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

PubMed Central

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

2014-01-01

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

Mechanistic investigation of the bypass of a bulky aromatic DNA adduct catalyzed by a Y-family DNA polymerase.

PubMed

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

2014-09-01

3-Nitrobenzanthrone (3-NBA), a nitropolyaromatic hydrocarbon (NitroPAH) pollutant in diesel exhaust, is a potent mutagen and carcinogen. After metabolic activation, the primary metabolites of 3-NBA react with DNA to form dG and dA adducts. One of the three major adducts identified is N-(2'-deoxyguanosin-8-yl)-3-aminobenzanthrone (dG(C8-N-ABA)). This bulky adduct likely stalls replicative DNA polymerases but can be traversed by lesion bypass polymerases in vivo. Here, we employed running start assays to show that a site-specifically placed dG(C8-N-ABA) is bypassed in vitro by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. However, the nucleotide incorporation rate of Dpo4 was significantly reduced opposite both the lesion and the template position immediately downstream from the lesion site, leading to two strong pause sites. To investigate the kinetic effect of dG(C8-N-ABA) on polymerization, we utilized pre-steady-state kinetic methods to determine the kinetic parameters for individual nucleotide incorporations upstream, opposite, and downstream from the dG(C8-N-ABA) lesion. Relative to the replication of the corresponding undamaged DNA template, both nucleotide incorporation efficiency and fidelity of Dpo4 were considerably decreased during dG(C8-N-ABA) lesion bypass and the subsequent extension step. The lower nucleotide incorporation efficiency caused by the lesion is a result of a significantly reduced dNTP incorporation rate constant and modestly weaker dNTP binding affinity. At both pause sites, nucleotide incorporation followed biphasic kinetics with a fast and a slow phase and their rates varied with nucleotide concentration. In contrast, only the fast phase was observed with undamaged DNA. A kinetic mechanism was proposed for the bypass of dG(C8-N-ABA) bypass catalyzed by Dpo4. Copyright © 2014 Elsevier B.V. All rights reserved.

Some quantitative indicators of postovulatory aging and its effect on larval and juvenile development of Atlantic salmon (Salmo salar).

PubMed

Mommens, Maren; Storset, Arne; Babiak, Igor

2015-07-01

Modern out-of-season egg production in Atlantic salmon (Salmo salar) increases the risk of postovulatory aging (POA) of oocytes. Postovulatory aging is known to influence oocyte quality in salmonids, but reliable tests for POA are lacking in Atlantic salmon egg production. To address this problem, we have collected oocytes from the same 20 Atlantic salmon females sequentially in approximately 1-week intervals, from the start of ovulation until 28 days postovulation (dpo), to determine the effect of natural retention of matured oocytes in body coelomic cavity on further performance of embryos and juveniles produced from those oocytes. Also, we investigated oocyte water hardening and several coelomic fluid parameters as potential quantitative indicators of POA. Oocyte quality decreased significantly from 22 dpo onward, as inferred from decrease in fertilization success and survival of embryos, alevins, and juveniles and increase in alevin and juvenile deformity rates. The occurrence of head deformities was significantly related to postovulatory age of oocytes. Coelomic fluid pH decreased significantly at 28 dpo and correlated positively with fertilization rates (r = 0.45), normal eyed embryo rates (r = 0.67), and alevin relative survival rates (r = 0.63) and negatively correlated with total alevin deformity rates (r = -0.59). Oocyte weight gain at 60 minutes decreased significantly at 28 dpo and correlated negatively with total alevin deformities and the occurrence of cranial nodules (r = -0.99). Generally, quality of ovulated oocytes remained stable for the first 2 weeks after ovulation. Later on, POA negatively influenced Atlantic salmon embryo, alevin, and juvenile performance. For the first time, we show a long-term effect of POA on salmonid juvenile performance. Standardized pH measurements of coelomic fluid could potentially improve embryo and juvenile production by identifying low-quality oocytes at an early stage during the production. Copyright © 2015 Elsevier Inc. All rights reserved.

Mitochondrial genome rearrangements in glomus species triggered by homologous recombination between distinct mtDNA haplotypes.

PubMed

Beaudet, Denis; Terrat, Yves; Halary, Sébastien; de la Providencia, Ivan Enrique; Hijri, Mohamed

2013-01-01

Comparative mitochondrial genomics of arbuscular mycorrhizal fungi (AMF) provide new avenues to overcome long-lasting obstacles that have hampered studies aimed at understanding the community structure, diversity, and evolution of these multinucleated and genetically polymorphic organisms.AMF mitochondrial (mt) genomes are homogeneous within isolates, and their intergenic regions harbor numerous mobile elements that have rapidly diverged, including homing endonuclease genes, small inverted repeats, and plasmid-related DNA polymerase genes (dpo), making them suitable targets for the development of reliable strain-specific markers. However, these elements may also lead to genome rearrangements through homologous recombination, although this has never previously been reported in this group of obligate symbiotic fungi. To investigate whether such rearrangements are present and caused by mobile elements in AMF, the mitochondrial genomes from two Glomeraceae members (i.e., Glomus cerebriforme and Glomus sp.) with substantial mtDNA synteny divergence,were sequenced and compared with available glomeromycotan mitochondrial genomes. We used an extensive nucleotide/protein similarity network-based approach to investigated podiversity in AMF as well as in other organisms for which sequences are publicly available. We provide strong evidence of dpo-induced inter-haplotype recombination, leading to a reshuffled mitochondrial genome in Glomus sp. These findings raise questions as to whether AMF single spore cultivations artificially underestimate mtDNA genetic diversity.We assessed potential dpo dispersal mechanisms in AMF and inferred a robust phylogenetic relationship with plant mitochondrial plasmids. Along with other indirect evidence, our analyses indicate that members of the Glomeromycota phylum are potential donors of mitochondrial plasmids to plants.

Structural and Functional Analysis of Sulfolobus solfataricus Y-Family DNA Polymerase Dpo4-Catalyzed Bypass of the Malondialdehyde−Deoxyguanosine Adduct

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

Eoff, Robert L.; Stafford, Jennifer B.; Szekely, Jozsef

2010-01-12

Oxidative stress can induce the formation of reactive electrophiles, such as DNA peroxidation products, e.g., base propenals, and lipid peroxidation products, e.g., malondialdehyde. Base propenals and malondialdehyde react with DNA to form adducts, including 3-(2'-deoxy-{beta}-d-erythro-pentofuranosyl)pyrimido[1,2-{alpha}]purin-10(3H)-one (M{sub 1}dG). When paired opposite cytosine in duplex DNA at physiological pH, M{sub 1}dG undergoes ring opening to form N{sup 2}-(3-oxo-1-propenyl)-dG (N{sup 2}-OPdG). Previous work has shown that M{sub 1}dG is mutagenic in bacteria and mammalian cells and that its mutagenicity in Escherichia coli is dependent on induction of the SOS response, indicating a role for translesion DNA polymerases in the bypass of M{sub 1}dG.more » To probe the mechanism by which translesion polymerases bypass M{sub 1}dG, kinetic and structural studies were conducted with a model Y-family DNA polymerase, Dpo4 from Sulfolobus solfataricus. The level of steady-state incorporation of dNTPs opposite M{sub 1}dG was reduced 260-2900-fold and exhibited a preference for dATP incorporation. Liquid chromatography-tandem mass spectrometry analysis of the full-length extension products revealed a spectrum of products arising principally by incorporation of dC or dA opposite M{sub 1}dG followed by partial or full-length extension. A greater proportion of -1 deletions were observed when dT was positioned 5' of M{sub 1}dG. Two crystal structures were determined, including a 'type II' frameshift deletion complex and another complex with Dpo4 bound to a dC-M{sub 1}dG pair located in the postinsertion context. Importantly, M{sub 1}dG was in the ring-closed state in both structures, and in the structure with dC opposite M{sub 1}dG, the dC residue moved out of the Dpo4 active site, into the minor groove. The results are consistent with the reported mutagenicity of M{sub 1}dG and illustrate how the lesion may affect replication events.« less

Structural Mechanism of Replication Stalling on a Bulky Amino-Polycyclic Aromatic Hydrocarbon DNA Adduct by a Y Family DNA Polymerase

PubMed Central

Kirouac, Kevin N.; Basu, Ashis K.; Ling, Hong

2013-01-01

Polycyclic aromatic hydrocarbons and their nitro derivatives are culprits of the detrimental health effects of environmental pollution. These hydrophobic compounds metabolize to reactive species and attach to DNA producing bulky lesions, such as N-[deoxyguanosine-8-yl]-1-aminopyrene (APG), in genomic DNA. The bulky adducts block DNA replication by high-fidelity polymerases and compromise replication fidelities and efficiencies by specialized lesion bypass polymerases. Here we present three crystal structures of the DNA polymerase Dpo4, a model translesion DNA polymerase of the Y family, in complex with APG-lesion-containing DNA in pre-insertion and extension stages. APG is captured in two conformations in the pre-insertion complex; one is highly exposed to the solvent, whereas the other is harbored in a shallow cleft between the finger and unique Y family little finger domain. In contrast, APG is in a single conformation at the extension stage, in which the pyrene ring is sandwiched between the little finger domain and a base from the turning back single-stranded template strand. Strikingly, a nucleotide intercalates the DNA helix to form a quaternary complex with Dpo4, DNA, and an incoming nucleotide, which stabilizes the distorted DNA structure at the extension stage. The unique APG DNA conformations in Dpo4 inhibit DNA translocation through the polymerase active site for APG bypass. We also modeled an insertion complex that illustrates a solvent-exposed pyrene ring contributing to an unstable insertion state. The structural work combined with our lesion replication assays provides a novel structural mechanism on bypass of DNA adducts containing polycyclic aromatic hydrocarbon moieties. PMID:23876706

Structural mechanism of replication stalling on a bulky amino-polycyclic aromatic hydrocarbon DNA adduct by a y family DNA polymerase.

PubMed

Kirouac, Kevin N; Basu, Ashis K; Ling, Hong

2013-11-15

Polycyclic aromatic hydrocarbons and their nitro derivatives are culprits of the detrimental health effects of environmental pollution. These hydrophobic compounds metabolize to reactive species and attach to DNA producing bulky lesions, such as N-[deoxyguanosine-8-yl]-1-aminopyrene (APG), in genomic DNA. The bulky adducts block DNA replication by high-fidelity polymerases and compromise replication fidelities and efficiencies by specialized lesion bypass polymerases. Here we present three crystal structures of the DNA polymerase Dpo4, a model translesion DNA polymerase of the Y family, in complex with APG-lesion-containing DNA in pre-insertion and extension stages. APG is captured in two conformations in the pre-insertion complex; one is highly exposed to the solvent, whereas the other is harbored in a shallow cleft between the finger and unique Y family little finger domain. In contrast, APG is in a single conformation at the extension stage, in which the pyrene ring is sandwiched between the little finger domain and a base from the turning back single-stranded template strand. Strikingly, a nucleotide intercalates the DNA helix to form a quaternary complex with Dpo4, DNA, and an incoming nucleotide, which stabilizes the distorted DNA structure at the extension stage. The unique APG DNA conformations in Dpo4 inhibit DNA translocation through the polymerase active site for APG bypass. We also modeled an insertion complex that illustrates a solvent-exposed pyrene ring contributing to an unstable insertion state. The structural work combined with our lesion replication assays provides a novel structural mechanism on bypass of DNA adducts containing polycyclic aromatic hydrocarbon moieties. © 2013.

Maintenance Downtime Sat. 3/11

Atmospheric Science Data Center

2017-03-09

... The DPO (archive 4,5,6) GPFS filesystem will be taken offline starting at 9am Saturday March 11, 2017 .  The Univa Grid ... at 8am Saturday March 11 and the dropbox will remain online for delivery of data only.  This Saturday’s work may potentially ...

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

Rechkoblit, Olga; Delaney, James C.; Essigmann, John M.

DNA is susceptible to alkylation damage by a number of environmental agents that modify the Watson-Crick edge of the bases. Such lesions, if not repaired, may be bypassed by Y-family DNA polymerases. The bypass polymerase Dpo4 is strongly inhibited by 1-methylguanine (m1G) and 3-methylcytosine (m3C), with nucleotide incorporation opposite these lesions being predominantly mutagenic. Further, extension after insertion of both correct and incorrect bases, introduces additional base substitution and deletion errors. Crystal structures of the Dpo4 ternary extension complexes with correct and mismatched 3'-terminal primer bases opposite the lesions reveal that both m1G and m3C remain positioned within the DNAmore » template/primer helix. However, both correct and incorrect pairing partners exhibit pronounced primer terminal nucleotide distortion, being primarily evicted from the DNA helix when opposite m1G or misaligned when pairing with m3C. Our studies provide insights into mechanisms related to hindered and mutagenic bypass of methylated lesions and models associated with damage recognition by repair demethylases.« less

32 CFR 701.125 - Computer matching program.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 32 National Defense 5 2012-07-01 2012-07-01 false Computer matching program. 701.125 Section 701... OF THE NAVY DOCUMENTS AFFECTING THE PUBLIC DON Privacy Program § 701.125 Computer matching program. The DPO has responsibility for coordinating the approval of DOD's participation in Computer Matching...

32 CFR 701.125 - Computer matching program.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 32 National Defense 5 2014-07-01 2014-07-01 false Computer matching program. 701.125 Section 701... OF THE NAVY DOCUMENTS AFFECTING THE PUBLIC DON Privacy Program § 701.125 Computer matching program. The DPO has responsibility for coordinating the approval of DOD's participation in Computer Matching...

32 CFR 701.125 - Computer matching program.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 32 National Defense 5 2013-07-01 2013-07-01 false Computer matching program. 701.125 Section 701... OF THE NAVY DOCUMENTS AFFECTING THE PUBLIC DON Privacy Program § 701.125 Computer matching program. The DPO has responsibility for coordinating the approval of DOD's participation in Computer Matching...

32 CFR 701.125 - Computer matching program.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 32 National Defense 5 2010-07-01 2010-07-01 false Computer matching program. 701.125 Section 701... OF THE NAVY DOCUMENTS AFFECTING THE PUBLIC DON Privacy Program § 701.125 Computer matching program. The DPO has responsibility for coordinating the approval of DOD's participation in Computer Matching...

32 CFR 701.125 - Computer matching program.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 32 National Defense 5 2011-07-01 2011-07-01 false Computer matching program. 701.125 Section 701... OF THE NAVY DOCUMENTS AFFECTING THE PUBLIC DON Privacy Program § 701.125 Computer matching program. The DPO has responsibility for coordinating the approval of DOD's participation in Computer Matching...

32 CFR 310.40 - Requirement for reports.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 32 National Defense 2 2010-07-01 2010-07-01 false Requirement for reports. 310.40 Section 310.40 National Defense Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED) PRIVACY PROGRAM DOD PRIVACY PROGRAM Reports § 310.40 Requirement for reports. The DPO shall establish requirements...

32 CFR 310.40 - Requirement for reports.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 32 National Defense 2 2013-07-01 2013-07-01 false Requirement for reports. 310.40 Section 310.40 National Defense Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED) PRIVACY PROGRAM DOD PRIVACY PROGRAM Reports § 310.40 Requirement for reports. The DPO shall establish requirements...

32 CFR 310.40 - Requirement for reports.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 32 National Defense 2 2014-07-01 2014-07-01 false Requirement for reports. 310.40 Section 310.40 National Defense Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED) PRIVACY PROGRAM DOD PRIVACY PROGRAM Reports § 310.40 Requirement for reports. The DPO shall establish requirements...

32 CFR 310.40 - Requirement for reports.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 32 National Defense 2 2012-07-01 2012-07-01 false Requirement for reports. 310.40 Section 310.40 National Defense Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED) PRIVACY PROGRAM DOD PRIVACY PROGRAM Reports § 310.40 Requirement for reports. The DPO shall establish requirements...

32 CFR 310.40 - Requirement for reports.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 32 National Defense 2 2011-07-01 2011-07-01 false Requirement for reports. 310.40 Section 310.40 National Defense Department of Defense (Continued) OFFICE OF THE SECRETARY OF DEFENSE (CONTINUED) PRIVACY PROGRAM DOD PRIVACY PROGRAM Reports § 310.40 Requirement for reports. The DPO shall establish requirements...

32 CFR 701.114 - PA enforcement actions.

Code of Federal Regulations, 2010 CFR

2010-07-01

... adequately addressed, the individual may contact CNO (DNS-36) or CMC (ARSF), for assistance. (b) Civil court... employee, the responsible DON activity shall promptly apprise CNO (DNS-36)) and provide a copy of all relevant documents. CNO (DNS-36) will in turn apprise the DPO, who will apprise the DOJ. When a court...

32 CFR 701.114 - PA enforcement actions.

Code of Federal Regulations, 2011 CFR

2011-07-01

... adequately addressed, the individual may contact CNO (DNS-36) or CMC (ARSF), for assistance. (b) Civil court... employee, the responsible DON activity shall promptly apprise CNO (DNS-36)) and provide a copy of all relevant documents. CNO (DNS-36) will in turn apprise the DPO, who will apprise the DOJ. When a court...

32 CFR 701.114 - PA enforcement actions.

Code of Federal Regulations, 2014 CFR

2014-07-01

... adequately addressed, the individual may contact CNO (DNS-36) or CMC (ARSF), for assistance. (b) Civil court... employee, the responsible DON activity shall promptly apprise CNO (DNS-36)) and provide a copy of all relevant documents. CNO (DNS-36) will in turn apprise the DPO, who will apprise the DOJ. When a court...

32 CFR 701.114 - PA enforcement actions.

Code of Federal Regulations, 2013 CFR

2013-07-01

... adequately addressed, the individual may contact CNO (DNS-36) or CMC (ARSF), for assistance. (b) Civil court... employee, the responsible DON activity shall promptly apprise CNO (DNS-36)) and provide a copy of all relevant documents. CNO (DNS-36) will in turn apprise the DPO, who will apprise the DOJ. When a court...

32 CFR 701.114 - PA enforcement actions.

Code of Federal Regulations, 2012 CFR

2012-07-01

... adequately addressed, the individual may contact CNO (DNS-36) or CMC (ARSF), for assistance. (b) Civil court... employee, the responsible DON activity shall promptly apprise CNO (DNS-36)) and provide a copy of all relevant documents. CNO (DNS-36) will in turn apprise the DPO, who will apprise the DOJ. When a court...

Application of the double paddle oscillator for quantifying environmental, surface mass variation

NASA Astrophysics Data System (ADS)

Wei, Haoyan; Pomeroy, Joshua

2016-04-01

Sub-monolayer sensitivity to controlled gas adsorption and desorption is demonstrated using a double paddle oscillator (DPO) installed within an ultra-high vacuum (UHV) environmental chamber equipped with in situ film deposition, (multi)gas admission and temperature control. This effort is intended to establish a robust framework for quantitatively comparing mass changes due to gas loading and unloading on different materials systems selected or considered for use as mass artefacts. Our apparatus is composed of a UHV chamber with gas introduction and temperature control and in situ materials deposition for future materials testing enabling in situ preparation of virgin surfaces that can be monitored during initial exposure to gasses of interest. These tools are designed to allow us to comparatively evaluate how different materials gain or lose mass due to precisely controlled environmental excursions, with a long term goal of measuring changes in absolute mass. Herein, we provide a detailed experimental description of the apparatus, an evaluation of the initial performance, and demonstration measurements using nitrogen adsorption and desorption directly on the DPO.

Application of the double paddle oscillator for quantifying environmental, surface mass variation

PubMed Central

Wei, Haoyan; Pomeroy, Joshua

2016-01-01

Sub-monolayer sensitivity to controlled gas adsorption and desorption is demonstrated using a double paddle oscillator (DPO) installed within an UHV (ultra-high vacuum) environmental chamber equipped with in situ film deposition, (multi)gas admission and temperature control. This effort is intended to establish a robust framework for quantitatively comparing mass changes due to gas loading and unloading on different materials systems selected or considered for use as mass artifacts. Our apparatus is composed of a UHV chamber with gas introduction and temperature control and in-situ materials deposition for future materials testing enabling in situ preparation of virgin surfaces that can be monitored during initial exposure to gasses of interest. These tools are designed to allow us to comparatively evaluate how different materials gain or lose mass due to precisely controlled environmental excursions, with a long term goal of measuring changes in absolute mass. Herein, we provide a detailed experimental description of the apparatus, an evaluation of the initial performance, and demonstration measurements using nitrogen adsorption and desorption directly on the DPO. PMID:27212736

Dual Priming Oligonucleotides for Broad-Range Amplification of the Bacterial 16S rRNA Gene Directly from Human Clinical Specimens

PubMed Central

Simmon, Keith; Karaca, Dilek; Langeland, Nina; Wiker, Harald G.

2012-01-01

Broad-range amplification and sequencing of the bacterial 16S rRNA gene directly from clinical specimens are offered as a diagnostic service in many laboratories. One major pitfall is primer cross-reactivity with human DNA which will result in mixed chromatograms. Mixed chromatograms will complicate subsequent sequence analysis and impede identification. In SYBR green real-time PCR assays, it can also affect crossing threshold values and consequently the status of a specimen as positive or negative. We evaluated two conventional primer pairs in common use and a new primer pair based on the dual priming oligonucleotide (DPO) principle. Cross-reactivity was observed when both conventional primer pairs were used, resulting in interpretation difficulties. No cross-reactivity was observed using the DPOs even in specimens with a high ratio of human to bacterial DNA. In addition to reducing cross-reactivity, the DPO principle also offers a high degree of flexibility in the design of primers and should be considered for any PCR assay intended for detection and identification of pathogens directly from human clinical specimens. PMID:22278843

Assessing environmental risks for high intensity agriculture using the material flow analysis method--a case study of the Dongting Lake basin in South Central China.

PubMed

Yin, Guanyi; Liu, Liming; Yuan, Chengcheng

2015-07-01

This study primarily examined the assessment of environmental risk in high intensity agricultural areas. Dongting Lake basin was taken as a case study, which is one of the major grain producing areas in China. Using data obtained from 1989 to 2012, we applied Material Flow Analysis (MFA) to show the material consumption, pollutant output and production storage in the agricultural-environmental system and assessed the environmental risk index on the basis of the MFA results. The results predicted that the status of the environmental quality of the Dongting Lake area is unsatisfactory for the foreseeable future. The direct material input (DMI) declined by 13.9%, the domestic processed output (DPO) increased by 28.21%, the intensity of material consumption (IMC) decreased by 36.7%, the intensity of material discharge (IMD) increased by 10%, the material productivity (MP) increased by 27 times, the environmental efficiency (EE) increased by 15.31 times, and the material storage (PAS) increased by 0.23%. The DMI and DPO was higher at rural places on the edge of cities, whereas the risk of urban agriculture has arisen due to the higher increasing rate of DMI and DPO in cities compared with the counties. The composite environmental risk index increased from 0.33 to 0.96, indicating that the total environmental risk changed gradually but seriously during the 24 years assessed. The driving factors that affect environmental risk in high intensity agriculture can be divided into five classes: social, economic, human, natural and disruptive incidents. This study discussed a number of effective measures for protecting the environment while ensuring food production yields. Additional research in other areas and certain improvements of this method in future studies may be necessary to develop a more effective method of managing and controlling agricultural-environmental interactions.

Contribution of KV1.5 Channel to H2O2-Induced Human Arteriolar Dilation and its Modulation by Coronary Artery Disease

PubMed Central

Nishijima, Yoshinori; Cao, Sheng; Chabowski, Dawid S.; Korishettar, Ankush; Ge, Alyce; Zheng, Xiaodong; Sparapani, Rodney; Gutterman, David D.; Zhang, David X.

2016-01-01

Rationale Hydrogen peroxide (H2O2) regulates vascular tone in the human microcirculation under physiological and pathophysiological conditions. It dilates arterioles by activating BKCa channels in subjects with coronary artery disease (CAD), but its mechanisms of action in subjects without CAD (non-CAD) as compared to those with CAD remain unknown. Objective We hypothesize that H2O2-elicited dilation involves different K+ channels in non-CAD versus CAD, resulting in an altered capacity for vasodilation during disease. Methods and Results H2O2 induced endothelium-independent vasodilation in non-CAD adipose arterioles, which was reduced by paxilline, a BKCa channel blocker, and by 4-AP, a KV channel blocker. Assays of mRNA transcripts, protein expression and subcellular localization revealed that KV1.5 is the major KV1 channel expressed in vascular smooth muscle cells (VSMCs) and is abundantly localized on the plasma membrane. The selective KV1.5 blocker DPO-1 and the KV1.3/1.5 blocker Psora-4 reduced H2O2-elicited dilation to a similar extent as 4-AP, but the selective KV1.3 blocker PAP-1 was without effect. In arterioles from CAD subjects, H2O2-induced dilation was significantly reduced and this dilation was inhibited by paxilline but not by 4-AP, DPO-1 or Psora-4. KV1.5 cell membrane localization and DPO-1-sensitive K+ currents were markedly reduced in isolated VSMCs from CAD arterioles, although mRNA or total cellular protein expression were largely unchanged. Conclusions In human arterioles, H2O2-induced dilation is impaired in CAD, which is associated with a transition from a combined BKCa- and KV (KV1.5)-mediated vasodilation toward a BKCa-predominant mechanism of dilation. Loss of KV1.5 vasomotor function may play an important role in microvascular dysfunction in CAD or other vascular diseases. PMID:27872049

Following DNA chain extension and protein conformational changes in crystals of a Y-family DNA polymerase via Raman crystallography.

PubMed

Espinoza-Herrera, Shirly J; Gaur, Vineet; Suo, Zucai; Carey, Paul R

2013-07-23

Y-Family DNA polymerases are known to bypass DNA lesions in vitro and in vivo. Sulfolobus solfataricus DNA polymerase (Dpo4) was chosen as a model Y-family enzyme for investigating the mechanism of DNA synthesis in single crystals. Crystals of Dpo4 in complexes with DNA (the binary complex) in the presence or absence of an incoming nucleotide were analyzed by Raman microscopy. (13)C- and (15)N-labeled d*CTP, or unlabeled dCTP, were soaked into the binary crystals with G as the templating base. In the presence of the catalytic metal ions, Mg(2+) and Mn(2+), nucleotide incorporation was detected by the disappearance of the triphosphate band of dCTP and the retention of *C modes in the crystal following soaking out of noncovalently bound C(or *C)TP. The addition of the second coded base, thymine, was observed by adding cognate dTTP to the crystal following a single d*CTP addition. Adding these two bases caused visible damage to the crystal that was possibly caused by protein and/or DNA conformational change within the crystal. When d*CTP is soaked into the Dpo4 crystal in the absence of Mn(2+) or Mg(2+), the primer extension reaction did not occur; instead, a ternary protein·template·d*CTP complex was formed. In the Raman difference spectra of both binary and ternary complexes, in addition to the modes of d(*C)CTP, features caused by ring modes from the template/primer bases being perturbed and from the DNA backbone appear, as well as features from perturbed peptide and amino acid side chain modes. These effects are more pronounced in the ternary complex than in the binary complex. Using standardized Raman intensities followed as a function of time, the C(*C)TP population in the crystal was maximal at ∼20 min. These remained unchanged in the ternary complex but declined in the binary complexes as chain incorporation occurred.

Hydrogen generation in CSP plants and maintenance of DPO/BP heat transfer fluids - A simulation approach

NASA Astrophysics Data System (ADS)

Kuckelkorn, Thomas; Jung, Christian; Gnädig, Tim; Lang, Christoph; Schall, Christina

2016-05-01

The ageing of diphenyl oxide/ biphenyl (DPO/BP) Heat Transfer Fluids (HTFs) implies challenging tasks for operators of parabolic trough power plants in order to find the economic optimum between plant performance and O&M costs. Focusing on the generation of hydrogen, which is effecting from the HTF ageing process, the balance of hydrogen pressure in the HTF is simulated for different operation scenarios. Accelerated build-up of hydrogen pressure in the HTF is causing increased permeation into the annular vacuum space of the installed receivers and must be avoided in order to maintain the performance of these components. Therefore, the effective hydrogen partial pressure in the HTF has to be controlled and limited according to the specified values so that the vacuum lifetime of the receivers and the overall plant performance can be ensured. In order to simulate and visualize the hydrogen balance of a typical parabolic trough plant, initially a simple model is used to calculate the balance of hydrogen in the system and this is described. As input data for the simulation, extrapolated hydrogen generation rates have been used, which were calculated from results of lab tests performed by DLR in Cologne, Germany. Hourly weather data, surface temperatures of the tubing system calculated by using the simulation tool from NREL, and hydrogen permeation rates for stainless steel and carbon steel grades taken from literature have been added to the model. In a first step the effect of HTF ageing, build-up of hydrogen pressure in the HTF and hydrogen loss rates through piping and receiver components have been modeled. In a second step a selective hydrogen removal process has been added to the model. The simulation results are confirming the need of active monitoring and controlling the effective hydrogen partial pressure in parabolic trough solar thermal power plants with DPO/BP HTF. Following the results of the simulation, the expected plant performance can only be achieved over lifetime, if the hydrogen partial pressure is actively controlled and limited.

Services provided by volunteer psychiatrists after 9/11 at the New York City family assistance center: September 12-November 20, 2001.

PubMed

Pandya, Anand; Katz, Craig L; Smith, Rebecca; Ng, Anthony T; Tafoya, Michael; Holmes, Anastasia; North, Carol S

2010-05-01

To characterize the experience of volunteer disaster psychiatrists who provided pro bono psychiatric services to 9/11 survivors in New York City, from September 12, 2001 to November 20, 2001. Disaster Psychiatry Outreach (DPO) is a non-profit organization founded in 1998 to provide volunteer psychiatric care to people affected by disasters and to promote education and research in support of this mission. Data for this study were collected from one-page clinical encounter forms completed by 268 DPO psychiatrists for 2 months after 9/11 concerning 848 patients served by the DPO 9/11 response program at the New York City Family Assistance Center. In this endeavor, 268 psychiatrist volunteers evaluated 848 individuals and provided appropriate interventions. The most commonly recorded clinical impressions indicated stress-related and adjustment disorders, but other conditions such as bereavement, major depression, and substance abuse/dependence were also observed. Free samples were available for one sedative and one anxiolytic agent; not surprisingly, these were the most commonly prescribed medications. Nearly half of those evaluated received psychotropic medications. In the acute aftermath of the attacks of September 11, 2001, volunteer psychiatrists were able to provide services in a disaster response setting, in which they were co-located with other disaster responders. These services included psychiatric assessment, provision of medication, psychological first aid, and referrals for ongoing care. Although systematic diagnoses could not be confirmed, the fact that most patients were perceived to have a psychiatric diagnosis and a substantial proportion received psychotropic medication suggests potential specific roles for psychiatrists that are unique and different from roles of other mental health professionals in the early post-disaster setting. In addition to further characterizing post-disaster mental health needs and patterns of service provision, future research should focus on the short- and long-term effects of psychiatric interventions, such as providing acute psychotropic medication services and assessing the effectiveness of traditional acute post-disaster interventions including crisis counseling and psychological first aid.

Radical Polymerization of Vinyl Acetate and Methyl Methacrylate Using Organochromium Initiators Complexed with Macrocyclic Polyamines

DTIC Science & Technology

1994-06-30

conversion, the increase was faster than at later stages. and therefore the steric strain enfosced by the 5gn backone dpdson Th efficiency of initation...BPOJ),- 0.25M. Cr(OAc)2jDPO, THF. 200CC IVAclo - SMK [Cr2io - [DPOJ~i 0.25M. (UI - (Cr2+’Jo (LIgl - (Cr2൒ 0 _ In the absence of any liganda, a

Delivery Time Variance Reduction in the Military Supply Chain

DTIC Science & Technology

2010-03-01

Donald Rumsfeld, designated “U.S. Transportation Command as the single Department of Defense Distribution Process Owner (DPO)” (USTRANSCOM, 2004...paragraphs explain OptQuest’s 54 functionality and capabilities as described by Laguna (1997) and Glover et al. (1999) as well as the OptQuest for ARENA...throughout the solution space ( Glover et al., 1999). Heuristics are strategies (in this case algorithms) that use different techniques and available

Universal detection of phytoplasmas and Xylella spp. by TaqMan singleplex and multiplex real-time PCR with dual priming oligonucleotides.

PubMed

Ito, Takao; Suzaki, Koichi

2017-01-01

Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR) assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO) with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays.

Universal detection of phytoplasmas and Xylella spp. by TaqMan singleplex and multiplex real-time PCR with dual priming oligonucleotides

PubMed Central

Suzaki, Koichi

2017-01-01

Phytoplasmas and Xylella spp. are bacteria that cause many economically important plant diseases worldwide. TaqMan probe-based quantitative real-time polymerase chain reaction (qPCR) assays have been utilized to universally detect phytoplasmas or Xylella fastidiosa. To develop a superior universal qPCR method, we used a dual priming oligonucleotide (DPO) with two annealing sites as a reverse primer to target the well-conserved bacterial 16S rDNA. The new qPCR assays universally detected various species of phytoplasmas and subspecies of X. fastidiosa as well as Xylella taiwanensis, and generally showed superior threshold cycle values when amplifying specific or non-specific products compared to current universal qPCR assays. The proposed qPCR assays were integrated to develop a multiplex qPCR assay that simultaneously detected phytoplasmas, Xylella spp., and an internal plant DNA positive control within 1 hour. This assay could detect a minimum of ten bacterial cells and was compatible with crude extractions used in the rapid screening of various plants. The amplicons were of sufficient lengths to be directly sequenced for preliminary identification, and the primers could be used in universal conventional PCR assays. Additionally, reverse DPO primers can be utilized to improve other probe-based qPCR assays. PMID:28957362

Clinical and epidemiologic characteristics of dengue and other etiologic agents among patients with acute febrile illness, Puerto Rico, 2012–2015

PubMed Central

Lorenzi, Olga D.; Andújar-Pérez, Doris A.; Torres-Velásquez, Brenda C.; Hunsperger, Elizabeth A.; Munoz-Jordan, Jorge Luis; Perez-Padilla, Janice; Rivera, Aidsa; Gonzalez-Zeno, Gladys E.; Sharp, Tyler M.; Galloway, Renee L.; Glass Elrod, Mindy; Mathis, Demetrius L.; Oberste, M. Steven; Nix, W. Allan; Henderson, Elizabeth; McQuiston, Jennifer; Singleton, Joseph; Kato, Cecilia; García Gubern, Carlos; Santiago-Rivera, William; Cruz-Correa, Jesús; Muns-Sosa, Robert; Ortiz-Rivera, Juan D.; Jiménez, Gerson; Galarza, Ivonne E.; Horiuchi, Kalanthe; Margolis, Harold S.; Alvarado, Luisa I.

2017-01-01

Identifying etiologies of acute febrile illnesses (AFI) is challenging due to non-specific presentation and limited availability of diagnostics. Prospective AFI studies provide a methodology to describe the syndrome by age and etiology, findings that can be used to develop case definitions and multiplexed diagnostics to optimize management. We conducted a 3-year prospective AFI study in Puerto Rico. Patients with fever ≤7 days were offered enrollment, and clinical data and specimens were collected at enrollment and upon discharge or follow-up. Blood and oro-nasopharyngeal specimens were tested by RT-PCR and immunodiagnostic methods for infection with dengue viruses (DENV) 1–4, chikungunya virus (CHIKV), influenza A and B viruses (FLU A/B), 12 other respiratory viruses (ORV), enterovirus, Leptospira spp., and Burkholderia pseudomallei. Clinical presentation and laboratory findings of participants infected with DENV were compared to those infected with CHIKV, FLU A/B, and ORV. Clinical predictors of laboratory-positive dengue compared to all other AFI etiologies were determined by age and day post-illness onset (DPO) at presentation. Of 8,996 participants enrolled from May 7, 2012 through May 6, 2015, more than half (54.8%, 4,930) had a pathogen detected. Pathogens most frequently detected were CHIKV (1,635, 18.2%), FLU A/B (1,074, 11.9%), DENV 1–4 (970, 10.8%), and ORV (904, 10.3%). Participants with DENV infection presented later and a higher proportion were hospitalized than those with other diagnoses (46.7% versus 27.3% with ORV, 18.8% with FLU A/B, and 11.2% with CHIKV). Predictors of dengue in participants presenting <3 DPO included leukopenia, thrombocytopenia, headache, eye pain, nausea, and dizziness, while negative predictors were irritability and rhinorrhea. Predictors of dengue in participants presenting 3–5 DPO were leukopenia, thrombocytopenia, facial/neck erythema, nausea, eye pain, signs of poor circulation, and diarrhea; presence of rhinorrhea, cough, and red conjunctiva predicted non-dengue AFI. By enrolling febrile patients at clinical presentation, we identified unbiased predictors of laboratory-positive dengue as compared to other common causes of AFI. These findings can be used to assist in early identification of dengue patients, as well as direct anticipatory guidance and timely initiation of correct clinical management. PMID:28902845

Clinical and epidemiologic characteristics of dengue and other etiologic agents among patients with acute febrile illness, Puerto Rico, 2012-2015.

PubMed

Tomashek, Kay M; Lorenzi, Olga D; Andújar-Pérez, Doris A; Torres-Velásquez, Brenda C; Hunsperger, Elizabeth A; Munoz-Jordan, Jorge Luis; Perez-Padilla, Janice; Rivera, Aidsa; Gonzalez-Zeno, Gladys E; Sharp, Tyler M; Galloway, Renee L; Glass Elrod, Mindy; Mathis, Demetrius L; Oberste, M Steven; Nix, W Allan; Henderson, Elizabeth; McQuiston, Jennifer; Singleton, Joseph; Kato, Cecilia; García Gubern, Carlos; Santiago-Rivera, William; Cruz-Correa, Jesús; Muns-Sosa, Robert; Ortiz-Rivera, Juan D; Jiménez, Gerson; Galarza, Ivonne E; Horiuchi, Kalanthe; Margolis, Harold S; Alvarado, Luisa I

2017-09-01

Identifying etiologies of acute febrile illnesses (AFI) is challenging due to non-specific presentation and limited availability of diagnostics. Prospective AFI studies provide a methodology to describe the syndrome by age and etiology, findings that can be used to develop case definitions and multiplexed diagnostics to optimize management. We conducted a 3-year prospective AFI study in Puerto Rico. Patients with fever ≤7 days were offered enrollment, and clinical data and specimens were collected at enrollment and upon discharge or follow-up. Blood and oro-nasopharyngeal specimens were tested by RT-PCR and immunodiagnostic methods for infection with dengue viruses (DENV) 1-4, chikungunya virus (CHIKV), influenza A and B viruses (FLU A/B), 12 other respiratory viruses (ORV), enterovirus, Leptospira spp., and Burkholderia pseudomallei. Clinical presentation and laboratory findings of participants infected with DENV were compared to those infected with CHIKV, FLU A/B, and ORV. Clinical predictors of laboratory-positive dengue compared to all other AFI etiologies were determined by age and day post-illness onset (DPO) at presentation. Of 8,996 participants enrolled from May 7, 2012 through May 6, 2015, more than half (54.8%, 4,930) had a pathogen detected. Pathogens most frequently detected were CHIKV (1,635, 18.2%), FLU A/B (1,074, 11.9%), DENV 1-4 (970, 10.8%), and ORV (904, 10.3%). Participants with DENV infection presented later and a higher proportion were hospitalized than those with other diagnoses (46.7% versus 27.3% with ORV, 18.8% with FLU A/B, and 11.2% with CHIKV). Predictors of dengue in participants presenting <3 DPO included leukopenia, thrombocytopenia, headache, eye pain, nausea, and dizziness, while negative predictors were irritability and rhinorrhea. Predictors of dengue in participants presenting 3-5 DPO were leukopenia, thrombocytopenia, facial/neck erythema, nausea, eye pain, signs of poor circulation, and diarrhea; presence of rhinorrhea, cough, and red conjunctiva predicted non-dengue AFI. By enrolling febrile patients at clinical presentation, we identified unbiased predictors of laboratory-positive dengue as compared to other common causes of AFI. These findings can be used to assist in early identification of dengue patients, as well as direct anticipatory guidance and timely initiation of correct clinical management.

First person: a mental health mission to post-earthquake El Salvador.

PubMed

Katz, Craig L

2013-09-01

In this article the author excerpts and discusses salient quotes or moments from the journal he compiled while visiting El Salvador in February 2001 as head of Disaster Psychiatry Outreach (DPO) to assist survivors of a major earthquake. This case discussion of a single disaster mental health response exemplifies key issues related to both short and long term mental health service delivery to disaster affected communities. Copyright © 2013 Elsevier Inc. All rights reserved.

Photon Stimulated Ion Desorption.

DTIC Science & Technology

1982-03-03

1978) 1997.(181 T. Shibaguchi, H . Onuki and R. Onaka, J. Phys. Soc. Contract DE.AC04.76-DPO0789. Experiments were Japan 42 (1977) S51. conducted at...University of California and the Naval Weapons Center. Sincerely COPY ovoikoble to DTIC doee 00t pe m i jully legible rep oductC r h Christopher C... H 20 is studied; only hydrogen ions are observed. Desorption of hydrogen ions from amorphous ice 7 is part of an ongoing study of condensed gases

Bone marrow stromal cells promote neuroplasticity of cerebral ischemic rats via a phosphorylated CRMP2-mediated mechanism.

PubMed

He, Xiang; Jiang, Ling; Dan, Qi-Qin; Lv, Qiang; Hu, Yue; Liu, Jia; Wang, Shu-Fen; Wang, Ting-Hua

2017-03-01

Collapsin response mediator protein 2 (CRMP2), an important protein involved in axonal growth and the maintenance of neuronal membrane integrity, has proved to be altered in nervous system diseases. This study was aimed to investigate the role of CRMP2 in bone marrow stromal cells (BMSCs) treating rats with cerebral ischemia. BMSCs were isolated from shaft of the femurs, tibiae, and humeri and were intra-carotid administrated immediately after middle cerebral artery occlusion (MCAO). Modified Neurological Severity Scores (mNSS) was conducted at 3, 7, 14dpo and the electrophysiologic evaluation was evaluated at 14dpo. Then all rats were sacrificed and brain tissues were harvested for RT-PCR, Western blot and Immunohistochemistry analysis. We found BMSCs treatment significantly improved the neurobehavioral performance impaired by ischemic brain injury, accompanied with the notably increasing levels of Synaptophysin (SYP) and Growth associated protein 43 (GAP43). We also found the protein level of phosphorylated CRMP2 (p-CRMP2) and phosphorylation-mediated protein including Glycogen synthase kinase 3 Beta (GSK3β), Cyclin-dependent kinase 5 (CDK5) were dramatically downregulated in ischemic rats following BMSCs transplant. Furthermore, the GSK3β-mediated factors including neurotrophic and signaling factors were all significantly upregulated in BMSCs-treated group. On the basis of these findings, we suggest that the neuroplasticity effect of BMSCs on cerebral ischemia may be associated with the phosphorylated modulation of CRMP2. Copyright © 2016 Elsevier B.V. All rights reserved.

Dendrites of medial olivocochlear neurons in mouse.

PubMed

Brown, M C; Levine, J L

2008-06-12

Stains for acetylcholinesterase (AChE) and retrograde labeling with Fluorogold (FG) were used to study olivocochlear neurons and their dendritic patterns in mice. The two methods gave similar results for location and number of somata. The total number of medial olivocochlear (MOC) neurons in the ventral nucleus of the trapezoid body (VNTB) is about 170 per side. An additional dozen large olivocochlear neurons are located in the dorsal periolivary nucleus (DPO). Dendrites of all of these neurons are long and extend in all directions from the cell bodies, a pattern that contrasts with the sharp frequency tuning of their responses. For VNTB neurons, there were greater numbers of dendrites directed medially than laterally and those directed medially were longer (on average, 25-50% longer). Dendrite extensions were most pronounced for neurons located in the rostral portion of the VNTB. When each dendrite from a single neuron was represented as a vector, and all the vectors summed, the result was also skewed toward the medial direction. DPO neurons, however, had more symmetric dendrites that projected into more dorsal parts of the trapezoid body, suggesting that this small group of olivocochlear neurons has very different physiological properties. Dendrites of both types of neurons were somewhat elongated rostrally, about 20% longer than those directed caudally. These results can be interpreted as extensions of dendrites of olivocochlear neurons toward their synaptic inputs: medially to meet crossing fibers from the cochlear nucleus that are part of the MOC reflex pathway, and rostrally to meet descending inputs from higher centers.

Achieving Ships Mission Flexibility Through Designing, Printing And Operating Unmanned Systems With Additive Manufacturing And Delayed Differentiation

DTIC Science & Technology

2016-09-01

the UAV’s reliability in fulfilling the mission as well as the build- time of the UAV. 14. SUBJECT TERMS design , print and operate, DPO...previously. There are opportunities to work on the design of the UAV to reduce the cognitive workload of the service member and time required to “print” and...the need arises to tailor the UAV for the specific mission. The modification of an existing design is expected to take a much shorter time than the

Immunoreactive Changes in the Hypoglossal Nucleus after Nerve Injury

DTIC Science & Technology

1991-07-25

m ^ •••.•(.•>a> j T ’ — ’ . - v - ; - . • . • " _ • , . - • ’ ^ v , > ’ --•̂ T f£is:j-i&. .i.’a^i:-:5 Figure 7 30...nerve injury. A. nerve crush B. nerve transection C. nerve resection 38 ’.. i" : ^ M ^’^’- B •Jysf:^.-^^ ^ . - . ><^^ ^^-:3??jFj\\T^ Figure 10...R) 330X. 40 0^ m •s ’^ " ^ ’ Figure 11 41 both sides of the nucleus. At 20 dpo, the CGRP-IR increase in neuronal cell bodies and

Engineering of Bacteriophages Y2::dpoL1-C and Y2::luxAB for Efficient Control and Rapid Detection of the Fire Blight Pathogen, Erwinia amylovora.

PubMed

Born, Yannick; Fieseler, Lars; Thöny, Valentin; Leimer, Nadja; Duffy, Brion; Loessner, Martin J

2017-06-15

Erwinia amylovora is the causative agent of fire blight, a devastating plant disease affecting members of the Rosaceae Alternatives to antibiotics for control of fire blight symptoms and outbreaks are highly desirable, due to increasing drug resistance and tight regulatory restrictions. Moreover, the available diagnostic methods either lack sensitivity, lack speed, or are unable to discriminate between live and dead bacteria. Owing to their extreme biological specificity, bacteriophages are promising alternatives for both aims. In this study, the virulent broad-host-range E. amylovora virus Y2 was engineered to enhance its killing activity and for use as a luciferase reporter phage, respectively. Toward these aims, a depolymerase gene of E. amylovora virus L1 ( dpoL1-C ) or a bacterial luxAB fusion was introduced into the genome of Y2 by homologous recombination. The genes were placed downstream of the major capsid protein orf68 , under the control of the native promoter. The modifications did not affect viability of infectivity of the recombinant viruses. Phage Y2:: dpoL1-C demonstrated synergistic activity between the depolymerase degrading the exopolysaccharide capsule and phage infection, which greatly enhanced bacterial killing. It also significantly reduced the ability of E. amylovora to colonize the surface of detached flowers. The reporter phage Y2:: luxAB transduced bacterial luciferase into host cells and induced synthesis of large amounts of a LuxAB luciferase fusion. After the addition of aldehyde substrate, bioluminescence could be readily monitored, and this enabled rapid and specific detection of low numbers of viable bacteria, without enrichment, both in vitro and in plant material. IMPORTANCE Fire blight, caused by Erwinia amylovora , is the major threat to global pome fruit production, with high economic losses every year. Bacteriophages represent promising alternatives to not only control the disease, but also for rapid diagnostics. To enhance biocontrol efficacy, we combined the desired properties of two phages, Y2 (broad host range) and L1 (depolymerase for capsule degradation) in a single recombinant phage. This phage showed enhanced biocontrol and could reduce E. amylovora on flowers. Phage Y2 was also genetically engineered into a luciferase reporter phage, which transduces bacterial bioluminescence into infected cells and allows detection of low numbers of viable target bacteria. The combination of speed, sensitivity, and specificity is superior to previously used diagnostic methods. In conclusion, genetic engineering could improve the properties of phage Y2 toward better killing efficacy and sensitive detection of E. amylovora cells. Copyright © 2017 American Society for Microbiology.

Engineering of Bacteriophages Y2::dpoL1-C and Y2::luxAB for Efficient Control and Rapid Detection of the Fire Blight Pathogen, Erwinia amylovora

PubMed Central

Born, Yannick; Fieseler, Lars; Thöny, Valentin; Leimer, Nadja; Duffy, Brion

2017-01-01

ABSTRACT Erwinia amylovora is the causative agent of fire blight, a devastating plant disease affecting members of the Rosaceae. Alternatives to antibiotics for control of fire blight symptoms and outbreaks are highly desirable, due to increasing drug resistance and tight regulatory restrictions. Moreover, the available diagnostic methods either lack sensitivity, lack speed, or are unable to discriminate between live and dead bacteria. Owing to their extreme biological specificity, bacteriophages are promising alternatives for both aims. In this study, the virulent broad-host-range E. amylovora virus Y2 was engineered to enhance its killing activity and for use as a luciferase reporter phage, respectively. Toward these aims, a depolymerase gene of E. amylovora virus L1 (dpoL1-C) or a bacterial luxAB fusion was introduced into the genome of Y2 by homologous recombination. The genes were placed downstream of the major capsid protein orf68, under the control of the native promoter. The modifications did not affect viability of infectivity of the recombinant viruses. Phage Y2::dpoL1-C demonstrated synergistic activity between the depolymerase degrading the exopolysaccharide capsule and phage infection, which greatly enhanced bacterial killing. It also significantly reduced the ability of E. amylovora to colonize the surface of detached flowers. The reporter phage Y2::luxAB transduced bacterial luciferase into host cells and induced synthesis of large amounts of a LuxAB luciferase fusion. After the addition of aldehyde substrate, bioluminescence could be readily monitored, and this enabled rapid and specific detection of low numbers of viable bacteria, without enrichment, both in vitro and in plant material. IMPORTANCE Fire blight, caused by Erwinia amylovora, is the major threat to global pome fruit production, with high economic losses every year. Bacteriophages represent promising alternatives to not only control the disease, but also for rapid diagnostics. To enhance biocontrol efficacy, we combined the desired properties of two phages, Y2 (broad host range) and L1 (depolymerase for capsule degradation) in a single recombinant phage. This phage showed enhanced biocontrol and could reduce E. amylovora on flowers. Phage Y2 was also genetically engineered into a luciferase reporter phage, which transduces bacterial bioluminescence into infected cells and allows detection of low numbers of viable target bacteria. The combination of speed, sensitivity, and specificity is superior to previously used diagnostic methods. In conclusion, genetic engineering could improve the properties of phage Y2 toward better killing efficacy and sensitive detection of E. amylovora cells. PMID:28389547

Biodiesel production from ethanolysis of DPO using deep eutectic solvent (DES) based choline chloride - ethylene glycol as co-solvent

NASA Astrophysics Data System (ADS)

Taslim, Indra, Leonardo; Manurung, Renita; Winarta, Agus; Ramadhani, Debbie Aditia

2017-03-01

Biodiesel is usually produced from transesterification using methanol or ethanol as alcohol. However, biodiesel produced using methanol has several disadvantages because methanol is toxic and not entirely bio-based as it is generally produced from petroleum, natural gas and coal. On the other hand, ethanol also has several disadvantages such as lower reactivity in transesterification process and formation of stable emulsion between ester and glycerol. To improve ethanolysis process, deep eutectic solvent (DES) was prepared from choline chloride and ethylene glycol to be used as co-solvent in ethanolysis. Deep eutectic solvent was prepared by mixing choline chloride and ethylene glycol at molar ratio of 1:2, temperature of 80 °C, and stirring speed of 300 rpm for 1 hour. The DES was characterized by its density and viscosity. The ethanolysis of DPO / Degummed Palm Oil was performed at 70 °C, ethanol to oil molar ratio of 9:1, catalyst (potassium hydroxide) concentration of 0.75 wt.% concentration, co-solvent (DES) concentration of 1, 2, 3, 4, 5 and 6 wt.%, stirring speed of 600 rpm, and reaction time of 1 hour. The obtained biodiesel was then characterized by its density, viscosity and ester content. The oil - ethanol phase condition was observed in reaction tube. The oil - ethanol phase with DES tends to form meniscus compared to that without DES. Which implied that oil and ethanol become more slightly miscible, which favours the reaction. Using DES as co-solvent in ethanolysis resulted in an increase in yield and easier purification. The esters properties met the international standards ASTM D6751, with highest yield achieved at 81.72 % with 99.35 % ethyl ester contents at 4% DES concentration.

Consequence of oxidant concentration on XPS properties of chemically synthesized polythiophene thin films

NASA Astrophysics Data System (ADS)

Kamat, Sandip V.; Chhabra, Jasvinder; Patil, V. S.; Yadav, J. B.; Puri, R. K.; Puri, Vijaya

2018-05-01

The polythiophene thin films were prepared by a wellknown chemical bath deposition technique. The deposited thin films were characterized for structural morphological properties and the adhesion of these thin films were measured by direct pull off (DPO) method, the effect of oxidant concentration on these thin films also studied. The FTIR spectra of chemically deposited polythiophene thin films shows the absorption peak at 836 cm-1 which represents c-s stretching vibrations, shifts to 869 cm-1 as the oxidant concentration increases. The band at 666 cm-1 representing c-s-c ring deformation becomes sharper and appears with a shoulder peak due to increase in oxidant concentration.

Transesterification of diethyl oxalate with phenol over sol-gel MoO(3)/TiO(2) catalysts.

PubMed

Kotbagi, Trupti; Nguyen, Duy Luan; Lancelot, Christine; Lamonier, Carole; Thavornprasert, Kaew-Arpha; Wenli, Zhu; Capron, Mickaël; Jalowiecki-Duhamel, Louise; Umbarkar, Shubhangi; Dongare, Mohan; Dumeignil, Franck

2012-08-01

The transesterification of diethyl oxalate (DEO) with phenol to form diphenyl oxalate (DPO) has been carried out in the liquid phase over very efficient MoO(3)/TiO(2) solid-acid sol-gel catalysts. A selectivity of 100 % with a remarkable maximum yield of 88 % were obtained, which opens the route to downstream phosgene-free processes for the synthesis of polycarbonates. Interpretation of the results of various acidity measurements (NH(3) and pyridine desorption, methanol oxidation as a probe reaction) allowed us to identify the catalytic sites as Lewis acid sites. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Toxicity of bovicin HC5 against mammalian cell lines and the role of cholesterol in bacteriocin activity.

PubMed

Paiva, Aline Dias; de Oliveira, Michelle Dias; de Paula, Sérgio Oliveira; Baracat-Pereira, Maria Cristina; Breukink, Eefjan; Mantovani, Hilário Cuquetto

2012-11-01

Bacteriocins are ribosomally synthesized antimicrobial peptides produced by Bacteria and some Archaea. The assessment of the toxic potential of antimicrobial peptides is important in order to apply these peptides on an industrial scale. The aim of the present study was to investigate the in vitro cytotoxic and haemolytic potential of bovicin HC5, as well as to determine whether cholesterol influences bacteriocin activity on model membranes. Nisin, for which the mechanism of action is well described, was used as a reference peptide in our assays. The viability of three distinct eukaryotic cell lines treated with bovicin HC5 or nisin was analysed by using the MTT assay and cellular morphological changes were determined by light microscopy. The haemolytic potential was evaluated by using the haemoglobin liberation assay and the role of cholesterol on bacteriocin activity was examined by using model membranes composed of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and DPoPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine). The IC(50) of bovicin HC5 and nisin against Vero cells was 65.42 and 13.48 µM, respectively. When the MTT assay was performed with MCF-7 and HepG2 cells, the IC(50) obtained for bovicin HC5 was 279.39 and 289.30 µM, respectively, while for nisin these values were 105.46 and 112.25 µM. The haemolytic activity of bovicin HC5 against eukaryotic cells was always lower than that determined for nisin. The presence of cholesterol did not influence the activity of either bacteriocin on DOPC model membranes, but nisin showed reduced carboxyfluorescein leakage in DPoPC membranes containing cholesterol. In conclusion, bovicin HC5 only exerted cytotoxic effects at concentrations that were greater than the concentration needed for its biological activity, and the presence of cholesterol did not affect its interaction with model membranes.

Pelvic modelling and the comparison between plate position for double pelvic osteotomy using artificial cancellous bone and finite element analysis.

PubMed

McCartney, William; MacDonald, Bryan; Ober, Ciprian Andrei; Lostado-Lorza, Rubén; Gómez, Fátima Somovilla

2018-03-20

Finite element analysis was used to compare fixation methods for double pelvic osteotomy (DPO). Using 3D scanning a stereolithography (stl) image was produced of a canine pelvis and this was subsequently refined in computer aided design (CAD). Using the CAD files, the images were imported in MSC Marc software to produce a working finite element (FE) model with 3 dimensional tetrahedral elements with linear shaped functions. The dimensions of a precontoured pelvic osteotomy plate with eight screws and a twisted seven screw straight plate were used to build the 2 fixations implants for the FE models. An equivalent load of 300 N was applied progressively on all FE models in order to facilitate its convergence. The load was applied in a distributed manner on the femur-hip joint contact area in order to simulate the actual behavior of the joint. The aim of the present study was to analyze the difference in stiffness and behavior under loading between a lateral vs ventral plate fixation, with unlocked screws and different gap scenarios, for stabilization of a pelvic osteotomy using finite element analysis. From both configurations the maximum displacement of the ventral plate with 7 screws without gap had a value of 1.988 mm, while in the DPO plate had a maximum displacement of 2.191 mm. The load applied for each of the different configurations studied when a gap of 1° was considered and also when a condition of no gap was considered. The ventral plate was stiffer than the lateral plate when a gap was not present. When the gap was closed in the ventral plate, the stiffness increased until a point that remained constant. Ventral plate fixation can be as or more stiff as lateral plate fixation and provides flexible fixation. This behavior should reduce screw loosening. Using ventral plate fixation is recommended to reduce screw loosening or failure.

Chemistry and Biology of Aflatoxin-DNA Adducts

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

Stone, Michael P.; Banerjee, Surajit; Brown, Kyle L.

Aspergillus flavus is a fungal contaminant of stored rice, wheat, corn, and other grainstuffs, and peanuts. This is of concern to human health because it produces the mycotoxin aflatoxin B{sub 1} (AFB{sub 1}), which is genotoxic and is implicated in the etiology of liver cancer. AFB{sub 1} is oxidized in vivo by cytochrome P450 to form aflatoxin B{sub 1} epoxide, which forms an N7-dG adduct (AFB{sub 1}-N7-dG) in DNA. The latter rearranges to a formamidopyrimidine (AFB{sub 1}-FAPY) derivative that equilibrates between {alpha} and {beta} anomers of the deoxyribose. In DNA, both the AFB{sub 1}-N7-dG and AFB{sub 1}-{beta}-FAPY adducts intercalate abovemore » the 5'-face of the damaged guanine. Each produces G {yields} T transversions in Escherichia coli, but the AFB{sub 1}-{beta}-FAPY adduct is more mutagenic. The Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) provides a model for understanding error-prone bypass of the AFB{sub 1}-N7-dG and AFB{sub 1}-{beta}-FAPY adducts. It bypasses the AFB{sub 1}-N7-dG adduct, but it conducts error-prone replication past the AFB{sub 1}-FAPY adduct, including mis-insertion of dATP, consistent with the G {yields} T mutations characteristic of AFB{sub 1} mutagenesis in E. coli. Crystallographic analyses of a series of binary and ternary complexes with the Dpo4 polymerase revealed differing orientations of the N7-C8 bond of the AFB{sub 1}-N7-dG adduct as compared to the N{sup 5}-C8 bond in the AFB{sub 1}-{beta}-FAPY adduct, and differential accommodation of the intercalated AFB{sub 1} moieties within the active site. These may modulate AFB{sub 1} lesion bypass by this polymerase.« less

Bypass of Aflatoxin B[subscript 1] Adducts by the Sulfolobus solfataricus DNA Polymerase IV

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

Banerjee, Surajit; Brown, Kyle L.; Egli, Martin

Aflatoxin B{sub 1} (AFB{sub 1}) is oxidized to an epoxide in vivo, which forms an N7-dG DNA adduct (AFB{sub 1}-N7-dG). The AFB{sub 1}-N7-dG can rearrange to a formamidopyrimidine (AFB{sub 1}-FAPY) derivative. Both AFB{sub 1}-N7-dG and the {beta}-anomer of the AFB{sub 1}-FAPY adduct yield G {yields} T transversions in Escherichia coli, but the latter is more mutagenic. We show that the Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) bypasses AFB{sub 1}-N7-dG in an error-free manner but conducts error-prone replication past the AFB{sub 1}-FAPY adduct, including misinsertion of dATP, consistent with the G {yields} T mutations observed in E. coli. Three ternarymore » (Dpo4-DNA-dNTP) structures with AFB{sub 1}-N7-dG adducted template:primers have been solved. These demonstrate insertion of dCTP opposite the AFB{sub 1}-N7-dG adduct, and correct vs incorrect insertion of dATP vs dTTP opposite the 5'-template neighbor dT from a primed AFB{sub 1}-N7-dG:dC pair. The insertion of dTTP reveals hydrogen bonding between the template N3 imino proton and the O{sup 2} oxygen of dTTP, and between the template T O{sup 4} oxygen and the N3 imino proton of dTTP, perhaps explaining why this polymerase does not efficiently catalyze phosphodiester bond formation from this mispair. The AFB{sub 1}-N7-dG maintains the 5'-intercalation of the AFB{sub 1} moiety observed in DNA. The bond between N7-dG and C8 of the AFB{sub 1} moiety remains in plane with the alkylated guanine, creating a 16{sup o} inclination of the AFB{sub 1} moiety with respect to the guanine. A binary (Dpo4-DNA) structure with an AFB{sub 1}-FAPY adducted template:primer also maintains 5'-intercalation of the AFB{sub 1} moiety. The {beta}-deoxyribose anomer is observed. Rotation about the FAPY C5-N{sup 5} bond orients the bond between N{sup 5} and C8 of the AFB{sub 1} moiety out of plane in the 5'-direction, with respect to the FAPY base. The formamide group extends in the 3'-direction. This improves stacking of the AFB{sub 1} moiety above the 5'-face of the FAPY base, as compared to the AFB{sub 1}-N7-dG adduct. Ternary structures with AFB{sub 1}-{beta}-FAPY adducted template:primers show correct vs incorrect insertion of dATP vs dTTP opposite the 5'-template neighbor dT from a primed AFB{sub 1}-{beta}-FAPY:dC pair. For dATP, the oxygen atom of the FAPY formamide group participates in a water-mediated hydrogen bond with Arg332. The insertion of dTTP yields a structure similar to that observed for the AFB{sub 1}-N7-dG adduct. The differential accommodation of these AFB{sub 1} adducts within the active site may, in part, modulate lesion bypass.« less

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

Wang, Yazhen; Musser, Sarah K.; Saleh, Sam

1,N{sup 2}-Propanodeoxyguanosine (PdG) is a stable structural analogue for the 3-(2'-deoxy-{beta}-d-erythro-pentofuranosyl)pyrimido[1,2-?]purin-10(3H)-one (M{sub 1}dG) adduct derived from exposure of DNA to base propenals and to malondialdehyde. The structures of ternary polymerase-DNA-dNTP complexes for three template-primer DNA sequences were determined, with the Y-family Sulfolobus solfataricus DNA polymerase IV (Dpo4), at resolutions between 2.4 and 2.7 {angstrom}. Three template 18-mer-primer 13-mer sequences, 5'-d(TCACXAAATCCTTCCCCC)-3'{center_dot}5'-d(GGGGGAAGGATTT)-3' (template I), 5'-d(TCACXGAATCCTTCCCCC)-3'{center_dot}5'-d(GGGGGAAGGATTC)-3' (template II), and 5'-d(TCATXGAATCCTTCCCCC)-3'{center_dot}5'-d(GGGGGAAGGATTC)-3' (template III), where X is PdG, were analyzed. With templates I and II, diffracting ternary complexes including dGTP were obtained. The dGTP did not pair with PdG, but instead with the 5'-neighboring templatemore » dC, utilizing Watson-Crick geometry. Replication bypass experiments with the template-primer 5?-TCACXAAATCCTTACGAGCATCGCCCCC-3'{center_dot}5'-GGGGGCGATGCTCGTAAGGATTT-3', where X is PdG, which includes PdG in the 5'-CXA-3' template sequence as in template I, showed that the Dpo4 polymerase inserted dGTP and dATP when challenged by the PdG adduct. For template III, in which the template sequence was 5'-TXG-3', a diffracting ternary complex including dATP was obtained. The dATP did not pair with PdG, but instead with the 5'-neighboring T, utilizing Watson-Crick geometry. Thus, all three ternary complexes were of the 'type II' structure described for ternary complexes with native DNA [Ling, H., Boudsocq, F., Woodgate, R., and Yang, W. (2001) Cell 107, 91--102]. The PdG adduct remained in the anti conformation about the glycosyl bond in each of these threee ternary complexes. These results provide insight into how -1 frameshift mutations might be generated for the PdG adduct, a structural model for the exocylic M{sub 1}dG adduct formed by malondialdehyde.« less

Quantitative Analysis of the Mutagenic Potential of 1-Aminopyrene-DNA Adduct Bypass Catalyzed by Y-Family DNA Polymerases

PubMed Central

Sherrer, Shanen M.; Taggart, David J.; Pack, Lindsey R.; Malik, Chanchal K.; Basu, Ashis K.; Suo, Zucai

2012-01-01

N- (deoxyguanosin-8-yl)-1-aminopyrene (dGAP) is the predominant nitro polyaromatic hydrocarbon product generated from the air pollutant 1-nitropyrene reacting with DNA. Previous studies have shown that dGAP induces genetic mutations in bacterial and mammalian cells. One potential source of these mutations is the error-prone bypass of dGAP lesions catalyzed by the low-fidelity Y-family DNA polymerases. To provide a comparative analysis of the mutagenic potential of the translesion DNA synthesis (TLS) of dGAP, we employed short oligonucleotide sequencing assays (SOSAs) with the model Y-family DNA polymerase from Sulfolobus solfataricus, DNA Polymerase IV (Dpo4), and the human Y-family DNA polymerases eta (hPolη), kappa (hPolκ), and iota (hPolι). Relative to undamaged DNA, all four enzymes generated far more mutations (base deletions, insertions, and substitutions) with a DNA template containing a site-specifically placed dGAP. Opposite dGAP and at an immediate downstream template position, the most frequent mutations made by the three human enzymes were base deletions and the most frequent base substitutions were dAs for all enzymes. Based on the SOSA data, Dpo4 was the least error-prone Y-family DNA polymerase among the four enzymes during the TLS of dGAP. Among the three human Y-family enzymes, hPolκ made the fewest mutations at all template positions except opposite the lesion site. hPolκ was significantly less error-prone than hPolι and hPolη during the extension of dGAP bypass products. Interestingly, the most frequent mutations created by hPolι at all template positions were base deletions. Although hRev1, the fourth human Y-family enzyme, could not extend dGAP bypass products in our standing start assays, it preferentially incorporated dCTP opposite the bulky lesion. Collectively, these mutagenic profiles suggest that hPolkk and hRev1 are the most suitable human Y-family DNA polymerases to perform TLS of dGAP in humans. PMID:22917544

Biomedical studies by PIXE

NASA Astrophysics Data System (ADS)

Afarideh, H.; Amirabadi, A.; Hadji-Saeid, S. M.; Mansourian, N.; Kaviani, K.; Zibafar, E.

1996-04-01

In the present biomedical research, PIXE a powerful technique for elemental analysis was employed to illustrate the importance of multi-elemental determination of serum trace elements in two cases of great medical interest. Those are evaluation of the desferroxamine drug (DPO), a widely used therapy for patient with β-thalassemia-Major (β-thal-M), and investigation of elemental variations in blood-serum in hyperbilirubinamia new-borns before and after blood transfusion (BT). The purpose of the work is to demonstrate the various aspects of PIXE analysis by some practical examples as well as to draw some general conclusions regarding the cure of those patients with the above mentioned disorders or diseases. To present in details each case, we divide the paper in two parts: part 1 and part 2 to consider the experimental procedure as well as the results individually.

General misincorporation frequency: Re-evaluation of the fidelity of DNA polymerases.

PubMed

Yang, Jie; Li, Bianbian; Liu, Xiaoying; Tang, Hong; Zhuang, Xiyao; Yang, Mingqi; Xu, Ying; Zhang, Huidong; Yang, Chun

2018-02-19

DNA replication in cells is performed in the presence of four dNTPs and four rNTPs. In this study, we re-evaluated the fidelity of DNA polymerases using the general misincorporation frequency consisting of three incorrect dNTPs and four rNTPs but not using the traditional special misincorporation frequency with only the three incorrect dNTPs. We analyzed both the general and special misincorporation frequencies of nucleotide incorporation opposite dG, rG, or 8-oxoG by Pseudomonas aeruginosa phage 1 (PaP1) DNA polymerase Gp90 or Sulfolobus solfataricus DNA polymerase Dpo4. Both misincorporation frequencies of other DNA polymerases published were also summarized and analyzed. The general misincorporation frequency is obviously higher than the special misincorporation frequency for many DNA polymerases, indicating the real fidelity of a DNA polymerase should be evaluated using the general misincorporation frequency. Copyright © 2018 Elsevier Inc. All rights reserved.

Biochemical investigations into the mutagenic potential of 8-oxo-2'-deoxyguanosine using nucleotide analogues.

PubMed

Hamm, Michelle L; Crowley, Kelly A; Ghio, Michael; Lindell, Maria A M; McFadden, Emily J; Silberg, Jordan S L; Weaver, Amelia M

2012-11-19

8-Oxo-2'-deoxyguanosine (OdG) is an abundant DNA lesion produced during oxidative damage to DNA. It can form relatively stable base pairs with both dC and dA that mimic natural dG:dC and dT:dA base pairs, respectively. Thus, when in the template strand, OdG can direct the insertion of either dCTP or dATP during replication, the latter of which can lead to a dG → T transversion. The potential for OdG to cause mutation is dependent on the preference for dCTP or dATP insertion opposite OdG, as well as the ability to extend past the resulting base pairs. The C2-amine and C8-oxygen could play major roles during these reactions since both would lie outside the Watson-Crick cognate base pairs shape in the major groove when OdG base pairs to dA and dC, respectively, and both have the ability to form strong interactions, like hydrogen bonds. To gain a more generalized understanding of how the C2-amine and C8-oxygen of OdG affect its mutagenic potential, the incorporation opposite and extension past seven analogues of dG/OdG that vary at C2 and/or C8 were characterized for three DNA polymerases, including an exonuclease-deficient version of the replicative polymerase from RB69 (RB69), human polymerase (pol) β, and polymerase IV from Sulfolobus solfataricus P2 (Dpo4). Based on the results from these studies, as well as those from previous studies with RB69, pol β, Dpo4, and two A-family polymerases, the influence of the C2-amine and C8-oxygen during each incorporation and extension reaction with each polymerase is discussed. In general, it appears that when the C2-amine and the C8-oxygen are in the minor groove, they allow OdG to retain interactions that are normally present during insertion and extension. However, when the two groups are in the major groove, they each tend to form novel active site interactions, both stabilizing and destabilizing, that are not present during insertion and extension with natural DNA.

Chloroplast DNA sequence of the green alga Oedogonium cardiacum (Chlorophyceae): Unique genome architecture, derived characters shared with the Chaetophorales and novel genes acquired through horizontal transfer

PubMed Central

Brouard, Jean-Simon; Otis, Christian; Lemieux, Claude; Turmel, Monique

2008-01-01

Background To gain insight into the branching order of the five main lineages currently recognized in the green algal class Chlorophyceae and to expand our understanding of chloroplast genome evolution, we have undertaken the sequencing of chloroplast DNA (cpDNA) from representative taxa. The complete cpDNA sequences previously reported for Chlamydomonas (Chlamydomonadales), Scenedesmus (Sphaeropleales), and Stigeoclonium (Chaetophorales) revealed tremendous variability in their architecture, the retention of only few ancestral gene clusters, and derived clusters shared by Chlamydomonas and Scenedesmus. Unexpectedly, our recent phylogenies inferred from these cpDNAs and the partial sequences of three other chlorophycean cpDNAs disclosed two major clades, one uniting the Chlamydomonadales and Sphaeropleales (CS clade) and the other uniting the Oedogoniales, Chaetophorales and Chaetopeltidales (OCC clade). Although molecular signatures provided strong support for this dichotomy and for the branching of the Oedogoniales as the earliest-diverging lineage of the OCC clade, more data are required to validate these phylogenies. We describe here the complete cpDNA sequence of Oedogonium cardiacum (Oedogoniales). Results Like its three chlorophycean homologues, the 196,547-bp Oedogonium chloroplast genome displays a distinctive architecture. This genome is one of the most compact among photosynthetic chlorophytes. It has an atypical quadripartite structure, is intron-rich (17 group I and 4 group II introns), and displays 99 different conserved genes and four long open reading frames (ORFs), three of which are clustered in the spacious inverted repeat of 35,493 bp. Intriguingly, two of these ORFs (int and dpoB) revealed high similarities to genes not usually found in cpDNA. At the gene content and gene order levels, the Oedogonium genome most closely resembles its Stigeoclonium counterpart. Characters shared by these chlorophyceans but missing in members of the CS clade include the retention of psaM, rpl32 and trnL(caa), the loss of petA, the disruption of three ancestral clusters and the presence of five derived gene clusters. Conclusion The Oedogonium chloroplast genome disclosed additional characters that bolster the evidence for a close alliance between the Oedogoniales and Chaetophorales. Our unprecedented finding of int and dpoB in this cpDNA provides a clear example that novel genes were acquired by the chloroplast genome through horizontal transfers, possibly from a mitochondrial genome donor. PMID:18558012

Economy-wide material input/output and dematerialization analysis of Jilin Province (China).

PubMed

Li, MingSheng; Zhang, HuiMin; Li, Zhi; Tong, LianJun

2010-06-01

In this paper, both direct material input (DMI) and domestic processed output (DPO) of Jilin Province in 1990-2006 were calculated and then based on these two indexes, a dematerialization model was established. The main results are summarized as follows: (1) both direct material input and domestic processed output increase at a steady rate during 1990-2006, with average annual growth rates of 4.19% and 2.77%, respectively. (2) The average contribution rate of material input to economic growth is 44%, indicating that the economic growth is visibly extensive. (3) During the studied period, accumulative quantity of material input dematerialization is 11,543 x 10(4) t and quantity of waste dematerialization is 5,987 x10(4) t. Moreover, dematerialization gaps are positive, suggesting that the potential of dematerialization has been well fulfilled. (4) In most years of the analyzed period, especially 2003-2006, the economic system of Jilin Province represents an unsustainable state. The accelerated economic growth relies mostly on excessive resources consumption after the Revitalization Strategy of Northeast China was launched.

A temperature-tolerant multiplex elements and genes screening system for genetically modified organisms based on dual priming oligonucleotide primers and capillary electrophoresis.

PubMed

Fu, Wei; Wei, Shuang; Wang, Chenguang; Du, Zhixin; Zhu, Pengyu; Wu, Xiyang; Wu, Gang; Zhu, Shuifang

2017-08-15

High throughput screening systems are the preferred solution to meet the urgent requirement of increasing number of genetically modified organisms (GMOs). In this study, we have successfully developed a multiplex GMO element screening system with dual priming oligonucleotide (DPO) primers. This system can detect the cauliflower mosaic virus 35S (CaMV 35S), terminator of nopaline synthase gene (NOS), figwort mosaic virus 35S (FMV 35S) promoter, neomycin phosphotransferaseII (NPTII), Bt Cry 1Ab, phosphinothricin acetyltransferase genes (bar) and Streptomyces viridochromogenes (pat) simultaneously, which covers more than 90% of all authorized GMO species worldwide. This system exhibits a high tolerance to annealing temperatures, high specificity and a limit of detection equal to conventional PCR. A total of 214 samples from markets, national entry-exit agencies, the Institute for Reference Materials and Measurement (IRMM) and the American Oil Chemists' Society (AOCS) were also tested for applicability. This screening system is therefore suitable for GMO screening. Copyright © 2017 Elsevier Ltd. All rights reserved.

Flavonoids as potent allosteric inhibitors of protein tyrosine phosphatase 1B: molecular dynamics simulation and free energy calculation.

PubMed

Zargari, Farshid; Lotfi, Maryam; Shahraki, Omolbanin; Nikfarjam, Zahra; Shahraki, Jafar

2017-12-11

Protein tyrosine phosphatase 1B (PTP1B) is a member of the PTP superfamily which is considered to be a negative regulator of insulin receptor (IR) signaling pathway. PTP1B is a promising drug target for the treatment of type 2 diabetes, obesity, and cancer. The existence of allosteric site in PTP1B has turned the researcher's attention to an alternate strategy for inhibition of this enzyme. Herein, the molecular interactions between the allosteric site of PTP1B with three non-competitive flavonoids, (MOR), (MOK), and (DPO) have been investigated. Three ligands were docked into allosteric site of the enzyme. The resulting protein-ligand complexes were used for molecular dynamics studies. Principal component and free-energy landscape (FEL) as well as cluster analyses were used to investigate the conformational and dynamical properties of the protein and identify representative enzyme substrates bounded to the inhibitors. Per residue energy decomposition analysis attributed dissimilar affinities of three inhibitors to the several hydrogen bonds and non-bonded interactions. In conclusion, our results exhibited an inhibitory pattern of the ligands against PTP1B.

Measurements of Enthalpy of Sublimation of Ne, N2, O2, Ar, CO2, Kr, Xe, and H2O using a Double Paddle Oscillator.

PubMed

Shakeel, Hamza; Wei, Haoyan; Pomeroy, Joshua M

2018-03-01

We report precise experimental values of the enthalpy of sublimation (Δ H s ) of quenched condensed films of neon (Ne), nitrogen (N 2 ), oxygen (O 2 ), argon (Ar), carbon dioxide (CO 2 ), krypton (Kr), xenon (Xe), and water (H 2 O) vapor using a single consistent measurement platform. The experiments are performed well below the triple point temperature of each gas and fall in the temperature range where existing experimental data is very limited. A 6 cm 2 and 400 µm thick double paddle oscillator (DPO) with high quality factor (Q ≈ 4 × 10 5 at 298K) and high frequency stability (33 parts per billion) is utilized for the measurements. The enthalpies of sublimation are derived by measuring the rate of mass loss during temperature programmed desorption. The mass change is detected due to change in the resonance frequency of the self-tracking oscillator. Our measurements typically remain within 10% of the available literature, theory, and National Institute of Standards and Technology (NIST) Web Thermo Tables ( WTT ) values, but are performed using an internally consistent method across different gases.

"Spoligoriftyping," a dual-priming-oligonucleotide-based direct-hybridization assay for tuberculosis control with a multianalyte microbead-based hybridization system.

PubMed

Gomgnimbou, Michel Kiréopori; Abadia, Edgar; Zhang, Jian; Refrégier, Guislaine; Panaiotov, Stefan; Bachiyska, Elizabeta; Sola, Christophe

2012-10-01

We developed "spoligoriftyping," a 53-plex assay based on two preexisting methods, the spoligotyping and "rifoligotyping" assays, by combining them into a single assay. Spoligoriftyping allows simultaneous spoligotyping (i.e., clustered regularly interspaced short palindromic repeat [CRISPR]-based genotyping) and characterization of the main rifampin drug resistance mutations on the rpoB hot spot region in a few hours. This test partly uses the dual-priming-oligonucleotide (DPO) principle, which allows simultaneous efficient amplifications of rpoB and the CRISPR locus in the same sample. We tested this method on a set of 114 previously phenotypically and genotypically characterized multidrug-resistant (MDR) Mycobacterium tuberculosis or drug-susceptible M. tuberculosis DNA extracted from clinical isolates obtained from patients from Bulgaria, Nigeria, and Germany. We showed that our method is 100% concordant with rpoB sequencing results and 99.95% (3,911/3,913 spoligotype data points) correlated with classical spoligotyping results. The sensitivity and specificity of our assay were 99 and 100%, respectively, compared to those of phenotypic drug susceptibility testing. Such assays pave the way to the implementation of locally and specifically adapted methods of performing in a single tube both drug resistance mutation detection and genotyping in a few hours.

Neutron/gamma pulse shape discrimination (PSD) in plastic scintillators with digital PSD electronics

NASA Astrophysics Data System (ADS)

Hutcheson, Anthony L.; Simonson, Duane L.; Christophersen, Marc; Phlips, Bernard F.; Charipar, Nicholas A.; Piqué, Alberto

2013-05-01

Pulse shape discrimination (PSD) is a common method to distinguish between pulses produced by gamma rays and neutrons in scintillator detectors. This technique takes advantage of the property of many scintillators that excitations by recoil protons and electrons produce pulses with different characteristic shapes. Unfortunately, many scintillating materials with good PSD properties have other, undesirable properties such as flammability, toxicity, low availability, high cost, and/or limited size. In contrast, plastic scintillator detectors are relatively low-cost, and easily handled and mass-produced. Recent studies have demonstrated efficient PSD in plastic scintillators using a high concentration of fluorescent dyes. To further investigate the PSD properties of such systems, mixed plastic scintillator samples were produced and tested. The addition of up to 30 wt. % diphenyloxazole (DPO) and other chromophores in polyvinyltoluene (PVT) results in efficient detection with commercial detectors. These plastic scintillators are produced in large diameters up to 4 inches by melt blending directly in a container suitable for in-line detector use. This allows recycling and reuse of materials while varying the compositions. This strategy also avoids additional sample handling and polishing steps required when using removable molds. In this presentation, results will be presented for different mixed-plastic compositions and compared with known scintillating materials

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

Banerjee, Surajit; Christov, Plamen P.; Kozekova, Albena

trans-4-Hydroxynonenal (HNE) is the major peroxidation product of {omega}-6 polyunsaturated fatty acids in vivo. Michael addition of the N{sub 2}-amino group of dGuo to HNE followed by ring closure of N1 onto the aldehyde results in four diastereomeric 1,N{sub 2}-dGuo (1,N{sub 2}-HNE-dGuo) adducts. The (6S,8R,11S)-HNE-1,N{sub 2}-dGuo adduct was incorporated into the 18-mer templates 5'-d(TCATXGAATCCTTCCCCC)-3' and d(TCACXGAATCCTTCCCCC)-3', where X = (6S,8R,11S)-HNE-1,N{sub 2}-dGuo adduct. These differed in the identity of the template 5'-neighbor base, which was either Thy or Cyt, respectively. Each of these templates was annealed with either a 13-mer primer 5'-d(GGGGGAAGGATTC)-3' or a 14-mer primer 5'-d(GGGGGAAGGATTCC)-3'. The addition of dNTPsmore » to the 13-mer primer allowed analysis of dNTP insertion opposite to the (6S,8R,11S)-HNE-1,N{sub 2}-dGuo adduct, whereas the 14-mer primer allowed analysis of dNTP extension past a primed (6S,8R,11S)-HNE-1,N{sub 2}-dGuo:dCyd pair. The Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) belongs to the Y-family of error-prone polymerases. Replication bypass studies in vitro reveal that this polymerase inserted dNTPs opposite the (6S,8R,11S)-HNE-1,N{sub 2}-dGuo adduct in a sequence-specific manner. If the template 5'-neighbor base was dCyt, the polymerase inserted primarily dGTP, whereas if the template 5'-neighbor base was dThy, the polymerase inserted primarily dATP. The latter event would predict low levels of Gua {yields} Thy mutations during replication bypass when the template 5'-neighbor base is dThy. When presented with a primed (6S,8R,11S)-HNE-1,N{sub 2}-dGuo:dCyd pair, the polymerase conducted full-length primer extension. Structures for ternary (Dpo4-DNA-dNTP) complexes with all four template-primers were obtained. For the 18-mer:13-mer template-primers in which the polymerase was confronted with the (6S,8R,11S)-HNE-1,N{sub 2}-dGuo adduct, the (6S,8R,11S)-1,N{sub 2}-dGuo lesion remained in the ring-closed conformation at the active site. The incoming dNTP, either dGTP or dATP, was positioned with Watson-Crick pairing opposite the template 5'-neighbor base, dCyt or dThy, respectively. In contrast, for the 18-mer:14-mer template-primers with a primed (6S,8R,11S)-HNE-1,N{sub 2}-dGuo:dCyd pair, ring opening of the adduct to the corresponding N{sub 2}-dGuo aldehyde species occurred. This allowed Watson-Crick base pairing at the (6S,8R,11S)-HNE-1,N{sub 2}-dGuo:dCyd pair.« less

Structural, optical and morphological studies of Cd2+ doping in CH3NH3PbI3 perovskite semiconductor at Pb2+ site for photovoltaic applications

NASA Astrophysics Data System (ADS)

Parrey, Khursheed; Warish, Mohd.; Devi, Nisha; Niazi, A.; Aziz, A.; Ansari, S. G.

2018-05-01

Doping of semiconductors in a controlled mannner have paramount technological importance as far as the optical and electronic properties of the devices are concerned. Hybrid organic-inorganic perovskites (HOPs) as intrinsic semiconductors have sensational properties required for both the solar photovoltaics and perovskite light emitting diodes. However, undoped and complexity in the dpoing process of HOPs have limited their exploitation in the field of elcronics. In this papper we present the synthesis of HOP semiconductor (CH3NH3PbI3) doped in Pb2+ position by Cd2+. We studied the effect of the incorporation of Cd2+ into the crystalline structure and analysed the changes in the properties like crystal structure, optical absorption and the surface morphology. The structure of HOPs confirmed by X-ray diffraction analysis is tetragonal perovskite type. It can be found that the crystallinity of the samples was enhanced with the doping concentration as the intensity of diffraction peaks were observed to increase with doping. The absorption spectra as obtained from UV-Visible spectrophotometry and Tauc plot analysis indicated that the band gap observed (1.73 eV) is direct type and gets reduced to 1.67 eV with the doping concentration. The red shift may be due to the increase in the size of nanocrystalline material with doping.

Disability inclusion in higher education in Uganda: Status and strategies.

PubMed

Emong, Paul; Eron, Lawrence

2016-01-01

Uganda has embraced inclusive education and evidently committed itself to bringing about disability inclusion at every level of education. Both legal and non-legal frameworks have been adopted and arguably are in line with the intent of the Convention on the Rights of Persons with Disabilities (CRPD) on education. The CRPD, in Article 24, requires states to attain a right to education for persons with disabilities without discrimination and on the basis of equal opportunities at all levels of education. Despite Uganda's robust disability legal and policy framework on education, there is evidence of exclusion and discrimination of students with disabilities in the higher education institutions. The main objective of this article is to explore the status of disability inclusion in higher education and strategies for its realisation, using evidence from Emong's study, workshop proceedings where the authors facilitated and additional individual interviews with four students with disabilities by the authors. The results show that there are discrimination and exclusion tendencies in matters related to admissions, access to lectures, assessment and examinations, access to library services, halls of residence and other disability support services. The article recommends that institutional policies and guidelines on support services for students with disabilities and special needs in higher education be developed, data on students with disabilities collected to help planning, collaboration between Disabled Peoples Organisations (DPO's) strengthened to ensure disability inclusion and the establishment of disability support centres.

Shaker-related voltage-gated K+ channel expression and vasomotor function in human coronary resistance arteries.

PubMed

Nishijima, Yoshinori; Korishettar, Ankush; Chabowski, Dawid S; Cao, Sheng; Zheng, Xiaodong; Gutterman, David D; Zhang, David X

2018-01-01

K V channels are important regulators of vascular tone, but the identity of specific K V channels involved and their regulation in disease remain less well understood. We determined the expression of K V 1 channel subunits and their role in cAMP-mediated dilation in coronary resistance arteries from subjects with and without CAD. HCAs from patients with and without CAD were assessed for mRNA and protein expression of K V 1 channel subunits with molecular techniques and for vasodilator response with isolated arterial myography. Assays of mRNA transcripts, membrane protein expression, and vascular cell-specific localization revealed abundant expression of K V 1.5 in vascular smooth muscle cells of non-CAD HCAs. Isoproterenol and forskolin, two distinct cAMP-mediated vasodilators, induced potent dilation of non-CAD arterioles, which was inhibited by both the general K V blocker 4-AP and the selective K V 1.5 blocker DPO-1. The cAMP-mediated dilation was reduced in CAD and was accompanied by a loss of or reduced contribution of 4-AP-sensitive K V channels. K V 1.5, as a major 4-AP-sensitive K V 1 channel expressed in coronary VSMCs, mediates cAMP-mediated dilation in non-CAD arterioles. The cAMP-mediated dilation is reduced in CAD coronary arterioles, which is associated with impaired 4-AP-sensitive K V channel function. © 2017 John Wiley & Sons Ltd.

The deficit of the isometric tetanic tension redeveloped after a release of frog muscle at a constant velocity

PubMed Central

1979-01-01

Frog sartorius muscles tetanized isometrically were released at a constant velocity from lengths lL to lS (delta l = lL -lS; Ls greater than lO). The tension PS redeveloped after the release was lower than the isometric tension PS at LS, and higher than the isometric tension PL at lL. The tension deficit D is defined as the difference PS-PS. The timing of the release during the tetanus did not influence D. D/PO was proportional to delta l/lO. The proportionality constant k was equal to 1.35 +/- 0.19 (n = 8) when the velocity of release was 2.5 mm/s. When the muscles were released the same delta l, D was found to be an exponential decreasing function of the velocity. The tension deficit was also found in experiments performed in the region lS less than lO. The proportionality constant k was smaller, but the influence of the velocity of the release on D was not modified. When the velocity of the release was changed during the release, D changed accordingly, showing that the effects of delta l and V are multiplicative. These facts suggest a working hypothesis based on the concept that the actin filaments which enter the overlap region during a release are strained by the tetanic stress and therefore unable to make normal cross-bridges. PMID:312915

[Simultaneous detection of respiratory viruses and influenza A virus subtypes using multiplex PCR].

PubMed

Ciçek, Candan; Bayram, Nuri; Anıl, Murat; Gülen, Figen; Pullukçu, Hüsnü; Saz, Eylem Ulaş; Telli, Canan; Cok, Gürsel

2014-10-01

This study was conducted to investigate the respiratory viruses and subtyping of influenza A virus when positive by multiplex PCR in patients with flu-like symptoms, after the pandemic caused by influenza A (H1N1)pdm09. Nasopharyngeal swab samples collected from 700 patients (313 female, 387 male; age range: 24 days-94 yrs, median age: 1 yr) between December 2010 - January 2013 with flu-like symptoms including fever, headache, sore throat, rhinitis, cough, myalgia as defined by the World Health Organization were included in the study. Nucleic acid extractions (Viral DNA/RNA Extraction Kit, iNtRON, South Korea) and cDNA synthesis (RevertAid First Strand cDNA Synthesis Kits, Fermentas, USA) were performed according to the manufacturer's protocol. Multiplex amplification of nucleic acids was performed using DPO (dual priming oligonucleotide) primers and RV5 ACE Screening Kit (Seegene, South Korea) in terms of the presence of influenza A (INF-A) virus, influenza B (INF-B) virus, respiratory syncytial virus (RSV), and the other respiratory viruses. PCR products were detected by automated polyacrylamide gel electrophoresis using Screen Tape multiple detection system. Specimens which were positive for viral nucleic acids have been further studied by using specific DPO primers, FluA ACE Subtyping and RV15 Screening (Seegene, South Korea) kits. Four INF-A virus subtypes [human H1 (hH1), human H3 (hH3), swine H1 (sH1), avian H5 (aH5)] and 11 other respiratory viruses [Adenovirus, parainfluenza virus (PIV) types 1-4, human bocavirus (HBoV), human metapneumovirus (HMPV), rhinovirus types A and B, human coronaviruses (HCoV) OC43, 229E/NL63] were investigated with those tests. In the study, 53.6% (375/700) of the patients were found to be infected with at least one virus and multiple respiratory virus infections were detected in 15.7% (59/375) of the positive cases, which were mostly (49/59, 83%) in pediatric patients. RSV and rhinovirus coinfections were the most prevalent (18/29, 62.7%) dual infections. The distribution of 436 respiratory viruses identified from 375 patients were as follows; 189 (43.3%) RSV, 93 (21.4%) rhinovirus, 86 (19.8%) INF-A, seven (1.6%) INF-B, 22 (5%) PIV types 1-3, 14 (3.2%) HMPV, 11 (2.5%) HCoV, nine (2%) HBoV, and five (1.2%) adenovirus. Fifty-five (64%) out of 86 INF-A viruses were subtyped as hH3, 24 (27.9%) were sH1 and seven (8.1%) were hH1. Avian H5 was not detected in any samples. The overall prevalence rates of INF-A, INF-B, RSV and other respiratory viruses were 12%, 1%, 27%, and 14.6%, respectively. RSV was the most prevalent respiratory agent in pediatric (161/313, 51%) cases, while INF-A virus in adult (24/62, 38.7%) patients. Influenza viruses were detected as responsible pathogens in 13.3% (93/700) of the patients with flu-like symptoms. Among the cases, a 1-month-old baby was infected with three virus strains (INF-A hH1+INF-A sH1+HCoV OC43) and a 82-year-old patient was infected with two INF-A virus subtypes (hH3 + sH1). INF-A viruses were mostly detected (79/86) in winter period, from December to March. INF-A virus sH1, was the most prevalent subtype in flu cases till February 2011 (22/86), after replaced by INF-A virus hH3. Beginning from February 2012, a significant increase observed in the cases infected with INF-A virus subtype hH3 (39/86). In conclusion, the identification and surveillance of influenza virus types and subtypes circulating in populations have importance both for epidemiological data and selection of vaccine strains.

Large scale real-time PCR analysis of mRNA abundance in rainbow trout eggs in relationship with egg quality and post-ovulatory ageing.

PubMed

Aegerter, Sandrine; Jalabert, Bernard; Bobe, Julien

2005-11-01

The mRNA levels of 39 target genes were monitored in unfertilized eggs of 14 rainbow trout sampled the day of ovulation and again 5, 14, and 21 days later. For all 56 collected egg batches, an egg sample was fertilized to estimate egg quality by monitoring embryonic development. Remaining eggs were used for RNA extraction and subsequent real-time PCR analysis. A significant drop of egg quality was observed when eggs were held in the body cavity for 14 or 21 days post-ovulation (dpo). During the same period, eight transcripts (nucleoplasmin or Npm2, ferritin H, tubulin beta, JNK1, cyclin A1, cyclin A2, cathepsin Z, and IGF2) exhibited a differential abundance at one or several collection time(s). Interestingly, we observed higher levels of cyclins A1 and A2 mRNAs in eggs taken 5 days post-ovulation than in eggs taken, from the same females, at the time of ovulation. In addition, seven transcripts exhibited a differential abundance between low quality and high quality eggs. Low quality eggs were characterized by lower levels of Npm2, tubulin beta, and IGF1 transcripts. In contrast, keratins 8 and 18, cathepsin Z, and prostaglandin synthase 2 were more abundant in low quality eggs than in high quality eggs. In this study, we have demonstrated differences in mRNA levels in the rainbow trout egg that are reflective of developmental competence differences induced by post-ovulatory ageing. The putative role of these transcripts in post-ovulatory ageing-induced egg quality defects is discussed with special attention for corresponding cellular functions.

Structural and Kinetic Analysis of Nucleoside Triphosphate Incorporation Opposite an Abasic Site by Human Translesion DNA Polymerase η

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

Patra, Amritaj; Zhang, Qianqian; Lei, Li

2015-02-09

The most prevalent lesion in DNA is an abasic site resulting from glycolytic cleavage of a base. In a number of cellular studies, abasic sites preferentially code for dATP insertion (the “A rule”). In some cases frameshifts are also common. X-ray structures with abasic sites in oligonucleotides have been reported for several microbial and human DNA polymerases (pols), e.g. Dpo4, RB69, KlenTaq, yeast pol ι, human (h) pol ι, and human pol β. We reported previously that hpol η is a major pol involved in abasic site bypass (Choi, J.-Y., Lim, S., Kim, E. J., Jo, A., and Guengerich, F.more » P. (2010 J. Mol. Biol. 404, 34–44). hpol η inserted all four dNTPs in steady-state and pre-steady-state assays, preferentially inserting A and G. In LC-MS analysis of primer-template pairs, A and G were inserted but little C or T was inserted. Frameshifts were observed when an appropriate pyrimidine was positioned 5' to the abasic site in the template. In x-ray structures of hpol η with a non-hydrolyzable analog of dATP or dGTP opposite an abasic site, H-bonding was observed between the phosphate 5' to the abasic site and water H-bonded to N1 and N6 of A and N1 and O6 of G nucleoside triphosphate analogs, offering an explanation for what appears to be a “purine rule.” A structure was also obtained for an A inserted and bonded in the primer opposite the abasic site, but it did not pair with a 5' T in the template. Finally, we conclude that hpol η, a major copying enzyme with abasic sites, follows a purine rule, which can also lead to frameshifts. The phenomenon can be explained with H-bonds.« less

SU-E-J-126: Respiratory Gating Quality Assurance: A Simple Method to Achieve Millisecond Temporal Resolution

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

McCabe, B; Wiersma, R

Purpose: Low temporal latency between a gating on/off signal and a linac beam on/off during respiratory gating is critical for patient safety. Although, a measurement of temporal lag is recommended by AAPM Task Group 142 for commissioning and annual quality assurance, there currently exists no published method. Here we describe a simple, inexpensive, and reliable method to precisely measure gating lag at millisecond resolutions. Methods: A Varian Real-time Position Management™ (RPM) gating simulator with rotating disk was modified with a resistive flex sensor (Spectra Symbol) attached to the gating box platform. A photon diode was placed at machine isocenter. Outputmore » signals of the flex sensor and diode were monitored with a multichannel oscilloscope (Tektronix™ DPO3014). Qualitative inspection of the gating window/beam on synchronicity were made by setting the linac to beam on/off at end-expiration, and the oscilloscope's temporal window to 100 ms to visually examine if the on/off timing was within the recommended 100-ms tolerance. Quantitative measurements were made by saving the signal traces and analyzing in MatLab™. The on and off of the beam signal were located and compared to the expected gating window (e.g. 40% to 60%). Four gating cycles were measured and compared. Results: On a Varian TrueBeam™ STx linac with RPM gating software, the average difference in synchronicity at beam on and off for four cycles was 14 ms (3 to 30 ms) and 11 ms (2 to 32 ms), respectively. For a Varian Clinac™ 21EX the average difference at beam on and off was 127 ms (122 to 133 ms) and 46 ms (42 to 49 ms), respectively. The uncertainty in the synchrony difference was estimated at ±6 ms. Conclusion: This new gating QA method is easy to implement and allows for fast qualitative inspection and quantitative measurements for commissioning and TG-142 annual QA measurements.« less

Structural basis for proficient incorporation of dTTP opposite O6-methylguanine by human DNA polymerase iota.

PubMed

Pence, Matthew G; Choi, Jeong-Yun; Egli, Martin; Guengerich, F Peter

2010-12-24

O(6)-methylguanine (O(6)-methylG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, even physiological ones (e.g. S-adenosylmethionine). The efficiency of a truncated, catalytic DNA polymerase ι core enzyme was determined for nucleoside triphosphate incorporation opposite O(6)-methylG, using steady-state kinetic analyses. The results presented here corroborate previous work from this laboratory using full-length pol ι, which showed that dTTP incorporation occurs with high efficiency opposite O(6)-methylG. Misincorporation of dTTP opposite O(6)-methylG occurred with ∼6-fold higher efficiency than incorporation of dCTP. Crystal structures of the truncated form of pol ι with O(6)-methylG as the template base and incoming dCTP or dTTP were solved and showed that O(6)-methylG is rotated into the syn conformation in the pol ι active site and that dTTP misincorporation by pol ι is the result of Hoogsteen base pairing with the adduct. Both dCTP and dTTP base paired with the Hoogsteen edge of O(6)-methylG. A single, short hydrogen bond formed between the N3 atom of dTTP and the N7 atom of O(6)-methylG. Protonation of the N3 atom of dCTP and bifurcation of the N3 hydrogen between the N7 and O(6) atoms of O(6)-methylG allow base pairing of the lesion with dCTP. We conclude that differences in the Hoogsteen hydrogen bonding between nucleotides is the main factor in the preferential selectivity of dTTP opposite O(6)-methylG by human pol ι, in contrast to the mispairing modes observed previously for O(6)-methylG in the structures of the model DNA polymerases Sulfolobus solfataricus Dpo4 and Bacillus stearothermophilus DNA polymerase I.

miR-1 is increased in pulmonary hypertension and downregulates Kv1.5 channels in rat pulmonary arteries.

PubMed

Mondejar-Parreño, Gema; Callejo, María; Barreira, Bianca; Morales-Cano, Daniel; Esquivel-Ruiz, Sergio; Moreno, Laura; Cogolludo, Angel; Perez-Vizcaino, Francisco

2018-05-02

■The expression of miR-1 is increased in lungs from the Hyp/Su5416 PAH rat model. ■PASMC from this animal model are more depolarised and show decreased expression and activity of Kv1.5. ■miR-1 directly targets Kv1.5 channels, reduces Kv1.5 activity and induces membrane depolarization. ■Antagomir-1 prevents Kv1.5 channel downregulation and the depolarization induced by hypoxia/Su5416 exposition. Impairment of voltage-dependent potassium channel (Kv) plays a central role in the development of cardiovascular diseases, including pulmonary arterial hypertension (PAH). MicroRNAs (miRNAs) are non-coding RNAs that regulate gene expression by binding to the 3'-UTR region of specific mRNAs. The aim of this study was to analyze the effects of miR-1 on Kv channel function in pulmonary arteries (PA). Kv channel activity was studied in PA from healthy animals transfected with miR-1 or scrambled-miR. Kv currents were studied using the whole-cell configuration of patch-clamp technique. The characterization of the Kv1.5 currents was performed with the selective inhibitor DPO-1. miR-1 expression was increased and Kv1.5 channels were decreased in lungs from a rat model of PAH induced by hypoxia and Su5416. miR-1 transfection increased cell capacitance, reduced Kv1.5 currents and induced membrane depolarization in isolated pulmonary artery smooth muscle cells (PASMCs). Luciferase reporter assay indicated that KCNA5, which encodes Kv1.5 channels, is a direct target gene of miR-1. Incubation of PA with Su5416 and hypoxia (3% O 2 ) increased miR-1 and induced a decline in Kv1.5 currents, which was prevented by antagomiR-1. In conclusion, these data indicate that miR-1 induces PASMC hypertrophy and reduces the activity and expression of Kv channels, suggesting a pathophysiological role in PAH. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Vaccination of metastatic melanoma patients with autologous dendritic cell (DC) derived-exosomes: results of thefirst phase I clinical trial

PubMed Central

Escudier, Bernard; Dorval, Thierry; Chaput, Nathalie; André, Fabrice; Caby, Marie-Pierre; Novault, Sophie; Flament, Caroline; Leboulaire, Christophe; Borg, Christophe; Amigorena, Sebastian; Boccaccio, Catherine; Bonnerot, Christian; Dhellin, Olivier; Movassagh, Mojgan; Piperno, Sophie; Robert, Caroline; Serra, Vincent; Valente, Nancy; Le Pecq, Jean-Bernard; Spatz, Alain; Lantz, Olivier; Tursz, Thomas; Angevin, Eric; Zitvogel, Laurence

2005-01-01

Background DC derived-exosomes are nanomeric vesicles harboring functional MHC/peptide complexes capable of promoting T cell immune responses and tumor rejection. Here we report the feasability and safety of the first Phase I clinical trial using autologous exosomes pulsed with MAGE 3 peptides for the immunization of stage III/IV melanoma patients. Secondary endpoints were the monitoring of T cell responses and the clinical outcome. Patients and methods Exosomes were purified from day 7 autologous monocyte derived-DC cultures. Fifteen patients fullfilling the inclusion criteria (stage IIIB and IV, HLA-A1+, or -B35+ and HLA-DPO4+ leukocyte phenotype, tumor expressing MAGE3 antigen) were enrolled from 2000 to 2002 and received four exosome vaccinations. Two dose levels of either MHC class II molecules (0.13 versus 0.40 × 1014 molecules) or peptides (10 versus 100 μg/ml) were tested. Evaluations were performed before and 2 weeks after immunization. A continuation treatment was performed in 4 cases of non progression. Results The GMP process allowed to harvest about 5 × 1014 exosomal MHC class II molecules allowing inclusion of all 15 patients. There was no grade II toxicity and the maximal tolerated dose was not achieved. One patient exhibited a partial response according to the RECIST criteria. This HLA-B35+/A2+ patient vaccinated with A1/B35 defined CTL epitopes developed halo of depigmentation around naevi, a MART1-specific HLA-A2 restricted T cell response in the tumor bed associated with progressive loss of HLA-A2 and HLA-BC molecules on tumor cells during therapy with exosomes. In addition, one minor, two stable and one mixed responses were observed in skin and lymph node sites. MAGE3 specific CD4+ and CD8+ T cell responses could not be detected in peripheral blood. Conclusion The first exosome Phase I trial highlighted the feasibility of large scale exosome production and the safety of exosome administration. PMID:15740633

Mechanisms of Insertion of dCTP and dTTP Opposite the DNA Lesion O6-Methyl-2'-deoxyguanosine by Human DNA Polymerase η.

PubMed

Patra, Amitraj; Zhang, Qianqian; Guengerich, F Peter; Egli, Martin

2016-11-11

O 6 -Methyl-2'-deoxyguanosine (O 6 -MeG) is a ubiquitous DNA lesion, formed not only by xenobiotic carcinogens but also by the endogenous methylating agent S-adenosylmethionine. It can introduce mutations during DNA replication, with different DNA polymerases displaying different ratios of correct or incorrect incorporation opposite this nucleoside. Of the "translesion" Y-family human DNA polymerases (hpols), hpol η is most efficient in incorporating equal numbers of correct and incorrect C and T bases. However, the mechanistic basis for this specific yet indiscriminate activity is not known. To explore this question, we report biochemical and structural analysis of the catalytic core of hpol η. Activity assays showed the truncated form displayed similar misincorporation properties as the full-length enzyme, incorporating C and T equally and extending from both. X-ray crystal structures of both dC and dT paired with O 6 -MeG were solved in both insertion and extension modes. The structures revealed a Watson-Crick-like pairing between O 6 -MeG and 2"-deoxythymidine-5"-[(α, β)-imido]triphosphate (approximating dT) at both the insertion and extension stages with formation of two H-bonds. Conversely, both the structures with O 6 - MeG opposite dCTP and dC display sheared configuration of base pairs but to different degrees, with formation of two bifurcated H-bonds and two single H-bonds in the structures trapped in the insertion and extension states, respectively. The structural data are consistent with the observed tendency of hpol η to insert both dC and dT opposite the O 6 -MeG lesion with similar efficiencies. Comparison of the hpol η active site configurations with either O 6 -MeG:dC or O 6 -MeG:dT bound compared with the corresponding situations in structures of complexes of Sulfolobus solfataricus Dpo4, a bypass pol that favors C relative to T by a factor of ∼4, helps rationalize the more error-prone synthesis opposite the lesion by hpol η. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Mechanisms of Insertion of dCTP and dTTP Opposite the DNA Lesion O6-Methyl-2′-deoxyguanosine by Human DNA Polymerase η*

PubMed Central

Patra, Amitraj; Zhang, Qianqian; Guengerich, F. Peter; Egli, Martin

2016-01-01

O6-Methyl-2′-deoxyguanosine (O6-MeG) is a ubiquitous DNA lesion, formed not only by xenobiotic carcinogens but also by the endogenous methylating agent S-adenosylmethionine. It can introduce mutations during DNA replication, with different DNA polymerases displaying different ratios of correct or incorrect incorporation opposite this nucleoside. Of the “translesion” Y-family human DNA polymerases (hpols), hpol η is most efficient in incorporating equal numbers of correct and incorrect C and T bases. However, the mechanistic basis for this specific yet indiscriminate activity is not known. To explore this question, we report biochemical and structural analysis of the catalytic core of hpol η. Activity assays showed the truncated form displayed similar misincorporation properties as the full-length enzyme, incorporating C and T equally and extending from both. X-ray crystal structures of both dC and dT paired with O6-MeG were solved in both insertion and extension modes. The structures revealed a Watson-Crick-like pairing between O6-MeG and 2"-deoxythymidine-5"-[(α, β)-imido]triphosphate (approximating dT) at both the insertion and extension stages with formation of two H-bonds. Conversely, both the structures with O6- MeG opposite dCTP and dC display sheared configuration of base pairs but to different degrees, with formation of two bifurcated H-bonds and two single H-bonds in the structures trapped in the insertion and extension states, respectively. The structural data are consistent with the observed tendency of hpol η to insert both dC and dT opposite the O6-MeG lesion with similar efficiencies. Comparison of the hpol η active site configurations with either O6-MeG:dC or O6-MeG:dT bound compared with the corresponding situations in structures of complexes of Sulfolobus solfataricus Dpo4, a bypass pol that favors C relative to T by a factor of ∼4, helps rationalize the more error-prone synthesis opposite the lesion by hpol η. PMID:27694439

Structural Basis for Proficient Incorporation of dTTP Opposite O[superscript 6]-Methylguanine by Human DNA Polymerase [iota

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

Pence, Matthew G.; Choi, Jeong-Yun; Egli, Martin

2012-03-15

O{sup 6}-Methylguanine (O{sup 6}-methylG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, even physiological ones (e.g. S-adenosylmethionine). The efficiency of a truncated, catalytic DNA polymerase L core enzyme was determined for nucleoside triphosphate incorporation opposite O{sup 6}-methylG, using steady-state kinetic analyses. The results presented here corroborate previous work from this laboratory using full-length pol L, which showed that dTTP incorporation occurs with high efficiency opposite O{sup 6}-methylG. Misincorporation of dTTP opposite O{sup 6}-methylG occurred with {approx}6-fold higher efficiency than incorporation of dCTP. Crystal structures of the truncated form of pol L with O{sup 6}-methylG asmore » the template base and incoming dCTP or dTTP were solved and showed that O{sup 6}-methylG is rotated into the syn conformation in the pol L active site and that dTTP misincorporation by pol L is the result of Hoogsteen base pairing with the adduct. Both dCTP and dTTP base paired with the Hoogsteen edge of O{sup 6}-methylG. A single, short hydrogen bond formed between the N3 atom of dTTP and the N7 atom of O{sup 6}-methylG. Protonation of the N3 atom of dCTP and bifurcation of the N3 hydrogen between the N7 and O{sup 6} atoms of O{sup 6}-methylG allow base pairing of the lesion with dCTP. We conclude that differences in the Hoogsteen hydrogen bonding between nucleotides is the main factor in the preferential selectivity of dTTP opposite O{sup 6}-methylG by human pol L, in contrast to the mispairing modes observed previously for O{sup 6}-methylG in the structures of the model DNA polymerases Sulfolobus solfataricus Dpo4 and Bacillus stearothermophilus DNA polymerase I.« less

Comparison of broiler performance and carcass yields when fed transgenic maize grain containing event DP-O9814O-6 and processed fractions from transgenic soybeans containing event DP-356O43-5.

PubMed

McNaughton, J; Roberts, M; Rice, D; Smith, B; Hinds, M; Delaney, B; Iiams, C; Sauber, T

2011-08-01

The performance of broilers fed diets containing maize grain from event DP-Ø9814Ø-6 (98140; gat4621 and zm-hra genes) and processed fractions (meal, hulls, and oil) from soybeans containing event DP-356Ø43-5 (356043; gat4601 and gm-hra genes) was evaluated in a 42-d feeding study. Diets were produced with nontransgenic maize grain and soybean fractions from controls with comparable genetic backgrounds to 98140 and 356043 (control), 98140 maize and 356043 soybean (98140 + 356043), or 3 commercially available nontransgenic maize and soybean combinations. Ross 708 broilers (n = 120/group; 50% male, 50% female) were fed diets in 3 phases: starter (d 0 to 21), grower (d 22 to 35), and finisher (d 36 to 42). Starter diets contained (on average) 63% maize and 28% soybean meal, grower diets 66% maize and 26% soybean meal, and finisher diets 72% maize and 21% soybean meal; soybean hulls and oils were held constant at 1.0 and 0.5%, respectively, across all diets in all phases. Weight gain, feed intake, and mortality-adjusted feed efficiency were calculated for d 0 to 42. Standard organ and carcass yield data were collected on d 42. Data were analyzed using a mixed model ANOVA with differences between control and 98140 + 356043 group means considered significant at P < 0.05. Reference group data were used only to estimate experimental variability and to generate tolerance intervals. No significant differences were observed in weight gain, mortality, mortality-adjusted feed efficiency, organ yields, or carcass yields between broilers consuming diets produced with 98140 + 356043 and those consuming diets produced with control maize and soybean fractions. All values of response variables evaluated in the control and 98140 + 356043 groups fell within calculated tolerance intervals. Based on these results, it was concluded that the combination of genetically modified 98140 maize and 356043 soybean fractions was nutritionally equivalent to nontransgenic maize and soybean controls with comparable genetic backgrounds.

Nutritional equivalency evaluation of transgenic maize grain from event DP-O9814O-6 and transgenic soybeans containing event DP-356O43-5: laying hen performance and egg quality measures.

PubMed

McNaughton, J; Roberts, M; Rice, D; Smith, B; Hinds, M; Delaney, B; Iiams, C; Sauber, T

2011-02-01

The objective of this study was to compare the nutritional performance of laying hens fed maize grain from event DP-Ø9814Ø-6 (98140; gat4621 and zm-hra genes) and processed soybean meal from soybeans containing event DP-356Ø43-5 (356043; gat4601 and gm-hra genes), individually or in combination, with the performance of hens fed diets containing nontransgenic maize and soybean meal. Healthy pullets (n = 216) placed in cages (3 hens/cage) were randomly assigned to 9 dietary treatments (8 cages/treatment): nontransgenic controls 1, 2, and 3 (comparable genetic background controls for 98140, 356043, and 98140 + 356043, respectively); reference 1, reference 2, and reference 3 (commercially available nontransgenic maize-soybean meal sources); and 98140 (test 1), 356043 (test 2), and 98140 + 356043 (test 3). The experiment was divided into three 4-wk phases (24 to 28 wk, 28 to 32 wk, and 32 to 36 wk of age), during which time hens were fed mash diets. Performance (BW, feed intake, and egg production) and egg quality data were collected. Data were analyzed using a mixed model ANOVA; differences between the control and respective test group means were considered significant at P < 0.05. Data generated from the reference groups were used only in the estimation of experimental variability and in generating the tolerance interval. Body weight and BW gain, egg production, and production efficiency for hens fed the test diets were similar to the respective values for hens fed the corresponding control diets. Haugh unit measures and egg component weights were similar between the respective test and control groups, and no differences were observed in quality grades or crack measures. All observed values of the control and test groups were within the calculated tolerance intervals. This research indicates that the performance and egg quality of hens fed diets containing 98140 maize grain, 356043 soybean meal, or a combination of the 2 was comparable with that of hens fed diets formulated with nontransgenic maize grain or soybean meal control diets with comparable genetic backgrounds.

A Combined Comparative Transcriptomic, Metabolomic, and Anatomical Analyses of Two Key Domestication Traits: Pod Dehiscence and Seed Dormancy in Pea (Pisum sp.)

PubMed Central

Hradilová, Iveta; Trněný, Oldřich; Válková, Markéta; Cechová, Monika; Janská, Anna; Prokešová, Lenka; Aamir, Khan; Krezdorn, Nicolas; Rotter, Björn; Winter, Peter; Varshney, Rajeev K.; Soukup, Aleš; Bednář, Petr; Hanáček, Pavel; Smýkal, Petr

2017-01-01

The origin of the agriculture was one of the turning points in human history, and a central part of this was the evolution of new plant forms, domesticated crops. Seed dispersal and germination are two key traits which have been selected to facilitate cultivation and harvesting of crops. The objective of this study was to analyze anatomical structure of seed coat and pod, identify metabolic compounds associated with water-impermeable seed coat and differentially expressed genes involved in pea seed dormancy and pod dehiscence. Comparative anatomical, metabolomics, and transcriptomic analyses were carried out on wild dormant, dehiscent Pisum elatius (JI64, VIR320) and cultivated, indehiscent Pisum sativum non-dormant (JI92, Cameor) and recombinant inbred lines (RILs). Considerable differences were found in texture of testa surface, length of macrosclereids, and seed coat thickness. Histochemical and biochemical analyses indicated genotype related variation in composition and heterogeneity of seed coat cell walls within macrosclereids. Liquid chromatography–electrospray ionization/mass spectrometry and Laser desorption/ionization–mass spectrometry of separated seed coats revealed significantly higher contents of proanthocyanidins (dimer and trimer of gallocatechin), quercetin, and myricetin rhamnosides and hydroxylated fatty acids in dormant compared to non-dormant genotypes. Bulk Segregant Analysis coupled to high throughput RNA sequencing resulted in identification of 770 and 148 differentially expressed genes between dormant and non-dormant seeds or dehiscent and indehiscent pods, respectively. The expression of 14 selected dormancy-related genes was studied by qRT-PCR. Of these, expression pattern of four genes: porin (MACE-S082), peroxisomal membrane PEX14-like protein (MACE-S108), 4-coumarate CoA ligase (MACE-S131), and UDP-glucosyl transferase (MACE-S139) was in agreement in all four genotypes with Massive analysis of cDNA Ends (MACE) data. In case of pod dehiscence, the analysis of two candidate genes (SHATTERING and SHATTERPROOF) and three out of 20 MACE identified genes (MACE-P004, MACE-P013, MACE-P015) showed down-expression in dorsal and ventral pod suture of indehiscent genotypes. Moreover, MACE-P015, the homolog of peptidoglycan-binding domain or proline-rich extensin-like protein mapped correctly to predicted Dpo1 locus on PsLGIII. This integrated analysis of the seed coat in wild and cultivated pea provides new insight as well as raises new questions associated with domestication and seed dormancy and pod dehiscence. PMID:28487704

Investigating the possibility of the CONSERT instrument operating as a bi-static RADAR sounder during the seperation, descent and landing phase of the ROSETTA mission

NASA Astrophysics Data System (ADS)

Statz, C.; Hegler, S.; Plettemeier, D.; Berquin, Y. P.; Herique, A.; Kofman, W. W.

2012-12-01

The main scientific objective of the Comet Nucleus Sounding Experiment by Radiowave Transmission (CONSERT) is to determine the dielectric properties of comet 67P/Chuyurmov-Gerasimenko's nucleus. This will be achieved by performing a sounding of the comet's core between the lander "Philae" launched on the comet's surface and the orbiter "Rosetta". For the sounding the lander will receive, process and retransmit the radio signal emitted by the CONSERT instrument aboard the orbiter. With data measured during the first science phase, a three-dimensional model of the material distribution with regard to the complex dielectric permittivity of the comet's nucleus is to be reconstructed. In order to increase the scientific outcome of the experiment and to collect data beneficial for the main scientific objective, it may be considered to operate the CONSERT instrument as a bi-static RADAR sounder during the non mission-critical parts of the separation, descent and landing (SDL) phase, i.e. when the lander is launched onto the comet's surface, of the ROSETTA mission. The data measured during this phase will be mainly echoes from the comet's surface and first meters of subsurface. Based on this data, we intent to create an initial dielectric permittivity mapping of the comet's surface at and around the landing site In order to estimate the performance of the instrument in this special operational mode, simulations of a sounding in SDL configuration were performed. The simulations are based on a hybrid method-of-moments physical-optics (EFIE-DPO) approach for large dielectric bodies with consideration of the behavior of the instrument's antennas and coupling with the spacecraft as well as polarization effects. The simulated results are furthermore processed in a system-level-instrument-simulator to include effects such as a realistic sounding signal, pulse-compression and analog digital conversion in the estimation of the sounding capabilities. The main objective of the simulations was to determine the influence of the orientation and position of lander and orbiter with respect to the comet on the received signal as well as the influence of the surfaces dielectric permittivity on the backscattered signal. Further investigations were carried out to determine the effects of different scales of surface roughness. First simulations validate the possibility of a CONSERT operation during the SDL phase. The results indicate the feasibility of a surface permittivity estimation of the landing site from the SDL data as well as the mapping of the surface permittivity and roughness around the landing site. Furthermore, the lander attitude and the deployment state of the lander's legs may also be reconstructed from the SDL measurements. The surface roughness and permittivity estimation and mapping, as well as the determination of the lander state will be subject of further investigations in this context.

Hypogonadism, diabetes mellitus, hypothyroidism, hypoparathyroidism: incidence and prevalence related to iron overload and chelation therapy in patients with thalassaemia major followed from 1980 to 2007 in the Ferrara Centre.

PubMed

Gamberini, Maria Rita; De Sanctis, Vincenzo; Gilli, Giuseppe

2008-10-01

273 patients with thalassaemia major followed from diagnosis in the Ferrara Centre were divided into 3 cohorts (C) according to the year of birth (C1=1954-1964, 85 patients; C2=1965-1974, 129 patients; C3=1975-2001, 59 patients) in order to study the trends of endocrine complications. Menarche occurred in 52 out of 112 patients (46%), without significant differences among the 3 groups, at the mean age of 13.9+/-1.4 years. Sixty-five percent of these patients had secondary amenorrhoea at the mean age of 18.8+/-3.7 years. In males complete pubertal development occurred in 48% of patients (C1:31%, C2: 44%, C3: 63%, p<0.05) followed by secondary hypogonadism in 24% of patients above 21 years of age. Primary (80%) and central 20%) hypothyroidism were diagnosed in 31% of patients (C1: 55%, C2: 31.5%, C3: 13.4%, p<0.05), diabetes mellitus (DM) in 17% of patients (C1: 28.6%, C2: 17.2%, C3: 3.4%, p<0.05), and hypoparathyroidism in 10.6% of cases (C1: 18.7%, C2: 10.1%, C3: 3.4%, p<0.05). No difference was found in patient mean age of diagnosis of hypothyroidism, DM or hypoparathyroidism (20.4+/-8.2 years, 19+/-5 yrs and 18.5+/-5.8 yrs respectively) but in all three groups age at diagnosis significantly increased over time (hypothyroidism and DM: p<0.001; hypoparathyroidism: p<0.01). Over time the prevalence of hypothyroidism, diabetes mellitus and hypoparathyroidism increased to 24.4%, 14.7%, and 6.7%, respectively, at the time of the study. Incidences peaked in the early 1980's, and declined in the following years (primary hypothyroidism from 6.5% in 1981 to 0.9% in 2007, p<0.01; DM from 3.9% in 1986 to 0.8% in 2007, p<0.05; hypoparathyroidism 2.4% in 1984 to 0% in 2007, p<0.01) and correlated with the decrease in annual mean serum ferritin levels in all patients (p<0.001). The main risk factors associated with endocrine complications were high serum ferritin levels, poor compliance with desferioxamine (DFO) therapy, early onset of transfusion therapy (only for hypogonadism) and splenectomy (only for hypothyroidism). Serum ferritin levels of approximately 2000 ng/ml were found to correlate with hypogonadism, and 3000 ng/ml for hypothyroidism, hypoparathyroidism and DM. The incidences of hypothyroidism, DM and hypoparathyroidism were not significantly different in 18 patients on long term treatment with deferiprone (DPO) compared with 64 patients continuously treated with DFO, from 1995 to 2007. In conclusion, our longitudinal study shows that in the last 30 years in the Ferrara Centre the incidences of hypothyroidism, diabetes mellitus, and hypoparathyroidism declined, and pubertal development in males with thalassemia major improved in patients, on DFO treatment, born after 1976. The efficacy of alternative chelation regimes with deferiprone or deferasirox to monotherapy with desferioxamine remains to be established.