Investigating Uranium Isotopic Distributions in Environmental Samples Using AMS and MC-ICPMS

Major, minor, and trace uranium isotopes were measured at Lawrence Livermore National Laboratory in environmentally acquired samples using different instruments to span large variations in concentrations. Multi-collector inductively-coupled plasma mass spectrometry (MC-ICPMS) can be used to measure major and minor isotopes: {sup 238}U, {sup 235}U, {sup 234}U and {sup 236}U. Accelerator mass spectrometry (AMS) can be used to measure minor and trace isotopes: {sup 234}U, {sup 236}U, and {sup 233}U. The main limit of quantification for minor or trace uranium isotopes is the abundance sensitivity of the measurement technique; i.e., the ability to measure a minor or trace isotope of mass M in the presence of a major isotope at M{+-}1 mass units. The abundance sensitivity for {sup 236}U/{sup 235}U isotope ratio measurements using MC-ICPMS is around {approx}2x10{sup -6}. This compares with a {sup 236}U/{sup 235}U abundance sensitivity of {approx}1x10{sup -7} for the current AMS system, with the expectation of 2-3 orders of magnitude improvement in sensitivity with the addition of another high energy filter. Comparing {sup 236}U/{sup 234}U from MC-ICPMS and AMS produced agreement within {approx}10% for samples at {sup 236}U levels high enough to be measurable by both techniques

Functional regulation of FEN1 nuclease and its link to cancer

Flap endonuclease-1 (FEN1) is a member of the Rad2 structure-specific nuclease family. FEN1 possesses FEN, 5′-exonuclease and gap-endonuclease activities. The multiple nuclease activities of FEN1 allow it to participate in numerous DNA metabolic pathways, including Okazaki fragment maturation, stalled replication fork rescue, telomere maintenance, long-patch base excision repair and apoptotic DNA fragmentation. Here, we summarize the distinct roles of the different nuclease activities of FEN1 in these pathways. Recent biochemical and genetic studies indicate that FEN1 interacts with more than 30 proteins and undergoes post-translational modifications. We discuss how FEN1 is regulated via these mechanisms. Moreover, FEN1 interacts with five distinct groups of DNA metabolic proteins, allowing the nuclease to be recruited to a specific DNA metabolic complex, such as the DNA replication machinery for RNA primer removal or the DNA degradosome for apoptotic DNA fragmentation. Some FEN1 interaction partners also stimulate FEN1 nuclease activities to further ensure efficient action in processing of different DNA structures. Post-translational modifications, on the other hand, may be critical to regulate protein–protein interactions and cellular localizations of FEN1. Lastly, we also review the biological significance of FEN1 as a tumor suppressor, with an emphasis on studies of human mutations and mouse models

The wonders of flap endonucleases: structure, function, mechanism and regulation.

Processing of Okazaki fragments to complete lagging strand DNA synthesis requires coordination among several proteins. RNA primers and DNA synthesised by DNA polymerase α are displaced by DNA polymerase δ to create bifurcated nucleic acid structures known as 5'-flaps. These 5'-flaps are removed by Flap Endonuclease 1 (FEN), a structure-specific nuclease whose divalent metal ion-dependent phosphodiesterase activity cleaves 5'-flaps with exquisite specificity. FENs are paradigms for the 5' nuclease superfamily, whose members perform a wide variety of roles in nucleic acid metabolism using a similar nuclease core domain that displays common biochemical properties and structural features. A detailed review of FEN structure is undertaken to show how DNA substrate recognition occurs and how FEN achieves cleavage at a single phosphate diester. A proposed double nucleotide unpairing trap (DoNUT) is discussed with regards to FEN and has relevance to the wider 5' nuclease superfamily. The homotrimeric proliferating cell nuclear antigen protein (PCNA) coordinates the actions of DNA polymerase, FEN and DNA ligase by facilitating the hand-off intermediates between each protein during Okazaki fragment maturation to maximise through-put and minimise consequences of intermediates being released into the wider cellular environment. FEN has numerous partner proteins that modulate and control its action during DNA replication and is also controlled by several post-translational modification events, all acting in concert to maintain precise and appropriate cleavage of Okazaki fragment intermediates during DNA replication

Cellular Active N-Hydroxyurea FEN1 Inhibitors Block Substrate Entry to the Active Site

The structure-specific nuclease human flap endonuclease-1 (hFEN1) plays a key role in DNA replication and repair and may be of interest as an oncology target. We present the first crystal structure of inhibitor-bound hFEN1 and show a cyclic N-hydroxyurea bound in the active site coordinated to two magnesium ions. Three such compounds had similar IC50 values but differed subtly in mode of action. One had comparable affinity for protein and protein– substrate complex and prevented reaction by binding to active site catalytic metal ions, blocking the unpairing of substrate DNA necessary for reaction. Other compounds were more competitive with substrate. Cellular thermal shift data showed engagement of both inhibitor types with hFEN1 in cells with activation of the DNA damage response evident upon treatment. However, cellular EC50s were significantly higher than in vitro inhibition constants and the implications of this for exploitation of hFEN1 as a drug target are discussed

Carbon nanotubes allow capture of krypton, barium and lead for multichannel biological X-ray fluorescence imaging

The desire to study biology in situ has been aided by many imaging techniques. Among these, X-ray fluorescence (XRF) mapping permits observation of elemental distributions in a multichannel manner. However, XRF imaging is underused, in part, because of the difficulty in interpreting maps without an underlying cellular ‘blueprint’; this could be supplied using contrast agents. Carbon nanotubes (CNTs) can be filled with a wide range of inorganic materials, and thus can be used as ‘contrast agents’ if biologically absent elements are encapsulated. Here we show that sealed single-walled CNTs filled with lead, barium and even krypton can be produced, and externally decorated with peptides to provide affinity for sub-cellular targets. The agents are able to highlight specific organelles in multiplexed XRF mapping, and are, in principle, a general and versatile tool for this, and other modes of biological imaging

Site-selective protein-modification chemistry for basic biology and drug development.

Nature has produced intricate machinery to covalently diversify the structure of proteins after their synthesis in the ribosome. In an attempt to mimic nature, chemists have developed a large set of reactions that enable post-expression modification of proteins at pre-determined sites. These reactions are now used to selectively install particular modifications on proteins for many biological and therapeutic applications. For example, they provide an opportunity to install post-translational modifications on proteins to determine their exact biological roles. Labelling of proteins in live cells with fluorescent dyes allows protein uptake and intracellular trafficking to be tracked and also enables physiological parameters to be measured optically. Through the conjugation of potent cytotoxicants to antibodies, novel anti-cancer drugs with improved efficacy and reduced side effects may be obtained. In this Perspective, we highlight the most exciting current and future applications of chemical site-selective protein modification and consider which hurdles still need to be overcome for more widespread use.We thank FCT Portugal (FCT Investigator to G.J.L.B.), the EU (Marie-Curie CIG to G.J.L.B. and Marie-Curie IEF to O.B.) and the EPSRC for funding. G.J.L.B. is a Royal Society University Research Fellow.This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/nchem.239

Radiocarbon dating of "old" charcoal using a wet oxidation, stepped-combustion procedure

We present results that validate a new wet oxidation, stepped-combustion procedure for dating "old" charcoal samples. An acid-base-wet oxidation (ABOX) pretreatment procedure has been developed that is used in place of the conventional acid-base-acid (ABA) pretreatment. Combustions and graphitizations are performed in a vacuum line that is insulated from the atmosphere by a second backing vacuum to eliminate the risk of atmospheric leakage into the line at any stage of the procedure. Combustions are performed at 3 temperatures (330 degrees C, 630 degrees C and 850 degrees C) with a graphite target produced from the CO2 evolved during each combustion step. In this way, the removal of any contamination can be monitored, and a high degree of confidence can be placed on the final age. The pretreatment, combustion, graphitization, and measurement blank for the procedure, based on the analysis of a "radiocarbon-dead" graphite, is 0.5 +/0.5 micrograms C (1sigma, n = 14), equivalent to 0.04 +/0.02 pMC or an "age" of approximately 60 ka for a 1 mg graphite target. Analyses of a "radiocarbon-dead" natural charcoal after ABOX pretreatment and stepped combustion suggest that the total blank (including contamination not removed by pretreatment) may be higher than for graphite, ranging up to 0.10 +/0.02 pMC. Additional experiments confirm good agreement with accepted values for the international low14C "New Kauri" standard (0.16-0.25 pMC). They also confirm excellent reproducibility, with 3 separate dates on different aliquots of a charcoal sample from Ngarrabullgan Cave (Queensland, Australia) ranging from 35.2 to 35.5 ka 14C BP. It is also demonstrated that the ABOX pretreatment, in conjunction with the new vacuum line described here, is able to remove contamination not removed by the conventional ABA pretreatment, suggesting that the technique can be used to produce reliable 14C dates on charcoal up to at least 50 ka.This material was digitized as part of a cooperative project between Radiocarbon and the University of Arizona Libraries.The Radiocarbon archives are made available by Radiocarbon and the University of Arizona Libraries. Contact [email protected] for further information.Migrated from OJS platform February 202