Focusing Capillary Optics for Use in Solution Small-Angle X-Ray Scattering

Measurements of the global conformation of macromolecules can be carried out using small-angle X-ray scattering (SAXS). Glass focusing capillaries, manufactured at the Cornell High Energy Synchrotron Source (CHESS), have been successfully employed for SAXS measurements on the heme protein cytochrome c. These capillaries provide high X-ray flux into a spot size of tens of micrometres, permitting short exposures of small-volume samples. Such a capability is ideal for use in conjunction with microfluidic mixers, where time resolution may be determined by beam size and sample volumes are kept small to facilitate mixing and conserve material

Spatial Distribution of Competing Ions around DNA in Solution

The competition of monovalent and divalent cations for proximity to negatively charged DNA is of biological importance and can provide strong constraints for theoretical treatments of polyelectrolytes. Resonant x-ray scattering experiments have allowed us to monitor the number and distribution of each cation in a mixed ion cloud around DNA. These measurements provide experimental evidence to support a general theoretical prediction: the normalized distribution of each ion around polyelectrolytes remains constant when ions are mixed at different ratios. In addition, the amplitudes of the scattering signals throughout the competition provide a measurement of the surface concentration parameter that predicts the competition behavior of these cations. The data suggest that ion size needs to be taken into account in applying Poisson-Boltzmann treatments to polyelectrolytes such as DNA

Positional effects of click cyclization on β-hairpin structure, stability, and function

The use of the copper (I)-assisted azide-alkyne cycloaddition (CuAAC, or “click” reaction) as a method of β-hairpin stabilization was investigated at several different positions to determine the impact on hairpin structure and function, including hydrogen bonded sites, non-hydrogen bonded sites, and at the peptide termini. The role of the turn sequence in the peptide and the chain length of the azied were also investigated. It was determined that the CuAAC reaction was a suitable method for locking in β-hairpin structure in peptides possessing either the type I’ turn, VNGO and the type II’ turn, VpGO. Moreover, all cyclic variants exhibited improved thermal stability and resistance to proteolysis as compared to the non-cyclic peptides, regardless of the position in the strand. Additionally, the function of the CuAAC cyclized peptides was not altered as exhibited by similar binding affinities for ATP as the WKWK peptide. These studies provided a comprehensive method for CuAAC cyclization of β-hairpin peptides, which could further be utilized in the inhibition of protein-protein and protein-nucleic acid interactions

Platinum-group element geochemistry of the Forest Reef Volcanics, southeastern Australia: Implications for porphyry Au-Cu mineralisation

Platinum-group element concentrations in felsic to intermediate rocks from the Forest Reef Volcanics, Cadia-Neville region, southeastern Australia have been analysed by the Ni-S fire assay-isotope dilution method. The Forest Reef Volcanics are shoshonitic to calc-alkaline in composition and fractionated to produce a wide range of compositions, with MgO varying between 9.7 and 1.8 wt.%. The interest in this suite is that it is coeval with Au-Cu porphyry-style mineralisation in the Cadia mineral district. This study uses PGE geochemistry to determine the timing of sulfide saturation, relative to volatile (ore-fluid) saturation, in the magma that gave rise to the Forest Reef Volcanics and, in turn, to assess how this timing affected the mineralisation potential of the evolving magmatic system. The Forest Reef Volcanics can be subdivided, on the basis of their contrasting PGE geochemistry, into high-Mg (>6.8 wt.% MgO) and low-Mg suites (≤6.8 wt.% MgO). Platinum, Pd and Re concentrations increase in the high-Mg samples, whereas Ir and Ru decrease and Rh concentrations remain steady, with decreasing MgO. The coupled Ir, Ru and Rh depletion is attributed to the partitioning of these elements into magnetite. The rate of Pt and Pd enrichment is not possible by closed-system fractional crystallisation alone, which suggests that the parent magma was replenished by a Pt-Pd-rich melt. In contrast, the PGE concentrations in the low-Mg samples decrease with decreasing MgO indicating the onset of sulfide saturation at 6.8 wt.% MgO, which is confirmed by the presence of spheroidal sulfide inclusions in liquidus crystals (i.e. clinopyroxene, plagioclase, magnetite). The rate of Pd depletion is appreciably less than for any other sulfide saturated felsic system for which data are available. This requires either that the amount of sulfide melt to have precipitated was unusually low, or that the rate of Pd depletion was limited by the mass of silicate melt the sulfide melt reached equilibrium with, or both. In any event, the fraction of sulfide melt that precipitated was too small to have had a significant effect on the Cu and Au content of the magma so that both Cu and Au were available to enter the ore-forming fluid when the magma became volatile saturated at, or shortly after, it reached ca. 2.9 wt.% MgO.This research was funded by a Newcrest Mining LTD Grant to Ian Campbell

Redesign of a WW Domain Peptide for Selective Recognition of Single-Stranded DNA

A β-sheet mini-protein based on the FBP11 WW1 domain sequence has been redesigned for the molecular recognition of ssDNA. A previous report showed that a β-hairpin peptide dimer, (WKWK)2, binds ssDNA with low micromolar affinity but with little selectivity over duplex DNA. This report extends those studies to a three-stranded β-sheet mini-protein designed to mimic the OB-fold. The new peptide binds ssDNA with low micromolar affinity and shows about 10-fold selectivity for ssDNA over duplex DNA. The redesigned peptide no longer binds its native ligand, the polyproline helix, confirming that the peptide has been redesigned for the function of binding ssDNA. Structural studies provide evidence that this peptide consists of a well structured β-hairpin made of Strands 2&3 with a less structured first strand that provides affinity for ssDNA but does not improve the stability of the full peptide. These studies provide insight into protein-DNA interactions as well as a novel example of protein-redesign

Adeno-Associated Virus-Mediated Gene Transfer to Renal Tubule Cells via a Retrograde Ureteral Approach

www.karger.com/nne This is an Open Access article licensed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License (www.karger.com/OA-license), applicable to the online version of the article only. Distribution for non-commercial purposes only

Ethanol and Reactive Species Increase Basal Sequence Heterogeneity of Hepatitis C Virus and Produce Variants with Reduced Susceptibility to Antivirals

Hepatitis C virus (HCV) exhibits a high level of genetic variability, and variants with reduced susceptibility to antivirals can occur even before treatment begins. In addition, alcohol decreases efficacy of antiviral therapy and increases sequence heterogeneity of HCV RNA but how ethanol affects HCV sequence is unknown. Ethanol metabolism and HCV infection increase the level of reactive species that can alter cell metabolism, modify signaling, and potentially act as mutagen to the viral RNA. Therefore, we investigated whether ethanol and reactive species affected the basal sequence variability of HCV RNA in hepatocytes. Human hepatoma cells supporting a continuous replication of genotype 1b HCV RNA (Con1, AJ242652) were exposed to ethanol, acetaldehyde, hydrogen peroxide, or L-buthionine-S,R-sulfoximine (BSO) that decreases intracellular glutathione as seen in patients. Then, NS5A region was sequenced and compared with genotype 1b HCV sequences in the database. Ethanol and BSO elevated nucleotide and amino acid substitution rates of HCV RNA by 4–18 folds within 48 hrs which were accompanied by oxidative RNA damage. Iron chelator and glutathione ester decreased both RNA damage and mutation rates. Furthermore, infectious HCV and HCV core gene were sufficient to induce oxidative RNA damage even in the absence of ethanol or BSO. Interestingly, the dn/ds ratio and percentage of sites undergoing positive selection increased with ethanol and BSO, resulting in an increased detection of NS5A variants with reduced susceptibility to interferon alpha, cyclosporine, and ribavirin and others implicated in immune tolerance and modulation of viral replication. Therefore, alcohol is likely to synergize with virus-induced oxidative/nitrosative stress to modulate the basal mutation rate of HCV. Positive selection induced by alcohol and reactive species may contribute to antiviral resistance

BCL6 enables Ph+ acute lymphoblastic leukaemia cells to survive BCR-ABL1 kinase inhibition.

Tyrosine kinase inhibitors (TKIs) are widely used to treat patients with leukaemia driven by BCR-ABL1 (ref. 1) and other oncogenic tyrosine kinases. Recent efforts have focused on developing more potent TKIs that also inhibit mutant tyrosine kinases. However, even effective TKIs typically fail to eradicate leukaemia-initiating cells (LICs), which often cause recurrence of leukaemia after initially successful treatment. Here we report the discovery of a novel mechanism of drug resistance, which is based on protective feedback signalling of leukaemia cells in response to treatment with TKI. We identify BCL6 as a central component of this drug-resistance pathway and demonstrate that targeted inhibition of BCL6 leads to eradication of drug-resistant and leukaemia-initiating subclones

Measuring Activity in the Ubiquitin–Proteasome System: From Large Scale Discoveries to Single Cells Analysis

The ubiquitin proteasome system (UPS) is the primary pathway responsible for the recognition and degradation of misfolded, damaged, or tightly regulated proteins in addition to performing essential roles in DNA repair, cell cycle regulation, cell migration, and the immune response. While traditional biochemical techniques have proven useful in the identification of key proteins involved in this pathway, the implementation of novel reporters responsible for measuring enzymatic activity of the UPS have provided valuable insight into the effectiveness of therapeutics and role of the UPS in various human diseases such as multiple myeloma and Huntington’s disease. These reporters, usually consisting of a recognition sequences fused to an analytical handle, are designed to specifically evaluate enzymatic activity of certain members of the UPS including the proteasome, E3 ubiquitin ligases, and deubiquitinating enzymes (DUBs). This review highlights the more commonly used reporters employed in a variety of scenarios ranging from high-throughput screening of novel inhibitors to single cell microscopy techniques measuring E3 ligase or proteasome activity. Finally, recent work is presented highlighting the development of novel degron-based substrate designed to overcome the limitations of current reporting techniques in measuring E3 ligase and proteasome activity in patient samples

The BAH domain of Rsc2 is a histone H3 binding domain

Bromo-adjacent homology (BAH) domains are commonly found in chromatin-associated proteins and fall into two classes; Remodels the Structure of Chromatin (RSC)-like or Sir3-like. Although Sir3-like BAH domains bind nucleosomes, the binding partners of RSC-like BAH domains are currently unknown. The Rsc2 subunit of the RSC chromatin remodeling complex contains an RSC-like BAH domain and, like the Sir3-like BAH domains, we find Rsc2 BAH also interacts with nucleosomes. However, unlike Sir3-like BAH domains, we find that Rsc2 BAH can bind to recombinant purified H3 in vitro, suggesting that the mechanism of nucleosome binding is not conserved. To gain insight into the Rsc2 BAH domain, we determined its crystal structure at 2.4 Å resolution. We find that it differs substantially from Sir3-like BAH domains and lacks the motifs in these domains known to be critical for making contacts with histones. We then go on to identify a novel motif in Rsc2 BAH that is critical for efficient H3 binding in vitro and show that mutation of this motif results in defective Rsc2 function in vivo. Moreover, we find this interaction is conserved across Rsc2-related proteins. These data uncover a binding target of the Rsc2 family of BAH domains and identify a novel motif that mediates this interaction