national control program aiming at national virus elimination was modeled. Complex herd and trade network structures suggest that PRRSV elimination in regions with intensive pig farming like that of middle Europe would have to involve legal regulation and be accompanied by important trade and animal movement restrictions. The proposed methodology of risk index mapping could be adapted to areas varying in size, herd structure and density. Interpreted in the regional context, this could help to classify the density of risk and to accordingly target resources and measures for elimination. Copyright © 2014 Elsevier B.V. All rights reserved.

DNA conformations in mismatch repair probed in solution by X-ray scattering from gold nanocrystals

PubMed Central

Hura, Greg L.; Tsai, Chi-Lin; Claridge, Shelley A.; Mendillo, Marc L.; Smith, Jessica M.; Williams, Gareth J.; Mastroianni, Alexander J.; Alivisatos, A. Paul; Putnam, Christopher D.; Kolodner, Richard D.; Tainer, John A.

2013-01-01

DNA metabolism and processing frequently require transient or metastable DNA conformations that are biologically important but challenging to characterize. We use gold nanocrystal labels combined with small angle X-ray scattering to develop, test, and apply a method to follow DNA conformations acting in the Escherichia coli mismatch repair (MMR) system in solution. We developed a neutral PEG linker that allowed gold-labeled DNAs to be flash-cooled and stored without degradation in sample quality. The 1,000-fold increased gold nanocrystal scattering vs. DNA enabled investigations at much lower concentrations than otherwise possible to avoid concentration-dependent tetramerization of the MMR initiation enzyme MutS. We analyzed the correlation scattering functions for the nanocrystals to provide higher resolution interparticle distributions not convoluted by the intraparticle distribution. We determined that mispair-containing DNAs were bent more by MutS than complementary sequence DNA (csDNA), did not promote tetramer formation, and allowed MutS conversion to a sliding clamp conformation that eliminated the DNA bends. Addition of second protein responder MutL did not stabilize the MutS-bent forms of DNA. Thus, DNA distortion is only involved at the earliest mispair recognition steps of MMR: MutL does not trap bent DNA conformations, suggesting migrating MutL or MutS/MutL complexes as a conserved feature of MMR. The results promote a mechanism of mismatch DNA bending followed by straightening in initial MutS and MutL responses in MMR. We demonstrate that small angle X-ray scattering with gold labels is an enabling method to examine protein-induced DNA distortions key to the DNA repair, replication, transcription, and packaging. PMID:24101514

Genetic programs can be compressed and autonomously decompressed in live cells

NASA Astrophysics Data System (ADS)

Lapique, Nicolas; Benenson, Yaakov

2018-04-01

Fundamental computer science concepts have inspired novel information-processing molecular systems in test tubes1-13 and genetically encoded circuits in live cells14-21. Recent research has shown that digital information storage in DNA, implemented using deep sequencing and conventional software, can approach the maximum Shannon information capacity22 of two bits per nucleotide23. In nature, DNA is used to store genetic programs, but the information content of the encoding rarely approaches this maximum24. We hypothesize that the biological function of a genetic program can be preserved while reducing the length of its DNA encoding and increasing the information content per nucleotide. Here we support this hypothesis by describing an experimental procedure for compressing a genetic program and its subsequent autonomous decompression and execution in human cells. As a test-bed we choose an RNAi cell classifier circuit25 that comprises redundant DNA sequences and is therefore amenable for compression, as are many other complex gene circuits15,18,26-28. In one example, we implement a compressed encoding of a ten-gene four-input AND gate circuit using only four genetic constructs. The compression principles applied to gene circuits can enable fitting complex genetic programs into DNA delivery vehicles with limited cargo capacity, and storing compressed and biologically inert programs in vivo for on-demand activation.

76 FR 29747 - State Program Requirements; Proposal To Approve Maine's Base National Pollutant Discharge...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-05-23

...'s Base National Pollutant Discharge Elimination System (NPDES) Permitting Program AGENCY... of Maine's Base NPDES Permitting Program in these territories as part of the administrative record to... Maine's base program as EPA approved it in 2001. Thus, the state's program would not include regulation...

Information Systems to Support Surveillance for Malaria Elimination

PubMed Central

Ohrt, Colin; Roberts, Kathryn W.; Sturrock, Hugh J. W.; Wegbreit, Jennifer; Lee, Bruce Y.; Gosling, Roly D.

2015-01-01

Robust and responsive surveillance systems are critical for malaria elimination. The ideal information system that supports malaria elimination includes: rapid and complete case reporting, incorporation of related data, such as census or health survey information, central data storage and management, automated and expert data analysis, and customized outputs and feedback that lead to timely and targeted responses. Spatial information enhances such a system, ensuring cases are tracked and mapped over time. Data sharing and coordination across borders are vital and new technologies can improve data speed, accuracy, and quality. Parts of this ideal information system exist and are in use, but have yet to be linked together coherently. Malaria elimination programs should support the implementation and refinement of information systems to support surveillance and response and ensure political and financial commitment to maintain the systems and the human resources needed to run them. National malaria programs should strive to improve the access and utility of these information systems and establish cross-border data sharing mechanisms through the use of standard indicators for malaria surveillance. Ultimately, investment in the information technologies that support a timely and targeted surveillance and response system is essential for malaria elimination. PMID:26013378

Information systems to support surveillance for malaria elimination.

PubMed

Ohrt, Colin; Roberts, Kathryn W; Sturrock, Hugh J W; Wegbreit, Jennifer; Lee, Bruce Y; Gosling, Roly D

2015-07-01

Robust and responsive surveillance systems are critical for malaria elimination. The ideal information system that supports malaria elimination includes: rapid and complete case reporting, incorporation of related data, such as census or health survey information, central data storage and management, automated and expert data analysis, and customized outputs and feedback that lead to timely and targeted responses. Spatial information enhances such a system, ensuring cases are tracked and mapped over time. Data sharing and coordination across borders are vital and new technologies can improve data speed, accuracy, and quality. Parts of this ideal information system exist and are in use, but have yet to be linked together coherently. Malaria elimination programs should support the implementation and refinement of information systems to support surveillance and response and ensure political and financial commitment to maintain the systems and the human resources needed to run them. National malaria programs should strive to improve the access and utility of these information systems and establish cross-border data sharing mechanisms through the use of standard indicators for malaria surveillance. Ultimately, investment in the information technologies that support a timely and targeted surveillance and response system is essential for malaria elimination. © The American Society of Tropical Medicine and Hygiene.

NPDES Stormwater Program

EPA Pesticide Factsheets

The National Pollutant Discharge Elimination System (NPDES) stormwater program regulates some stormwater discharges from three potential sources: municipal separate storm sewer systems (MS4s), construction activities, and industrial activities.

National Pollutant Discharge Elimination System (NPDES)

EPA Pesticide Factsheets

Provides information about how the permit program interacts with other CWA programs to protect and improve water quality, and provides resources for professionals working in the program at the federal, state, local, and firm level, and concerned public.

Learn About the Water Pollution Control (Section 106) Grant Program

EPA Pesticide Factsheets

Under CWA Section 106, EPA is authorized to provide grants to states, eligible interstate agencies, and eligible tribes to establish and administer programs, including enforcement programs,for the prevention, reduction, and elimination of water pollution.

Do adjusted clinical groups eliminate incentives for HMOs to avoid substance abusers? Evidence from the Maryland Medicaid HealthChoice program.

PubMed

Ettner, Susan L; Johnson, Steven

2003-01-01

The adequacy of risk adjustment to eliminate incentives for managed care organizations (MCOs) to avoid enrolling costly patients had been questioned. This study explored systematic differences in expenditures between beneficiaries with and without substance disorders assigned to the same capitation rate group under the Maryland Medicaid HealthChoice program. The investigators used fiscal year (FY) 1995 to 1997 Medicaid data to assign beneficiaries to rate cells based on FY 1995 diagnoses and compared the distribution of expenditures for beneficiaries with and without substance disorders, defined using FY 1997 and FY 1995 diagnoses. Results showed that differences in FY 1997 expenditures between beneficiaries with and without FY 1995 substance disorders were negligible. However, MCOs could expect greater average losses and lower average profits on beneficiaries with FY 1997 substance disorders. Thus, the adjusted clinical groups methodology used to adjust capitation payments in the HealthChoice program attenuated, but did not eliminate, financial incentives for MCOs to avoid substance abusers.

Near elimination of genital warts in Australia predicted with extension of human papillomavirus vaccination to males.

PubMed

Korostil, Igor A; Ali, Hammad; Guy, Rebecca J; Donovan, Basil; Law, Matthew G; Regan, David G

2013-11-01

The National Human Papillomavirus (HPV) Vaccination Program for females delivering the quadrivalent vaccine Gardasil has been included in the National Immunisation Program in Australia since 2007. Sentinel surveillance data show that genital wart incidence has been steadily declining since then. The objective of this study was to estimate the additional impact on genital warts as a result of male vaccination, which was approved by the Australian government in 2012 and commenced in 2013. We use a mathematical model of HPV transmission in the Australian heterosexual population to predict the impact of male vaccination on the incidence of genital warts. Our model produced results that are consistent with the actual observed decline in genital warts and predicted a much lower incidence, approaching elimination, in coming decades with the introduction of male vaccination. Results from our model indicate that the planned extension of the National HPV Vaccination Program to males will lead to the near elimination of genital warts in both the female and male heterosexual populations in Australia.

'DNA Strider': a 'C' program for the fast analysis of DNA and protein sequences on the Apple Macintosh family of computers.

PubMed Central

Marck, C

1988-01-01

DNA Strider is a new integrated DNA and Protein sequence analysis program written with the C language for the Macintosh Plus, SE and II computers. It has been designed as an easy to learn and use program as well as a fast and efficient tool for the day-to-day sequence analysis work. The program consists of a multi-window sequence editor and of various DNA and Protein analysis functions. The editor may use 4 different types of sequences (DNA, degenerate DNA, RNA and one-letter coded protein) and can handle simultaneously 6 sequences of any type up to 32.5 kB each. Negative numbering of the bases is allowed for DNA sequences. All classical restriction and translation analysis functions are present and can be performed in any order on any open sequence or part of a sequence. The main feature of the program is that the same analysis function can be repeated several times on different sequences, thus generating multiple windows on the screen. Many graphic capabilities have been incorporated such as graphic restriction map, hydrophobicity profile and the CAI plot- codon adaptation index according to Sharp and Li. The restriction sites search uses a newly designed fast hexamer look-ahead algorithm. Typical runtime for the search of all sites with a library of 130 restriction endonucleases is 1 second per 10,000 bases. The circular graphic restriction map of the pBR322 plasmid can be therefore computed from its sequence and displayed on the Macintosh Plus screen within 2 seconds and its multiline restriction map obtained in a scrolling window within 5 seconds. PMID:2832831

A type III-B CRISPR-Cas effector complex mediating massive target DNA destruction.

PubMed

Han, Wenyuan; Li, Yingjun; Deng, Ling; Feng, Mingxia; Peng, Wenfang; Hallstrøm, Søren; Zhang, Jing; Peng, Nan; Liang, Yun Xiang; White, Malcolm F; She, Qunxin

2017-02-28

The CRISPR (clustered regularly interspaced short palindromic repeats) system protects archaea and bacteria by eliminating nucleic acid invaders in a crRNA-guided manner. The Sulfolobus islandicus type III-B Cmr-α system targets invading nucleic acid at both RNA and DNA levels and DNA targeting relies on the directional transcription of the protospacer in vivo. To gain further insight into the involved mechanism, we purified a native effector complex of III-B Cmr-α from S. islandicus and characterized it in vitro. Cmr-α cleaved RNAs complementary to crRNA present in the complex and its ssDNA destruction activity was activated by target RNA. The ssDNA cleavage required mismatches between the 5΄-tag of crRNA and the 3΄-flanking region of target RNA. An invader plasmid assay showed that mutation either in the histidine-aspartate acid (HD) domain (a quadruple mutation) or in the GGDD motif of the Cmr-2α protein resulted in attenuation of the DNA interference in vivo. However, double mutation of the HD motif only abolished the DNase activity in vitro. Furthermore, the activated Cmr-α binary complex functioned as a highly active DNase to destroy a large excess DNA substrate, which could provide a powerful means to rapidly degrade replicating viral DNA. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Detection of DNA–protein crosslinks (DPCs) by novel direct fluorescence labeling methods: distinct stabilities of aldehyde and radiation-induced DPCs

PubMed Central

Shoulkamy, Mahmoud I.; Nakano, Toshiaki; Ohshima, Makiko; Hirayama, Ryoichi; Uzawa, Akiko; Furusawa, Yoshiya; Ide, Hiroshi

2012-01-01

Proteins are covalently trapped on DNA to form DNA–protein crosslinks (DPCs) when cells are exposed to DNA-damaging agents. DPCs interfere with many aspects of DNA transactions. The current DPC detection methods indirectly measure crosslinked proteins (CLPs) through DNA tethered to proteins. However, a major drawback of such methods is the non-linear relationship between the amounts of DNA and CLPs, which makes quantitative data interpretation difficult. Here we developed novel methods of DPC detection based on direct CLP measurement, whereby CLPs in DNA isolated from cells are labeled with fluorescein isothiocyanate (FITC) and quantified by fluorometry or western blotting using anti-FITC antibodies. Both formats successfully monitored the induction and elimination of DPCs in cultured cells exposed to aldehydes and mouse tumors exposed to ionizing radiation (carbon-ion beams). The fluorometric and western blotting formats require 30 and 0.3 μg of DNA, respectively. Analyses of the isolated genomic DPCs revealed that both aldehydes and ionizing radiation produce two types of DPC with distinct stabilities. The stable components of aldehyde-induced DPCs have half-lives of up to days. Interestingly, that of radiation-induced DPCs has an infinite half-life, suggesting that the stable DPC component exerts a profound effect on DNA transactions over many cell cycles. PMID:22730301

40 CFR 122.1 - Purpose and scope.

Code of Federal Regulations, 2011 CFR

2011-07-01

... Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) WATER PROGRAMS EPA ADMINISTERED PERMIT PROGRAMS: THE NATIONAL POLLUTANT DISCHARGE ELIMINATION SYSTEM Definitions and General Program Requirements...) Program under sections 318, 402, and 405 of the Clean Water Act (CWA) (Public Law 92-500, as amended, 33 U...

14 CFR § 1253.115 - Assurance required.

Code of Federal Regulations, 2014 CFR

2014-01-01

... BASIS OF SEX IN EDUCATION PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Introduction... such assurance applies fails to commit itself to take whatever remedial action is necessary in accordance with § 1253.110(a) to eliminate existing discrimination on the basis of sex or to eliminate the...

14 CFR 1253.115 - Assurance required.

Code of Federal Regulations, 2011 CFR

2011-01-01

... OF SEX IN EDUCATION PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Introduction § 1253... such assurance applies fails to commit itself to take whatever remedial action is necessary in accordance with § 1253.110(a) to eliminate existing discrimination on the basis of sex or to eliminate the...

14 CFR 1253.115 - Assurance required.

Code of Federal Regulations, 2010 CFR

2010-01-01

... OF SEX IN EDUCATION PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Introduction § 1253... such assurance applies fails to commit itself to take whatever remedial action is necessary in accordance with § 1253.110(a) to eliminate existing discrimination on the basis of sex or to eliminate the...

6 CFR 17.115 - Assurance required.

Code of Federal Regulations, 2011 CFR

2011-01-01

... OF SEX IN EDUCATION PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Introduction § 17... such assurance applies fails to commit itself to take whatever remedial action is necessary in accordance with § 17.110(a) to eliminate existing discrimination on the basis of sex or to eliminate the...

6 CFR 17.115 - Assurance required.

Code of Federal Regulations, 2013 CFR

2013-01-01

... OF SEX IN EDUCATION PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Introduction § 17... such assurance applies fails to commit itself to take whatever remedial action is necessary in accordance with § 17.110(a) to eliminate existing discrimination on the basis of sex or to eliminate the...

31 CFR 28.115 - Assurance required.

Code of Federal Regulations, 2010 CFR

2010-07-01

... OF SEX IN EDUCATION PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Introduction § 28... such assurance applies fails to commit itself to take whatever remedial action is necessary in accordance with § 28.110(a) to eliminate existing discrimination on the basis of sex or to eliminate the...

31 CFR 28.115 - Assurance required.

Code of Federal Regulations, 2013 CFR

2013-07-01

... OF SEX IN EDUCATION PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Introduction § 28... such assurance applies fails to commit itself to take whatever remedial action is necessary in accordance with § 28.110(a) to eliminate existing discrimination on the basis of sex or to eliminate the...

6 CFR 17.115 - Assurance required.

Code of Federal Regulations, 2010 CFR

2010-01-01

... OF SEX IN EDUCATION PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Introduction § 17... such assurance applies fails to commit itself to take whatever remedial action is necessary in accordance with § 17.110(a) to eliminate existing discrimination on the basis of sex or to eliminate the...

6 CFR 17.115 - Assurance required.

Code of Federal Regulations, 2012 CFR

2012-01-01

... OF SEX IN EDUCATION PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Introduction § 17... such assurance applies fails to commit itself to take whatever remedial action is necessary in accordance with § 17.110(a) to eliminate existing discrimination on the basis of sex or to eliminate the...

31 CFR 28.115 - Assurance required.

Code of Federal Regulations, 2012 CFR

2012-07-01

... OF SEX IN EDUCATION PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Introduction § 28... such assurance applies fails to commit itself to take whatever remedial action is necessary in accordance with § 28.110(a) to eliminate existing discrimination on the basis of sex or to eliminate the...

31 CFR 28.115 - Assurance required.

Code of Federal Regulations, 2014 CFR

2014-07-01

... OF SEX IN EDUCATION PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Introduction § 28... such assurance applies fails to commit itself to take whatever remedial action is necessary in accordance with § 28.110(a) to eliminate existing discrimination on the basis of sex or to eliminate the...

31 CFR 28.115 - Assurance required.

Code of Federal Regulations, 2011 CFR

2011-07-01

... OF SEX IN EDUCATION PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Introduction § 28... such assurance applies fails to commit itself to take whatever remedial action is necessary in accordance with § 28.110(a) to eliminate existing discrimination on the basis of sex or to eliminate the...

14 CFR 1253.115 - Assurance required.

Code of Federal Regulations, 2012 CFR

2012-01-01

... OF SEX IN EDUCATION PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Introduction § 1253... such assurance applies fails to commit itself to take whatever remedial action is necessary in accordance with § 1253.110(a) to eliminate existing discrimination on the basis of sex or to eliminate the...

6 CFR 17.115 - Assurance required.

Code of Federal Regulations, 2014 CFR

2014-01-01

... OF SEX IN EDUCATION PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Introduction § 17... such assurance applies fails to commit itself to take whatever remedial action is necessary in accordance with § 17.110(a) to eliminate existing discrimination on the basis of sex or to eliminate the...

14 CFR 1253.115 - Assurance required.

Code of Federal Regulations, 2013 CFR

2013-01-01

... OF SEX IN EDUCATION PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Introduction § 1253... such assurance applies fails to commit itself to take whatever remedial action is necessary in accordance with § 1253.110(a) to eliminate existing discrimination on the basis of sex or to eliminate the...

Oligo Design: a computer program for development of probes for oligonucleotide microarrays.

PubMed

Herold, Keith E; Rasooly, Avraham

2003-12-01

Oligonucleotide microarrays have demonstrated potential for the analysis of gene expression, genotyping, and mutational analysis. Our work focuses primarily on the detection and identification of bacteria based on known short sequences of DNA. Oligo Design, the software described here, automates several design aspects that enable the improved selection of oligonucleotides for use with microarrays for these applications. Two major features of the program are: (i) a tiling algorithm for the design of short overlapping temperature-matched oligonucleotides of variable length, which are useful for the analysis of single nucleotide polymorphisms and (ii) a set of tools for the analysis of multiple alignments of gene families and related short DNA sequences, which allow for the identification of conserved DNA sequences for PCR primer selection and variable DNA sequences for the selection of unique probes for identification. Note that the program does not address the full genome perspective but, instead, is focused on the genetic analysis of short segments of DNA. The program is Internet-enabled and includes a built-in browser and the automated ability to download sequences from GenBank by specifying the GI number. The program also includes several utilities, including audio recital of a DNA sequence (useful for verifying sequences against a written document), a random sequence generator that provides insight into the relationship between melting temperature and GC content, and a PCR calculator.

Structural Determinants of DNA Binding by a P. falciparum ApiAP2 Transcriptional Regulator

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

Lindner, Scott E.; De Silva, Erandi K.; Keck, James L.

2010-11-05

Putative transcription factors have only recently been identified in the Plasmodium spp., with the major family of regulators comprising the Apicomplexan Apetala2 (AP2) proteins. To better understand the DNA-binding mechanisms of these transcriptional regulators, we characterized the structure and in vitro function of an AP2 DNA-binding domain from a prototypical Apicomplexan AP2 protein, PF14{_}0633 from Plasmodium falciparum. The X-ray crystal structure of the PF14{_}0633 AP2 domain bound to DNA reveals a {beta}-sheet fold that binds the DNA major groove through base-specific and backbone contacts; a prominent {alpha}-helix supports the {beta}-sheet structure. Substitution of predicted DNA-binding residues with alanine weakened ormore » eliminated DNA binding in solution. In contrast to plant AP2 domains, the PF14{_}0633 AP2 domain dimerizes upon binding to DNA through a domain-swapping mechanism in which the {alpha}-helices of the AP2 domains pack against the {beta}-sheets of the dimer mates. DNA-induced dimerization of PF14{_}0633 may be important for tethering two distal DNA loci together in the nucleus and/or for inducing functional rearrangements of its domains to facilitate transcriptional regulation. Consistent with a multisite binding mode, at least two copies of the consensus sequence recognized by PF14{_}0633 are present upstream of a previously identified group of sporozoite-stage genes. Taken together, these findings illustrate how Plasmodium has adapted the AP2 DNA-binding domain for genome-wide transcriptional regulation.« less

High-throughput multiplex cpDNA resequencing clarifies the genetic diversity and genetic relationships among Brassica napus, Brassica rapa and Brassica oleracea.

PubMed

Qiao, Jiangwei; Cai, Mengxian; Yan, Guixin; Wang, Nian; Li, Feng; Chen, Binyun; Gao, Guizhen; Xu, Kun; Li, Jun; Wu, Xiaoming

2016-01-01

Brassica napus (rapeseed) is a recent allotetraploid plant and the second most important oilseed crop worldwide. The origin of B. napus and the genetic relationships with its diploid ancestor species remain largely unresolved. Here, chloroplast DNA (cpDNA) from 488 B. napus accessions of global origin, 139 B. rapa accessions and 49 B. oleracea accessions were populationally resequenced using Illumina Solexa sequencing technologies. The intraspecific cpDNA variants and their allelic frequencies were called genomewide and further validated via EcoTILLING analyses of the rpo region. The cpDNA of the current global B. napus population comprises more than 400 variants (SNPs and short InDels) and maintains one predominant haplotype (Bncp1). Whole-genome resequencing of the cpDNA of Bncp1 haplotype eliminated its direct inheritance from any accession of the B. rapa or B. oleracea species. The distribution of the polymorphism information content (PIC) values for each variant demonstrated that B. napus has much lower cpDNA diversity than B. rapa; however, a vast majority of the wild and cultivated B. oleracea specimens appeared to share one same distinct cpDNA haplotype, in contrast to its wild C-genome relatives. This finding suggests that the cpDNA of the three Brassica species is well differentiated. The predominant B. napus cpDNA haplotype may have originated from uninvestigated relatives or from interactions between cpDNA mutations and natural/artificial selection during speciation and evolution. These exhaustive data on variation in cpDNA would provide fundamental data for research on cpDNA and chloroplasts. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Aptamer-based turn-on fluorescent four-branched quaternary ammonium pyrazine probe for selective thrombin detection.

PubMed

Yan, Shengyong; Huang, Rong; Zhou, Yangyang; Zhang, Ming; Deng, Minggang; Wang, Xiaolin; Weng, Xiaocheng; Zhou, Xiang

2011-01-28

In this thrombin detection system, the bright fluorescence of TASPI is almost eliminated by the DNA aptamer TBA (turn-off); however, in the presence of thrombin, it specifically binds to TBA by folding unrestricted TBA into an anti-parallel G-quadruplex structure and then releasing TASPI molecules, resulting in vivid and facile fluorescence recovery (turn-on).

Community Attitudes Toward Mass Drug Administration for Control and Elimination of Neglected Tropical Diseases After the 2014 Outbreak of Ebola Virus Disease in Lofa County, Liberia.

PubMed

Bogus, Joshua; Gankpala, Lincoln; Fischer, Kerstin; Krentel, Alison; Weil, Gary J; Fischer, Peter U; Kollie, Karsor; Bolay, Fatorma K

2016-03-01

The recent outbreak of Ebola virus disease (EVD) interrupted mass drug administration (MDA) programs to control and eliminate neglected tropical diseases in Liberia. MDA programs treat entire communities with medication regardless of infection status to interrupt transmission and eliminate lymphatic filariasis and onchocerciasis. Following reports of hostilities toward health workers and fear that they might be spreading EVD, it was important to determine whether attitudes toward MDA might have changed after the outbreak. We surveyed 140 community leaders from 32 villages in Lofa County, Liberia, that had previously participated in MDA and are located in an area that was an early epicenter of the EVD outbreak. Survey respondents reported a high degree of community trust in the MDA program, and 97% thought their communities were ready to resume MDA. However, respondents predicted that fewer people would comply with MDA after the EVD epidemic than before. The survey also uncovered fears in the community that EVD and MDA might be linked. Respondents suggested that MDA programs emphasize to people that the medications are identical to those previously distributed and that MDA programs have nothing to do with EVD. © The American Society of Tropical Medicine and Hygiene.

Community-wide reduction in prevalence and intensity of intestinal helminths as a collateral benefit of lymphatic filariasis elimination programs.

PubMed

De Rochars, Madsen Beau; Direny, Abdel N; Roberts, Jacquelin M; Addiss, David G; Radday, Jeanne; Beach, Michael J; Streit, Thomas G; Dardith, Desire; Lafontant, Jack Guy; Lammie, Patrick J

2004-10-01

Annual mass treatment with antifilarial drugs is the cornerstone of the global program to eliminate lymphatic filariasis (LF). Although the primary goal of the program is to interrupt transmission of LF, additional public health benefits also are expected because of the known anthelminthic properties of these drugs. Since rapid re-infection with intestinal helminths occurs following treatment, annual de-worming may not be sufficient to produce a lasting reduction in the prevalence and intensity of these infections. We conducted stool examinations in four sentinel communities before and approximately nine months after each of two rounds of mass drug administration (MDA) with diethylcarbamazine and albendazole in the context of an LF elimination program in Leogane, Haiti. At baseline, overall Ascaris, Trichuris, and hookworm infection prevalences were 20.9%, 34.0%, and 11.2%, respectively (n = 2,716 stools). Nine months after the second MDA, Ascaris, Trichuris and hookworm prevalences had decreased significantly, to 14.1%, 14.6%, and 2.0%, respectively (n = 814 stools). Infection intensity decreased significantly for all three parasites as well. These results demonstrate that substantial reductions in intestinal helminth infections are associated with mass treatment of filariasis in Haiti and are consistent with the conclusion that high levels of coverage for the LF program can decrease transmission of geohelminths.

Plasmodium vivax Malaria in Cambodia

PubMed Central

Siv, Sovannaroth; Roca-Feltrer, Arantxa; Vinjamuri, Seshu Babu; Bouth, Denis Mey; Lek, Dysoley; Rashid, Mohammad Abdur; By, Ngau Peng; Popovici, Jean; Huy, Rekol; Menard, Didier

2016-01-01

The Cambodian National Strategic Plan for Elimination of Malaria aims to move step by step toward elimination of malaria across Cambodia with an initial focus on Plasmodium falciparum malaria before achieving elimination of all forms of malaria, including Plasmodium vivax in 2025. The emergence of artemisinin-resistant P. falciparum in western Cambodia over the last decade has drawn global attention to support the ultimate goal of P. falciparum elimination, whereas the control of P. vivax lags much behind, making the 2025 target gradually less achievable unless greater attention is given to P. vivax elimination in the country. The following review presents in detail the past and current situation regarding P. vivax malaria, activities of the National Malaria Control Program, and interventional measures applied. Constraints and obstacles that can jeopardize our efforts to eliminate this parasite species are discussed. PMID:27708187

Evaluation of the Removal of Indicator Bacteria from Domestic Sludge Processed by Autothermal Thermophilic Aerobic Digestion (ATAD)

PubMed Central

Piterina, Anna V.; Bartlett, John; Pembroke, Tony J.

2010-01-01

The degradation of sludge solids in an insulated reactor during Autothermal Thermophilic Aerobic Digestion (ATAD) processing results in auto-heating, thermal treatment and total solids reduction, however, the ability to eliminate pathogenic organisms has not been analysed under large scale process conditions. We evaluated the ATAD process over a period of one year in a two stage, full scale Irish ATAD plant established in Killarney and treating mixed primary and secondary sludge, by examining the sludge microbiologically at various stages during and following ATAD processing to determine its ability to eliminate indicator organisms. Salmonella spp. (pathogen) and fecal-coliform (indicator) densities were well below the limits used to validate class A biosolids in the final product. Enteric pathogens present at inlet were deactivated during the ATAD process and were not detected in the final product using both traditional microbial culture and molecular phylogenetic techniques. A high DNase activity was detected in the bulk sludge during the thermophilic digestion stage which may be responsible for the rapid turn over of DNA from lysed cells and the removal of mobile DNA. These results offer assurance for the safe use of ATAD sludge as a soil supplement following processing. PMID:20948933

Evaluation of the removal of indicator bacteria from domestic sludge processed by Autothermal Thermophilic Aerobic Digestion (ATAD).

PubMed

Piterina, Anna V; Bartlett, John; Pembroke, Tony J

2010-09-01

The degradation of sludge solids in an insulated reactor during Autothermal Thermophilic Aerobic Digestion (ATAD) processing results in auto-heating, thermal treatment and total solids reduction, however, the ability to eliminate pathogenic organisms has not been analysed under large scale process conditions. We evaluated the ATAD process over a period of one year in a two stage, full scale Irish ATAD plant established in Killarney and treating mixed primary and secondary sludge, by examining the sludge microbiologically at various stages during and following ATAD processing to determine its ability to eliminate indicator organisms. Salmonella spp. (pathogen) and fecal-coliform (indicator) densities were well below the limits used to validate class A biosolids in the final product. Enteric pathogens present at inlet were deactivated during the ATAD process and were not detected in the final product using both traditional microbial culture and molecular phylogenetic techniques. A high DNase activity was detected in the bulk sludge during the thermophilic digestion stage which may be responsible for the rapid turn over of DNA from lysed cells and the removal of mobile DNA. These results offer assurance for the safe use of ATAD sludge as a soil supplement following processing.

Impact of Practice-Based Instruction on Graduate Programs in the Pharmaceutical Sciences.

ERIC Educational Resources Information Center

Schumacher, Gerald E.

1979-01-01

A practice- and science-based program of graduate education and scholarship for pharmaceutical science is proposed. Recommendations include the elimination of weak graduate programs, increased industrial support, and development of the clinical scientist. (SF)

Finding Protein and Nucleotide Similarities with FASTA

PubMed Central

Pearson, William R.

2016-01-01

The FASTA programs provide a comprehensive set of rapid similarity searching tools ( fasta36, fastx36, tfastx36, fasty36, tfasty36), similar to those provided by the BLAST package, as well as programs for slower, optimal, local and global similarity searches ( ssearch36, ggsearch36) and for searching with short peptides and oligonucleotides ( fasts36, fastm36). The FASTA programs use an empirical strategy for estimating statistical significance that accommodates a range of similarity scoring matrices and gap penalties, improving alignment boundary accuracy and search sensitivity (Unit 3.5). The FASTA programs can produce “BLAST-like” alignment and tabular output, for ease of integration into existing analysis pipelines, and can search small, representative databases, and then report results for a larger set of sequences, using links from the smaller dataset. The FASTA programs work with a wide variety of database formats, including mySQL and postgreSQL databases (Unit 9.4). The programs also provide a strategy for integrating domain and active site annotations into alignments and highlighting the mutational state of functionally critical residues. These protocols describe how to use the FASTA programs to characterize protein and DNA sequences, using protein:protein, protein:DNA, and DNA:DNA comparisons. PMID:27010337

Finding Protein and Nucleotide Similarities with FASTA.

PubMed

Pearson, William R

2016-03-24

The FASTA programs provide a comprehensive set of rapid similarity searching tools (fasta36, fastx36, tfastx36, fasty36, tfasty36), similar to those provided by the BLAST package, as well as programs for slower, optimal, local, and global similarity searches (ssearch36, ggsearch36), and for searching with short peptides and oligonucleotides (fasts36, fastm36). The FASTA programs use an empirical strategy for estimating statistical significance that accommodates a range of similarity scoring matrices and gap penalties, improving alignment boundary accuracy and search sensitivity. The FASTA programs can produce "BLAST-like" alignment and tabular output, for ease of integration into existing analysis pipelines, and can search small, representative databases, and then report results for a larger set of sequences, using links from the smaller dataset. The FASTA programs work with a wide variety of database formats, including mySQL and postgreSQL databases. The programs also provide a strategy for integrating domain and active site annotations into alignments and highlighting the mutational state of functionally critical residues. These protocols describe how to use the FASTA programs to characterize protein and DNA sequences, using protein:protein, protein:DNA, and DNA:DNA comparisons. Copyright © 2016 John Wiley & Sons, Inc.

DNA replication checkpoint promotes G1-S transcription by inactivating the MBF repressor Nrm1

PubMed Central

de Bruin, R. A. M.; Kalashnikova, T. I.; Aslanian, A.; Wohlschlegel, J.; Chahwan, C.; Yates, J. R.; Russell, P.; Wittenberg, C.

2008-01-01

The cell cycle transcriptional program imposes order on events of the cell-cycle and is a target for signals that regulate cell-cycle progression, including checkpoints required to maintain genome integrity. Neither the mechanism nor functional significance of checkpoint regulation of the cell-cycle transcription program are established. We show that Nrm1, an MBF-specific transcriptional repressor acting at the transition from G1 to S phase of the cell cycle, is at the nexus between the cell cycle transcriptional program and the DNA replication checkpoint in fission yeast. Phosphorylation of Nrm1 by the Cds1 (Chk2) checkpoint protein kinase, which is activated in response to DNA replication stress, promotes its dissociation from the MBF transcription factor. This leads to the expression of genes encoding components that function in DNA replication and repair pathways important for cell survival in response to arrested DNA replication. PMID:18682565

40 CFR 122.4 - Prohibitions (applicable to State NPDES programs, see § 123.25).

Code of Federal Regulations, 2011 CFR

2011-07-01

... AGENCY (CONTINUED) WATER PROGRAMS EPA ADMINISTERED PERMIT PROGRAMS: THE NATIONAL POLLUTANT DISCHARGE ELIMINATION SYSTEM Definitions and General Program Requirements § 122.4 Prohibitions (applicable to State... imposition of conditions cannot ensure compliance with the applicable water quality requirements of all...

40 CFR 122.4 - Prohibitions (applicable to State NPDES programs, see § 123.25).

Code of Federal Regulations, 2010 CFR

2010-07-01

... AGENCY (CONTINUED) WATER PROGRAMS EPA ADMINISTERED PERMIT PROGRAMS: THE NATIONAL POLLUTANT DISCHARGE ELIMINATION SYSTEM Definitions and General Program Requirements § 122.4 Prohibitions (applicable to State... imposition of conditions cannot ensure compliance with the applicable water quality requirements of all...

Bacterial DNA in water and dialysate: detection and significance for patient outcomes.

PubMed

Handelman, Garry J; Megdal, Peter A; Handelman, Samuel K

2009-01-01

The fluid used for hemodialysis may contain DNA fragments from bacteria, which could be harmful for patient outcomes. DNA fragments from bacteria, containing the nonmethylated CpG motif, can trigger inflammation through the monocyte and lymphocyte Toll-like receptor 9, and these DNA fragments have been observed in dialysate. The fragments may transfer across the dialyzer into the patient's bloodstream during hemodialysis treatment. During hemodiafiltration, the fragments would be introduced directly into the bloodstream. The DNA fragments may arise from biofilm in the pipes of the water system, from growth of bacteria in the water, or as contaminants in the bicarbonate and salt mixture used for preparation of dialysate. Current filtration methods, such as Diasafe filters, are not able to remove these fragments. It would be prudent to seek to reduce or eliminate these contaminants. However, the cost and effort of decreasing bacterial DNA content may ultimately require substantial facility improvements; we therefore need to fund research studies to determine if modifications to reduce bacterial DNA content are clinically warranted. This research will require methods to accurately determine the species of bacteria that contribute the DNA, since this information will allow the source to be established as biofilm, bicarbonate mixtures, or other problems in the dialysis system such as bacterial growth or leakage during water preparation. In this review, the evidence for bacterial DNA fragments will be examined and suggestions for further studies will be described.

Zinc-binding Domain of the Bacteriophage T7 DNA Primase Modulates Binding to the DNA Template*

PubMed Central

Lee, Seung-Joo; Zhu, Bin; Akabayov, Barak; Richardson, Charles C.

2012-01-01

The zinc-binding domain (ZBD) of prokaryotic DNA primases has been postulated to be crucial for recognition of specific sequences in the single-stranded DNA template. To determine the molecular basis for this role in recognition, we carried out homolog-scanning mutagenesis of the zinc-binding domain of DNA primase of bacteriophage T7 using a bacterial homolog from Geobacillus stearothermophilus. The ability of T7 DNA primase to catalyze template-directed oligoribonucleotide synthesis is eliminated by substitution of any five-amino acid residue-long segment within the ZBD. The most significant defect occurs upon substitution of a region (Pro-16 to Cys-20) spanning two cysteines that coordinate the zinc ion. The role of this region in primase function was further investigated by generating a protein library composed of multiple amino acid substitutions for Pro-16, Asp-18, and Asn-19 followed by genetic screening for functional proteins. Examination of proteins selected from the screening reveals no change in sequence-specific recognition. However, the more positively charged residues in the region facilitate DNA binding, leading to more efficient oligoribonucleotide synthesis on short templates. The results suggest that the zinc-binding mode alone is not responsible for sequence recognition, but rather its interaction with the RNA polymerase domain is critical for DNA binding and for sequence recognition. Consequently, any alteration in the ZBD that disturbs its conformation leads to loss of DNA-dependent oligoribonucleotide synthesis. PMID:23024359

Multiplexed Elimination of Wild-Type DNA and High-Resolution Melting Prior to Targeted Resequencing of Liquid Biopsies.

PubMed

Ladas, Ioannis; Fitarelli-Kiehl, Mariana; Song, Chen; Adalsteinsson, Viktor A; Parsons, Heather A; Lin, Nancy U; Wagle, Nikhil; Makrigiorgos, G Mike

2017-10-01

The use of clinical samples and circulating cell-free DNA (cfDNA) collected from liquid biopsies for diagnostic and prognostic applications in cancer is burgeoning, and improved methods that reduce the influence of excess wild-type (WT) portion of the sample are desirable. Here we present enrichment of mutation-containing sequences using enzymatic degradation of WT DNA. Mutation enrichment is combined with high-resolution melting (HRM) performed in multiplexed closed-tube reactions as a rapid, cost-effective screening tool before targeted resequencing. We developed a homogeneous, closed-tube approach to use a double-stranded DNA-specific nuclease for degradation of WT DNA at multiple targets simultaneously. The No Denaturation Nuclease-assisted Minor Allele Enrichment with Probe Overlap (ND-NaME-PrO) uses WT oligonucleotides overlapping both strands on putative DNA targets. Under conditions of partial denaturation (DNA breathing), the oligonucleotide probes enhance double-stranded DNA-specific nuclease digestion at the selected targets, with high preference toward WT over mutant DNA. To validate ND-NaME-PrO, we used multiplexed HRM, digital PCR, and MiSeq targeted resequencing of mutated genomic DNA and cfDNA. Serial dilution of KRAS mutation-containing DNA shows mutation enrichment by 10- to 120-fold and detection of allelic fractions down to 0.01%. Multiplexed ND-NaME-PrO combined with multiplexed PCR-HRM showed mutation scanning of 10-20 DNA amplicons simultaneously. ND-NaME-PrO applied on cfDNA from clinical samples enables mutation enrichment and HRM scanning over 10 DNA targets. cfDNA mutations were enriched up to approximately 100-fold (average approximately 25-fold) and identified via targeted resequencing. Closed-tube homogeneous ND-NaME-PrO combined with multiplexed HRM is a convenient approach to efficiently enrich for mutations on multiple DNA targets and to enable prescreening before targeted resequencing. © 2017 American Association for Clinical Chemistry.

College Choice in a Brand Elimination Framework: The Administrator's Perspective.

ERIC Educational Resources Information Center

Rosen, Deborah E.; Curran, James M.; Greenlee, Timothy B.

1998-01-01

Through a survey of business programs, a study examined the nature and extent of student recruiting activities and classified them according to a "brand elimination" model. Timing and methods of recruiting were then compared to reports of enrollment changes. Results suggest that targeted recruitment activities aimed at creating awareness…

An Effective and Benign Treatment of Rumination. Brief Report.

ERIC Educational Resources Information Center

Barton, Lyle E.; Barton, Carolyn L.

1985-01-01

A simple and effective program for the reduction or elimination of ruminative behavior in four school-aged students with multiple handicaps used a combination of peanut butter, reduced fluid consumption at meals, and fading. In each of the cases, the ruminative behavior was significantly reduced or eliminated. (Author/CL)

15 CFR 8a.115 - Assurance required.

Code of Federal Regulations, 2012 CFR

2012-01-01

... SEX IN EDUCATION PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Introduction § 8a.115... assurance applies fails to commit itself to take whatever remedial action is necessary in accordance with § 8a.110(a) to eliminate existing discrimination on the basis of sex or to eliminate the effects of...

10 CFR 1042.115 - Assurance required.

Code of Federal Regulations, 2012 CFR

2012-01-01

... DEPARTMENT OF ENERGY (GENERAL PROVISIONS) NONDISCRIMINATION ON THE BASIS OF SEX IN EDUCATION PROGRAMS OR... fails to commit itself to take whatever remedial action is necessary in accordance with § 1042.110(a) to eliminate existing discrimination on the basis of sex or to eliminate the effects of past discrimination...

10 CFR 1042.115 - Assurance required.

Code of Federal Regulations, 2013 CFR

2013-01-01

... DEPARTMENT OF ENERGY (GENERAL PROVISIONS) NONDISCRIMINATION ON THE BASIS OF SEX IN EDUCATION PROGRAMS OR... fails to commit itself to take whatever remedial action is necessary in accordance with § 1042.110(a) to eliminate existing discrimination on the basis of sex or to eliminate the effects of past discrimination...

15 CFR 8a.115 - Assurance required.

Code of Federal Regulations, 2014 CFR

2014-01-01

... SEX IN EDUCATION PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Introduction § 8a.115... assurance applies fails to commit itself to take whatever remedial action is necessary in accordance with § 8a.110(a) to eliminate existing discrimination on the basis of sex or to eliminate the effects of...

15 CFR 8a.115 - Assurance required.

Code of Federal Regulations, 2011 CFR

2011-01-01

... SEX IN EDUCATION PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Introduction § 8a.115... assurance applies fails to commit itself to take whatever remedial action is necessary in accordance with § 8a.110(a) to eliminate existing discrimination on the basis of sex or to eliminate the effects of...

15 CFR 8a.115 - Assurance required.

Code of Federal Regulations, 2010 CFR

2010-01-01

... SEX IN EDUCATION PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Introduction § 8a.115... assurance applies fails to commit itself to take whatever remedial action is necessary in accordance with § 8a.110(a) to eliminate existing discrimination on the basis of sex or to eliminate the effects of...

10 CFR 1042.115 - Assurance required.

Code of Federal Regulations, 2014 CFR

2014-01-01

... DEPARTMENT OF ENERGY (GENERAL PROVISIONS) NONDISCRIMINATION ON THE BASIS OF SEX IN EDUCATION PROGRAMS OR... fails to commit itself to take whatever remedial action is necessary in accordance with § 1042.110(a) to eliminate existing discrimination on the basis of sex or to eliminate the effects of past discrimination...

10 CFR 1042.115 - Assurance required.

Code of Federal Regulations, 2010 CFR

2010-01-01

... DEPARTMENT OF ENERGY (GENERAL PROVISIONS) NONDISCRIMINATION ON THE BASIS OF SEX IN EDUCATION PROGRAMS OR... fails to commit itself to take whatever remedial action is necessary in accordance with § 1042.110(a) to eliminate existing discrimination on the basis of sex or to eliminate the effects of past discrimination...

10 CFR 1042.115 - Assurance required.

Code of Federal Regulations, 2011 CFR

2011-01-01

... DEPARTMENT OF ENERGY (GENERAL PROVISIONS) NONDISCRIMINATION ON THE BASIS OF SEX IN EDUCATION PROGRAMS OR... fails to commit itself to take whatever remedial action is necessary in accordance with § 1042.110(a) to eliminate existing discrimination on the basis of sex or to eliminate the effects of past discrimination...

15 CFR 8a.115 - Assurance required.

Code of Federal Regulations, 2013 CFR

2013-01-01

... SEX IN EDUCATION PROGRAMS OR ACTIVITIES RECEIVING FEDERAL FINANCIAL ASSISTANCE Introduction § 8a.115... assurance applies fails to commit itself to take whatever remedial action is necessary in accordance with § 8a.110(a) to eliminate existing discrimination on the basis of sex or to eliminate the effects of...

Comprehensive restriction enzyme lists to update any DNA sequence computer program.

PubMed

Raschke, E

1993-04-01

Restriction enzyme lists are presented for the practical working geneticist to update any DNA computer program. These lists combine formerly scattered information and contain all presently known restriction enzymes with a unique recognition sequence, a cut site, or methylation (in)sensitivity. The lists are in the shortest possible form to also be functional with small DNA computer programs, and will produce clear restriction maps without any redundancy or loss of information. The lists discern between commercial and noncommercial enzymes, and prototype enzymes and different isoschizomers are cross-referenced. Differences in general methylation sensitivities and (in)sensitivities against Dam and Dcm methylases of Escherichia coli are indicated. Commercial methylases and intron-encoded endonucleases are included. An address list is presented to contact commercial suppliers. The lists are constantly updated and available in electronic form as pure US ASCII files, and in formats for the DNA computer programs DNA-Strider for Apple Macintosh, and DNAsis for IBM personal computers or compatibles via e-mail from the internet address: NETSERV@EMBL-HEIDELBERG.DE by sending only the message HELP RELIBRARY.

Production, Service and Trade Enterprise EKOREX Co. Ltd.

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

Wlodkowski, A.

1995-12-31

In the first period of its activity the business employed skilled and experienced specialists from the ex-Military College for Army Chemical Engineers in Cracow; therefore, the enterprise dealt chiefly with the elimination of environmental contamination. Nowadays, the enterprise`s operational range comprises: consulting and training services related with ecology; study on environmental contamination; participation in the US program of low emission elimination in Cracow; designing, consulting in the realization of projects {open_quotes}GEF{close_quotes} (Global Environmental Facility); designing, construction, servicing, operating the sewerage and water treatment plants, boiler-houses, incinerators etc.; and designing of heat networks, exchanger junctions, central heating and household hot watermore » installations. Since 1991 employees have individually participated in making the program and in testing boilers and fuels verified in the boiler houses covered by the Polish - US program of reduction of low emission sources in Cracow. We have actively joined the program of elimination of heating network boiler houses (industrial and local) by designing (for the Cracow cogeneration plant and MPEC) new connections among some structures and the municiple thermal distribution network and exchangers stations. In 1994, 47 such designs were made and have been working on successive projects to be carried out in Cracow.« less

The Computer Revolution and Physical Chemistry.

ERIC Educational Resources Information Center

O'Brien, James F.

1989-01-01

Describes laboratory-oriented software programs that are short, time-saving, eliminate computational errors, and not found in public domain courseware. Program availability for IBM and Apple microcomputers is included. (RT)

Improvements to the adaptive maneuvering logic program

NASA Technical Reports Server (NTRS)

Burgin, George H.

1986-01-01

The Adaptive Maneuvering Logic (AML) computer program simulates close-in, one-on-one air-to-air combat between two fighter aircraft. Three important improvements are described. First, the previously available versions of AML were examined for their suitability as a baseline program. The selected program was then revised to eliminate some programming bugs which were uncovered over the years. A listing of this baseline program is included. Second, the equations governing the motion of the aircraft were completely revised. This resulted in a model with substantially higher fidelity than the original equations of motion provided. It also completely eliminated the over-the-top problem, which occurred in the older versions when the AML-driven aircraft attempted a vertical or near vertical loop. Third, the requirements for a versatile generic, yet realistic, aircraft model were studied and implemented in the program. The report contains detailed tables which make the generic aircraft to be either a modern, high performance aircraft, an older high performance aircraft, or a previous generation jet fighter.

32 CFR 272.5 - Responsibilities.

Code of Federal Regulations, 2011 CFR

2011-07-01

... for plans and programs; develop policies; conduct analyses and studies; and make recommendations for... research programs and projects to eliminate unpromising or unnecessarily duplicative programs, and to... of Defense, appropriate funding levels for DoD basic research. (4) Develop and maintain a metrics...

24 CFR 761.10 - Definitions.

Code of Federal Regulations, 2010 CFR

2010-04-01

... THE SECRETARY, DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT (HOUSING ASSISTANCE PROGRAMS AND PUBLIC AND INDIAN HOUSING PROGRAMS) DRUG ELIMINATION PROGRAMS General § 761.10 Definitions. The definitions Department, HUD, and Public Housing Agency (PHA) are defined in part 5 of this title. Controlled substance...

24 CFR 761.10 - Definitions.

Code of Federal Regulations, 2011 CFR

2011-04-01

... THE SECRETARY, DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT (HOUSING ASSISTANCE PROGRAMS AND PUBLIC AND INDIAN HOUSING PROGRAMS) DRUG ELIMINATION PROGRAMS General § 761.10 Definitions. The definitions Department, HUD, and Public Housing Agency (PHA) are defined in part 5 of this title. Controlled substance...

24 CFR 761.10 - Definitions.

Code of Federal Regulations, 2013 CFR

2013-04-01

... THE SECRETARY, DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT (HOUSING ASSISTANCE PROGRAMS AND PUBLIC AND INDIAN HOUSING PROGRAMS) DRUG ELIMINATION PROGRAMS General § 761.10 Definitions. The definitions Department, HUD, and Public Housing Agency (PHA) are defined in part 5 of this title. Controlled substance...

24 CFR 761.10 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-04-01

... THE SECRETARY, DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT (HOUSING ASSISTANCE PROGRAMS AND PUBLIC AND INDIAN HOUSING PROGRAMS) DRUG ELIMINATION PROGRAMS General § 761.10 Definitions. The definitions Department, HUD, and Public Housing Agency (PHA) are defined in part 5 of this title. Controlled substance...

24 CFR 761.10 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-04-01

... THE SECRETARY, DEPARTMENT OF HOUSING AND URBAN DEVELOPMENT (HOUSING ASSISTANCE PROGRAMS AND PUBLIC AND INDIAN HOUSING PROGRAMS) DRUG ELIMINATION PROGRAMS General § 761.10 Definitions. The definitions Department, HUD, and Public Housing Agency (PHA) are defined in part 5 of this title. Controlled substance...

44 CFR 78.1 - Purpose.

Code of Federal Regulations, 2013 CFR

2013-10-01

... INSURANCE AND HAZARD MITIGATION National Flood Insurance Program FLOOD MITIGATION ASSISTANCE § 78.1 Purpose... of the Flood Mitigation Assistance (FMA) program, authorized by Sections 1366 and 1367 of the... eliminate claims under the National Flood Insurance Program (NFIP) through mitigation activities. The...

44 CFR 78.1 - Purpose.

Code of Federal Regulations, 2014 CFR

2014-10-01

... INSURANCE AND HAZARD MITIGATION National Flood Insurance Program FLOOD MITIGATION ASSISTANCE § 78.1 Purpose... of the Flood Mitigation Assistance (FMA) program, authorized by Sections 1366 and 1367 of the... eliminate claims under the National Flood Insurance Program (NFIP) through mitigation activities. The...

44 CFR 78.1 - Purpose.

Code of Federal Regulations, 2012 CFR

2012-10-01

... INSURANCE AND HAZARD MITIGATION National Flood Insurance Program FLOOD MITIGATION ASSISTANCE § 78.1 Purpose... of the Flood Mitigation Assistance (FMA) program, authorized by Sections 1366 and 1367 of the... eliminate claims under the National Flood Insurance Program (NFIP) through mitigation activities. The...

44 CFR 78.1 - Purpose.

Code of Federal Regulations, 2011 CFR

2011-10-01

... INSURANCE AND HAZARD MITIGATION National Flood Insurance Program FLOOD MITIGATION ASSISTANCE § 78.1 Purpose... of the Flood Mitigation Assistance (FMA) program, authorized by Sections 1366 and 1367 of the... eliminate claims under the National Flood Insurance Program (NFIP) through mitigation activities. The...

Program for creating an operating system generation cross reference index (SGINDEX)

NASA Technical Reports Server (NTRS)

Barth, C. W.

1972-01-01

Computer program to collect key data from Stage Two input of OS/360 system and to prepare formatted listing of index entries collected is discussed. Program eliminates manual paging through system output by providing comprehensive cross reference.

Elimination of African onchocerciasis: modeling the impact of increasing the frequency of ivermectin mass treatment.

PubMed

Coffeng, Luc E; Stolk, Wilma A; Hoerauf, Achim; Habbema, Dik; Bakker, Roel; Hopkins, Adrian D; de Vlas, Sake J

2014-01-01

The African Programme for Onchocerciasis Control (APOC) is currently shifting its focus from morbidity control to elimination of infection. To enhance the likelihood of elimination and speed up its achievement, programs may consider to increase the frequency of ivermectin mass treatment from annual to 6-monthly or even higher. In a computer simulation study, we examined the potential impact of increasing the mass treatment frequency for different settings. With the ONCHOSIM model, we simulated 92,610 scenarios pertaining to different assumptions about transmission conditions, history of mass treatment, the future mass treatment strategy, and ivermectin efficacy. Simulation results were used to determine the minimum remaining program duration and number of treatment rounds required to achieve 99% probability of elimination. Doubling the frequency of treatment from yearly to 6-monthly or 3-monthly was predicted to reduce remaining program duration by about 40% or 60%, respectively. These reductions come at a cost of additional treatment rounds, especially in case of 3-monthly mass treatment. Also, aforementioned reductions are highly dependent on maintained coverage, and could be completely nullified if coverage of mass treatment were to fall in the future. In low coverage settings, increasing treatment coverage is almost just as effective as increasing treatment frequency. We conclude that 6-monthly mass treatment may only be worth the effort in situations where annual treatment is expected to take a long time to achieve elimination in spite of good treatment coverage, e.g. because of unfavorable transmission conditions or because mass treatment started recently.

12 CFR 268.102 - Board program for equal employment opportunity.

Code of Federal Regulations, 2014 CFR

2014-01-01

... Opportunity § 268.102 Board program for equal employment opportunity. (a) The Board shall maintain a continuing affirmative program to promote equal opportunity and to identify and eliminate discriminatory... 12 Banks and Banking 4 2014-01-01 2014-01-01 false Board program for equal employment opportunity...

Program Elimination, Financial Emergency, and Student Rights.

ERIC Educational Resources Information Center

Olswang, Steven Glenn; And Others

1982-01-01

The rights of students to complete programs into which they have matriculated and the obligations of colleges and universities to maintain these programs for sufficient periods of time to fulfill any existing contracts with students are discussed. Contract principles are applied in protecting a student's right to complete degree programs. (MLW)

Trends toward the Future in Physical Activity Programming

ERIC Educational Resources Information Center

Charles, John M.; Charles, P. Kelly

2016-01-01

In the wake of multiple activity program eliminations and frequent downgrading of activity programs nationwide, the question to be addressed is how we can draw upon our interdisciplinary, intergenerational, and intercultural understandings of human movement to construct physical activity programs for the future that might withstand the test of…

Redox polymer and probe DNA tethered to gold electrodes for enzyme-amplified amperometric detection of DNA hybridization.

PubMed

Kavanagh, Paul; Leech, Dónal

2006-04-15

The detection of nucleic acids based upon recognition surfaces formed by co-immobilization of a redox polymer mediator and DNA probe sequences on gold electrodes is described. The recognition surface consists of a redox polymer, [Os(2,2'-bipyridine)2(polyvinylimidazole)(10)Cl](+/2+), and a model single DNA strand cross-linked and tethered to a gold electrode via an anchoring self-assembled monolayer (SAM) of cysteamine. Hybridization between the immobilized probe DNA of the recognition surface and a biotin-conjugated target DNA sequence (designed from the ssrA gene of Listeria monocytogenes), followed by addition of an enzyme (glucose oxidase)-avidin conjugate, results in electrical contact between the enzyme and the mediating redox polymer. In the presence of glucose, the current generated due to the catalytic oxidation of glucose to gluconolactone is measured, and a response is obtained that is binding-dependent. The tethering of the probe DNA and redox polymer to the SAM improves the stability of the surface to assay conditions of rigorous washing and high salt concentration (1 M). These conditions eliminate nonspecific interaction of both the target DNA and the enzyme-avidin conjugate with the recognition surfaces. The sensor response increases linearly with increasing concentration of target DNA in the range of 1 x 10(-9) to 2 x 10(-6) M. The detection limit is approximately 1.4 fmol, (corresponding to 0.2 nM of target DNA). Regeneration of the recognition surface is possible by treatment with 0.25 M NaOH solution. After rehybridization of the regenerated surface with the target DNA sequence, >95% of the current is recovered, indicating that the redox polymer and probe DNA are strongly bound to the surface. These results demonstrate the utility of the proposed approach.

Dangers resulting from DNA profiling of biological materials derived from patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) with regard to forensic genetic analysis.

PubMed

Jacewicz, R; Lewandowski, K; Rupa-Matysek, J; Jędrzejczyk, M; Berent, J

The study documents the risk that comes with DNA analysis of materials derived from patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in forensic genetics. DNA chimerism was studied in 30 patients after allo-HSCT, based on techniques applied in contemporary forensic genetics, i.e. real-time PCR and multiplex PCR-STR with the use of autosomal DNA as well as Y-DNA markers. The results revealed that the DNA profile of the recipient's blood was identical with the donor's in the majority of cases. Therefore, blood analysis can lead to false conclusions in personal identification as well as kinship analysis. An investigation of buccal swabs revealed a mixture of DNA in the majority of recipients. Consequently, personal identification on the basis of stain analysis of the same origin may be impossible. The safest (but not ideal) material turned out to be the hair root. Its analysis based on autosomal DNA revealed 100% of the recipient's profile. However, an analysis based on Y-chromosome markers performed in female allo-HSCT recipients with male donors demonstrated the presence of donor DNA in hair cells - similarly to the blood and buccal swabs. In the light of potential risks arising from DNA profiling of biological materials derived from persons after allotransplantation in judicial aspects, certain procedures were proposed to eliminate such dangers. The basic procedures include abandoning the approach based exclusively on blood collection, both for kinship analysis and personal identification; asking persons who are to be tested about their history of allo-HSCT before sample collection and profile entry in the DNA database, and verification of DNA profiling based on hair follicles in uncertain cases.

75 FR 33704 - Loan Guaranty: Elimination of Redundant Regulations

Federal Register 2010, 2011, 2012, 2013, 2014

2010-06-15

... budgetary impact of entitlements, grants, user fees, or loan programs or the rights and obligations of..., Handicapped, Housing, Indians, Individuals with disabilities, Loan programs--housing and community development...

An Analysis of Current Organizational Development Efforts in the United States Coast Guard

DTIC Science & Technology

1978-09-01

the goal is to eliminate racism and sexism , the program is questionable. B. How much progress have we made ensuring that all Coast... the chain-of-command’s ability to eliminate racism and sexism . We do not believe that racism and sexism can be eliminated from outside a command - it...INIZODOCTIO0 This chapter deals with the related literature and ccncepts pertinent to the comparison of service theory and historical antecedents of OD; The

The role of necroptosis in pulmonary diseases.

PubMed

Mizumura, Kenji; Maruoka, Shuichiro; Gon, Yasuhiro; Choi, Augustine M K; Hashimoto, Shu

2016-11-01

By regulating the cell number and eliminating harmful cells, programmed cell death plays a critical role in development, homeostasis, and disease. While apoptosis is a recognized form of programmed cell death, necrosis was considered a type of uncontrolled cell death induced by extreme physical or chemical stress. However, recent studies have revealed the existence of a genetically programmed and regulated form of necrosis, termed necroptosis. Necroptosis is defined as necrotic cell death that is dependent on receptor-interacting protein kinase 3 (RIPK3). RIPK3, receptor-interacting protein kinase 1 (RIPK1), and a mixed-lineage kinase domain-like protein (MLKL) form a multiprotein complex called a necrosome. Although necroptosis generally provides a cell-autonomous host defense, on the other hand, cell rupture caused by necroptosis induces inflammation through the release of damage-associated molecular patterns, such as mitochondrial DNA, HMGB1, and IL-1. Previously, necroptosis was considered an alternative to apoptosis, but it is becoming increasingly clear that necroptosis itself is relevant to clinical disease, independent of apoptosis. According to some recent studies, autophagy, a cellular process for organelle and protein turnover, regulates necroptosis. This review outlines the principal components of necroptosis and provides an overview of the emerging importance of necroptosis in the pathogenesis of pulmonary disease, including chronic obstructive pulmonary disease, lung cancer, infection, and sepsis. We also discuss the molecular relationship between necroptosis and autophagy. Strategies targeting necroptosis may yield novel therapies for pulmonary diseases. Copyright © 2016 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.

Unique helicase determinants in the essential conjugative TraI factor from Salmonella enterica serovar Typhimurium plasmid pCU1.

PubMed

McLaughlin, Krystle J; Nash, Rebekah P; Redinbo, Mathew R

2014-09-01

The widespread development of multidrug-resistant bacteria is a major health emergency. Conjugative DNA plasmids, which harbor a wide range of antibiotic resistance genes, also encode the protein factors necessary to orchestrate the propagation of plasmid DNA between bacterial cells through conjugative transfer. Successful conjugative DNA transfer depends on key catalytic components to nick one strand of the duplex DNA plasmid and separate the DNA strands while cell-to-cell transfer occurs. The TraI protein from the conjugative Salmonella plasmid pCU1 fulfills these key catalytic roles, as it contains both single-stranded DNA-nicking relaxase and ATP-dependent helicase domains within a single, 1,078-residue polypeptide. In this work, we unraveled the helicase determinants of Salmonella pCU1 TraI through DNA binding, ATPase, and DNA strand separation assays. TraI binds DNA substrates with high affinity in a manner influenced by nucleic acid length and the presence of a DNA hairpin structure adjacent to the nick site. TraI selectively hydrolyzes ATP, and mutations in conserved helicase motifs eliminate ATPase activity. Surprisingly, the absence of a relatively short (144-residue) domain at the extreme C terminus of the protein severely diminishes ATP-dependent strand separation. Collectively, these data define the helicase motifs of the conjugative factor TraI from Salmonella pCU1 and reveal a previously uncharacterized C-terminal functional domain that uncouples ATP hydrolysis from strand separation activity. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Acute loss of TET function results in aggressive myeloid cancer in mice

PubMed Central

An, Jungeun; González-Avalos, Edahí; Chawla, Ashu; Jeong, Mira; López-Moyado, Isaac F.; Li, Wei; Goodell, Margaret A.; Chavez, Lukas; Ko, Myunggon; Rao, Anjana

2015-01-01

TET-family dioxygenases oxidize 5-methylcytosine (5mC) in DNA, and exert tumour suppressor activity in many types of cancers. Even in the absence of TET coding region mutations, TET loss-of-function is strongly associated with cancer. Here we show that acute elimination of TET function induces the rapid development of an aggressive, fully-penetrant and cell-autonomous myeloid leukaemia in mice, pointing to a causative role for TET loss-of-function in this myeloid malignancy. Phenotypic and transcriptional profiling shows aberrant differentiation of haematopoietic stem/progenitor cells, impaired erythroid and lymphoid differentiation and strong skewing to the myeloid lineage, with only a mild relation to changes in DNA modification. We also observe progressive accumulation of phospho-H2AX and strong impairment of DNA damage repair pathways, suggesting a key role for TET proteins in maintaining genome integrity. PMID:26607761

Helix Unwinding and Base Flipping Enable Human MTERF1 to Terminate Mitochondrial Transcription

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

Yakubovskaya, E.; Mejia, E; Byrnes, J

2010-01-01

Defects in mitochondrial gene expression are associated with aging and disease. Mterf proteins have been implicated in modulating transcription, replication and protein synthesis. We have solved the structure of a member of this family, the human mitochondrial transcriptional terminator MTERF1, bound to dsDNA containing the termination sequence. The structure indicates that upon sequence recognition MTERF1 unwinds the DNA molecule, promoting eversion of three nucleotides. Base flipping is critical for stable binding and transcriptional termination. Additional structural and biochemical results provide insight into the DNA binding mechanism and explain how MTERF1 recognizes its target sequence. Finally, we have demonstrated that themore » mitochondrial pathogenic G3249A and G3244A mutations interfere with key interactions for sequence recognition, eliminating termination. Our results provide insight into the role of mterf proteins and suggest a link between mitochondrial disease and the regulation of mitochondrial transcription.« less

Enhanced translocation of single DNA molecules through α-hemolysin nanopores by manipulation of internal charge

PubMed Central

Maglia, Giovanni; Restrepo, Marcela Rincon; Mikhailova, Ellina; Bayley, Hagan

2008-01-01

Both protein and solid-state nanopores are under intense investigation for the analysis of nucleic acids. A crucial advantage of protein nanopores is that site-directed mutagenesis permits precise tuning of their properties. Here, by augmenting the internal positive charge within the α-hemolysin pore and varying its distribution, we increase the frequency of translocation of a 92-nt single-stranded DNA through the pore at +120 mV by ≈10-fold over the wild-type protein and dramatically lower the voltage threshold at which translocation occurs, e.g., by 50 mV for 1 event·s−1·μM−1. Further, events in which DNA enters the pore, but is not immediately translocated, are almost eliminated. These experiments provide a basis for improved nucleic acid analysis with protein nanopores, which might be translated to solid-state nanopores by using chemical surface modification. PMID:19060213

A Novel Method for Rapid Hybridization of DNA to a Solid Support

PubMed Central

Pettersson, Erik; Ahmadian, Afshin; Ståhl, Patrik L.

2013-01-01

Here we present a novel approach entitled Magnetic Forced Hybridization (MFH) that provides the means for efficient and direct hybridization of target nucleic acids to complementary probes immobilized on a glass surface in less than 15 seconds at ambient temperature. In addition, detection is carried out instantly since the beads become visible on the surface. The concept of MFH was tested for quality control of array manufacturing, and was combined with a multiplex competitive hybridization (MUCH) approach for typing of Human Papilloma Virus (HPV). Magnetic Forced Hybridization of bead-DNA constructs to a surface achieves a significant reduction in diagnostic testing time. In addition, readout of results by visual inspection of the unassisted eye eliminates the need for additional expensive instrumentation. The method uses the same set of beads throughout the whole process of manipulating and washing DNA constructs prior to detection, as in the actual detection step itself. PMID:23950946

Cytoprotective Effect of Peptide Sedatin, an Agonist of μ/δ-Opioid Receptors, on Primary Culture of Pulmonary Fibroblasts of Albino Rats under Conditions of Oxidative Stress.

PubMed

Sazonova, E N; Samarina, E Yu; Lebed'ko, O A; Maltseva, I M; Timoshin, S S

2016-05-01

We studied the effects of a synthetic analogue of dermorphin peptide sedatin on DNA synthesis, nucleolar apparatus, and parameters of free radical oxidation in the primary culture of pulmonary fibroblasts under conditions of oxidative stress. Oxidative stress significantly enhanced production of superoxide anion radical in the culture, sufficiently inhibited DNA synthesis in fibroblasts, and reduced the size of cell nuclei and parameters of the nucleolar apparatus. Sedatin prevented accumulation of free radical oxidation products and changes in karyometry parameters induced by oxidative stress. The peptide completely eliminated changes in the parameters of fibroblast nucleolar apparatus and abolished the inhibitory effect of oxidative stress on the number of DNA-synthesizing cells. Pretreatment with non-selective opioid receptor antagonist naloxone hydrochloride partially abolished the effects of sedatin in the primary culture of pulmonary fibroblasts.

Development and validation of the RapidHIT™ 200 utilising NGMSElect™ Express for the processing of buccal swabs.

PubMed

Shackleton, David; Pagram, Jenny; Ives, Lesley; Vanhinsbergh, Des

2018-06-02

The RapidHIT™ 200 System is a fully automated sample-to-DNA profile system designed to produce high quality DNA profiles within 2h. The use of RapidHIT™ 200 System within the United Kingdom Criminal Justice System (UKCJS) has required extensive development and validation of methods with a focus on AmpFℓSTR ® NGMSElect™ Express PCR kit to comply with specific regulations for loading to the UK National DNA Database (NDNAD). These studies have been carried out using single source reference samples to simulate live reference samples taken from arrestees and victims for elimination. The studies have shown that the system is capable of generating high quality profile and has achieved the accreditations necessary to load to the NDNAD; a first for the UK. Copyright © 2018 Elsevier B.V. All rights reserved.

Activation induced deaminase C-terminal domain links DNA breaks to end protection and repair during class switch recombination

PubMed Central

Zahn, Astrid; Eranki, Anil K.; Patenaude, Anne-Marie; Methot, Stephen P.; Fifield, Heather; Cortizas, Elena M.; Foster, Paul; Imai, Kohsuke; Durandy, Anne; Larijani, Mani; Verdun, Ramiro E.; Di Noia, Javier M.

2014-01-01

Activation-induced deaminase (AID) triggers antibody class switch recombination (CSR) in B cells by initiating DNA double strand breaks that are repaired by nonhomologous end-joining pathways. A role for AID at the repair step is unclear. We show that specific inactivation of the C-terminal AID domain encoded by exon 5 (E5) allows very efficient deamination of the AID target regions but greatly impacts the efficiency and quality of subsequent DNA repair. Specifically eliminating E5 not only precludes CSR but also, causes an atypical, enzymatic activity-dependent dominant-negative effect on CSR. Moreover, the E5 domain is required for the formation of AID-dependent Igh-cMyc chromosomal translocations. DNA breaks at the Igh switch regions induced by AID lacking E5 display defective end joining, failing to recruit DNA damage response factors and undergoing extensive end resection. These defects lead to nonproductive resolutions, such as rearrangements and homologous recombination that can antagonize CSR. Our results can explain the autosomal dominant inheritance of AID variants with truncated E5 in patients with hyper-IgM syndrome 2 and establish that AID, through the E5 domain, provides a link between DNA damage and repair during CSR. PMID:24591601

Micromechanical study of protein-DNA interactions and chromosomes

NASA Astrophysics Data System (ADS)

Marko, John

I will discuss micromechanics experiments that our group has used to analyze protein-DNA interactions and chromosome organization. In single-DNA experiments we have found that a feature of protein-DNA complexes is that their dissociation rates can depend strikingly on bulk solution concentrations of other proteins and DNA segments; I will describe experiments which demonstrate this effect, which can involve tens-fold changes in off-rates with submicromolar changes in solution concentrations. Second, I will discuss experiments aimed at analyzing large-scale human chromosome structure; we isolate metaphase chromosomes, which in their native form behave as remarkably elastic networks of chromatin. Exposure to DNA-cutting restriction enzymes completely eliminates this elasticity, indicating that there is not a mechanically contiguous protein ''scaffold'' from which the chromosome gains its stability. I will show results of siRNA experiments indicating that depletion of condensin proteins leads to destabilization of chromosome mechanics, indicating condensin's role as the major chromatin ''cross-linker'' in metaphase chromosomes. Finally I will discuss similar experiments on human G1 nuclei, where we use genetic and chemical modifications to separate the contributions of the nuclear lamina and chromatin to the mechanical stiffness of the nucleus as a whole. Supported by the NSF (DMR-1206868, MCB-1022117) and the NIH (GM105847, CA193419).