Measurement of 8-hydroxy-2′-deoxyguanosine in DNA by high-performance liquid chromatography-mass spectrometry: comparison with measurement by gas chromatography-mass spectrometry

Measurement of 8-hydroxy-2′-deoxyguanosine (8-OH-dGuo) in DNA by high-performance liquid chromatography/mass spectrometry (LC/MS) was studied. A methodology was developed for separation by LC of 8-OH-dGuo from intact and modified nucleosides in DNA hydrolyzed by a combination of four enzymes: DNase I, phosphodiesterases I and II and alkaline phosphatase. The atmospheric pressure ionization-electrospray process was used for mass spectral measurements. A stable isotope-labeled analog of 8-OH-dGuo was used as an internal standard for quantification by isotope-dilution MS (IDMS). Results showed that LC/IDMS with selected ion-monitoring (SIM) is well suited for identification and quantification of 8-OH-dGuo in DNA at background levels and in damaged DNA. The sensitivity level of LC/IDMS-SIM was found to be comparable to that reported previously using LC-tandem MS (LC/MS/MS). It was found that approximately five lesions per 106 DNA bases can be detected using amounts of DNA as low as 2 µg. The results also suggest that this lesion may be quantified in DNA at levels of one lesion per 106 DNA bases, or even lower, when more DNA is used. Up to 50 µg of DNA per injection were used without adversely affecting the measurements. Gas chromatography/isotope-dilution MS with selected-ion monitoring (GC/IDMS-SIM) was also used to measure this compound in DNA following its removal from DNA by acidic hydrolysis or by hydrolysis with Escherichia coli Fpg protein. The background levels obtained by LC/IDMS-SIM and GC/IDMS-SIM were almost identical. Calf thymus DNA and DNA isolated from cultured HeLa cells were used for this purpose. This indicates that these two techniques can provide similar results in terms of the measurement of 8-OH-dGuo in DNA. In addition, DNA in buffered aqueous solution was damaged by ionizing radiation at different radiation doses and analyzed by LC/IDMS-SIM and GC/IDMS-SIM. Again, similar results were obtained by the two techniques. The sensitivity of GC/MS-SIM for 7,8-dihydro-8-oxoguanine was also examined and found to be much greater than that of LC/MS-SIM and the reported sensitivity of LC/MS/MS for 8-OH-dGuo. Taken together, the results unequivocally show that LC/IDMS-SIM is well suited for sensitive and accurate measurement of 8-OH-dGuo in DNA and that both LC/IDMS-SIM and GC/IDMS-SIM can provide similar results.

Fluorescence correlation spectroscopy: Diagnostics for sparse molecules

The robust glow of molecular fluorescence renders even sparse molecules detectable and susceptible to analysis for concentration, mobility, chemistry, and photophysics. Correlation spectroscopy, a statistical-physics-based tool, gleans quantitative information from the spontaneously fluctuating fluorescence signals obtained from small molecular ensembles. This analytical power is available for studying molecules present at minuscule concentrations in liquid solutions (less than one nanomolar), or even on the surfaces of living cells at less than one macromolecule per square micrometer. Indeed, routines are becoming common to detect, locate, and examine individual molecules under favorable conditions.

Stat3-mediated Myc expression is required for Src transformation and PDGF-induced mitogenesis

Signal transducer and activator of transcription (STAT) proteins perform key roles in mediating signaling by cytokines and growth factors, including platelet-derived growth factor (PDGF). In addition, Src family kinases activate STAT signaling and are required for PDGF-induced mitogenesis in normal cells. One STAT family member, Stat3, has been shown to have an essential role in cell transformation by the Src oncoprotein. However, the mechanisms by which STAT-signaling pathways contribute to mitogenesis and transformation are not fully defined. We show here that disruption of Stat3 signaling by using dominant-negative Stat3β protein in NIH 3T3 fibroblasts suppresses c-Myc expression concomitant with inhibition of v-Src-induced transformation. Ectopic expression of c-Myc is able to partially reverse this inhibition, suggesting that c-Myc is a downstream effector of Stat3 signaling in v-Src transformation. Furthermore, c-myc gene knockout fibroblasts are refractory to transformation by v-Src, consistent with a requirement for c-Myc protein in v-Src transformation. In normal NIH 3T3 cells, disruption of Stat3 signaling with dominant-negative Stat3β protein inhibits PDGF-induced mitogenesis in a manner that is reversed by ectopic c-Myc expression. Moreover, inhibition of Src family kinases with the pharmacologic agent, SU6656, blocks Stat3 activation by PDGF. These findings, combined together, delineate the signaling pathway, PDGF → Src → Stat3 → Myc, that is important in normal PDGF-induced mitogenesis and subverted in Src transformation.

Characterization of repetitive DNA in the Mycoplasma genitalium genome: possible role in the generation of antigenic variation.

We have characterized a family of repetitive DNA elements with homology to the MgPa cellular adhesion operon of Mycoplasma genitalium, a bacterium that has the smallest known genome of any free-living organism. One element, 2272 bp in length and flanked by DNA with no homology to MgPa, was completely sequenced. At least four others were partially sequenced. The complete element is a composite of six regions. Five of these regions show sequence similarity with nonadjacent segments of genes of the MgPa operon. The sixth region, located near the center of the element, is an A+T-rich sequence that has only been found in this repeat family. Open reading frames are present within the five individual regions showing sequence homology to MgPa and the adjacent open reading frame 3 (ORF3) gene. However, termination codons are found between adjacent regions of homology to the MgPa operon and in the A+T-rich sequence. Thus, these repetitive elements do not appear to be directly expressible protein coding sequences. The sequence of one region from five different repetitive elements was compared with the homologous region of the MgPa gene from the type strain G37 and four newly isolated M. genitalium strains. Recombination between repetitive elements of strain G37 and the MgPa operon can explain the majority of polymorphisms within our partial sequences of the MgPa genes of the new isolates. Therefore, we propose that the repetitive elements of M. genitalium provide a reservoir of sequence that contributes to antigenic variation in proteins of the MgPa cellular adhesion operon.

Intersubunit contacts made by tryptophan 120 with biotin are essential for both strong biotin binding and biotin-induced tighter subunit association of streptavidin.

In natural streptavidin, tryptophan 120 of each subunit makes contacts with the biotin bound by an adjacent subunit through the dimer-dimer interface. To understand quantitatively the role of tryptophan 120 and its intersubunit communication in the properties of streptavidin, a streptavidin mutant in which tryptophan 120 is converted to phenylalanine was produced and characterized. The streptavidin mutant forms a tetrameric molecule and binds one biotin per subunit, as does natural streptavidin, indicating that the mutation of tryptophan 120 to phenylalanine has no significant effect on the basic properties of streptavidin. However, its biotin-binding affinity was reduced substantially, to approximately 10(8) M-1, indicating that the contact made by tryptophan 120 to biotin has a considerable contribution to the extremely tight biotin binding by streptavidin. The mutant retained bound biotin over a wide pH range or with the addition of urea up to 6 M at neutral pH. However, bound biotin was efficiently released by the addition of excess free biotin due, presumably, to exchange reactions. Electrophoretic analysis revealed that the intersubunit contact made by tryptophan 120 to biotin through the dimer-dimer interface is the major interaction responsible for the biotin-induced, tighter subunit association of streptavidin. In addition, the mutant has weaker subunit association than natural streptavidin even in the absence of biotin, indicating that tryptophan 120 also contributes to the subunit association of tetramers in the absence of biotin.

Migrantes centroamericanos en tránsito por México: trayectorias, redes y riesgos

The main interest of this research project focuses on transit migration, one element of the more general international migration process, where undocumented migrants find themselves in a condition of irregularity and have to face a migratory route under conditions of severe vulnerability. The main question guiding this research is: How to study and define the Transit Migration phenomenon from its specific context, considering its conditions and consequences? This implies also to respond three more questions: How do migrants perceive their irregular transit through Mexico? What are the networks and social links that articulate the trajectories of irregular Transit Migration through Mexico? and what is the meaning of these networks and links from the perspective of those individuals undergoing irregular Transit Migration processes?...

3XMM J185246.6+003317: another low magnetic field magnetar

We study the outburst of the newly discovered X-ray transient 3XMMJ185246.6+003317, re-analyzing all available XMM-Newton observations of the source to perform a phase-coherent timing analysis, and derive updated values of the period and period derivative. We find the source rotating at P = 11.55871346(6) s (90% confidence level; at epoch MJD 54728.7) but no evidence for a period derivative in the seven months of outburst decay spanned by the observations. This translates to a 3σ upper limit for the period derivative of ˙ P <1.4×10−13 s s−1, which, assuming the classical magneto-dipolar braking model, gives a limit on the dipolar magnetic field of Bdip < 4.1×1013 G. The X-ray outburst and spectral characteristics of 3XMM J185246.6+003317 confirm its identification as a magnetar, but the magnetic field upper limit we derive defines it as the third “low-B” magnetar discovered in the past 3 yr, after SGR 0418+5729 and Swift J1822.3−1606. We have also obtained an upper limit to the quiescent luminosity (<4×1033 erg s−1), in line with the expectations for an old magnetar. The discovery of this new low field magnetar reaffirms the prediction of about one outburst per year from the hidden population of aged magnetars.

Orbscan Topography in Primary Open-Angle Glaucoma

Purpose: To compare anterior and posterior corneal curvatures between eyes with primary open-angle glaucoma (POAG) and healthy eyes. Methods: This is a prospective, cross-sectional, observer-masked study. A total of 138 white subjects (one eye per patient) were consecutively recruited; 69 eyes had POAG (study group), and the other 69 comprised a group of healthy control eyes matched for age and central corneal pachymetry with the study ones. Exclusion criteria included any corneal or ocular inflammatory disease, previous ocular surgery, or treatment with carbonic anhydrase inhibitors. The same masked observer performed Goldmann applanation tonometry, ultrasound pachymetry, and Orbscan II topography in all cases. Central corneal thickness, intraocular pressure, and anterior and posterior topographic elevation maps were analyzed and compared between both groups. Results: Patients with POAG had greater forward shifting of the posterior corneal surface than that in healthy control eyes (p < 0.01). Significant differences in anterior corneal elevation between controls and POAG eyes were also found (p < 0.01). Conclusions: Primary open-angle glaucoma eyes have a higher elevation of the posterior corneal surface than that in central corneal thickness–matched nonglaucomatous eyes.

O Serviço Europeu de Acção Externa

Com esta dissertação pretendemos analisar o Serviço Europeu de Ação Externa como uma das principais inovações do Tratado de Lisboa. O Serviço Europeu de Ação Externa é o corpo diplomático da União Europeia que se define pela coesão, cooperação, e divulgação da Politica Externa da União Europeia. No entanto, os seus primeiros anos não foram fáceis, iniciou a sua vigência em plena crise económica e suscitou várias dúvidas aos Estados-Membros, no que diz respeito à soberania. Contudo, defendemos que o SEAE mostra a verdadeira força da União, pois permite a coesão dos Estados-Membros que falam a uma só voz perante outros Estados e organizações. Para além disso, o papel do SEAE afigura-se relevante pelo número de cidadãos que residem em Estados terceiros, cabendo assim à União Europeia através do SEAE a proteção destes cidadãos europeus.

Russia's interest in the Mistral: the political and military aspects. OSW Commentary No. 41, 2010-10-11

The negotiations over Russia’s purchase of French Mistral-class multipurpose assault ships, which were intensified in 2010, have gained a significant political dimension. The prospects of such a spectacular acquisition of large and expensive assault ships from one of NATO’s member states are being used by Russia to demonstrate that it has opened a new stage of relations with Western Europe. Paris has welcomed Russia’s desire to embark on military cooperation; for France, the Mistral deal has become a convenient tool to prove that relations with Russia are becoming increasingly normal, and that Russia poses no threat to the European security.

Citizenship Deprivation: A Normative Analysis. Liberty and Security in Europe No. 82, 19 March 2015

Most critical analyses assess citizenship-deprivation policies against international human rights and domestic rule of law standards, such as prevention of statelessness, non-arbitrariness with regard to justifications and judicial remedies, or non-discrimination between different categories of citizens. This report considers instead from a political theory perspective how deprivation policies reflect specific conceptions of political community. We distinguish four normative conceptions of the grounds of membership in a political community that apply to decisions on acquisition and loss of citizenship status: i) a ‘State discretion’ view, according to which governments should be as free as possible in pursuing State interests when determining citizenship status; ii) an ‘individual choice’ view, according to which individuals should be as free as possible in choosing their citizenship status; iii) an ‘ascriptive community’ view, according to which both State and individual choices should be minimised through automatic determination of membership based on objective criteria such as the circumstances of birth; and iv) a ‘genuine link’ view, according to which the ties of individuals to particular States determine their claims to inclusion and against deprivation while providing at the same time objections against including individuals without genuine links. We argue that most citizenship laws combine these four normative views in different ways, but that from a democratic perspective the ‘genuine link’ view is normatively preferable to the others. The report subsequently examines five general grounds for citizenship withdrawal – threats to public security, non-compliance with citizenship duties, flawed acquisition, derivative loss and loss of genuine links – and considers how the four normative views apply to withdrawal provision motivated by these concerns. The final section of the report examines whether EU citizenship provides additional reasons for protection against Member States’ powers of citizenship deprivation. We suggest that, in addition to fundamental rights protection through EU law and protection of free movement rights, three further arguments could be invoked: toleration of dual citizenship in a political union, prevention of unequal conditions for loss among EU citizens, and the salience of genuine links to the EU itself rather than merely to one of its Member States.

Sediment color variation in laminated Holocene sediments of ODP Site 178-1098

Continuous sediment color records with a resolution of one measurement per millimeter were generated for Site 1098 (Palmer Deep, Antarctic Peninsula) from digital images of the core surfaces to test if the laminated intervals at this site will allow for analysis of high-frequency climate variability in the Circum-Antarctic. Long-term variation in color values correlates with gamma-ray attenuation bulk density. Darker colors are found in laminated intervals with lower bulk density, high biogenic silica, and high total organic carbon content. Darker color values result from the addition of dark laminae to background sediments that show little variation in color. The thicknesses of dark and light laminae were measured in the top 25 meters composite depth to determine the temporal resolution of the laminae. The alternation between dark, biogenic-rich laminae and background sediment essentially represents an annual cycle, but the sediment is not consistently varved. The modal thickness of light laminae is close to the long-term average annual accumulation rate, and results indicate that approximately half of the dark/light couplets in distinctly laminated intervals represent a single year. Missing biogenic laminae are interpreted to represent reduced primary productivity during cold years with delayed breakup of the sea-ice cover.

Paleomagnetic age assigments of ODP Site 113-697 (Table 1)

At Site 697 a 320 m thick Pleistocene and Pliocene section was recovered, consisting of hemipelagic terrigenous mud with varying amounts of diatoms, thin altered ash layers, and ice-rafted debris (IRD). Sedimentation rates range from 41 m/m.y. (upper Pleistocene) to 150 m/m.y. (lower Pliocene). Diatom percentage and sediment grain-size have been measured for the whole section with approximately one sample per 5,000 yr. IRD is most abundant in the lower Pliocene (sediments older than 4.5 Ma) following the first major West Antarctic glaciation. A decrease in IRD to near-zero above 3.2 Ma may record a transition from valley glaciers to a grounded ice-sheet on West Antarctica. Bottom current flow, recorded in sediments as the proportion of silt, was at a maximum around 3.0-3.3 Ma then gradually decreased until 0.5 Ma. In the upper Pleistocene, maxima in diatom percentage are assumed to occur during interglacials, implying reduced sea-ice cover; maxima in silt percentage correspond to diatom maxima, implying stronger bottom water flow during interglacials.

Monthly catalog of United States Government publications /

One issue per year, 1977 (no. 988)-1984 (no. 1074), 2001 (no. 1294)- has title: Serials supplement; one issue per year, 1985-1991, has title: Periodicals supplement; one issue each year, 1992-2000, has title: Periodicals supplement for ... (varies slightly)

Transactions of the American Institute of Mining and Metallurgical Engineers.

Consists of transactions of various Branches and Divisions of the Institute.