Softening of the stiffness of bottlebrush polymers by mutual interaction

We study bottlebrush macromolecules in a good solvent by small-angle neutron scattering (SANS), static light scattering (SLS), and dynamic light scattering (DLS). These polymers consist of a linear backbone to which long side chains are chemically grafted. The backbone contains about 1600 monomer units (weight average) and every second monomer unit carries side-chains with ca. 60 monomer units. The SLS- and SANS data extrapolated to infinite dilution lead to the form factor of the polymer that can be described in terms of a worm-like chain with a contour length of 380 nm and a persistence length of 17.5 nm. An analysis of the DLS data confirm these model parameters. The scattering intensities taken at finite concentration can be modeled using the polymer reference interaction site model. It reveals a softening of the bottlebrush polymers caused by their mutual interaction. We demonstrate that the persistence decreases from 17.5 nm down to 5 nm upon increasing the concentration from dilute solution to the highest concentration 40.59 g/l under consideration. The observed softening of the chains is comparable to the theoretically predicted decrease of the electrostatic persistence length of linear polyelectrolyte chains at finite concentrations.Comment: 4 pages, 4 figure

Biological Surface Coating and Molting Inhibition as Mechanisms of TiO2 Nanoparticle Toxicity in Daphnia magna

The production and use of nanoparticles (NP) has steadily increased within the last decade; however, knowledge about risks of NP to human health and ecosystems is still scarce. Common knowledge concerning NP effects on freshwater organisms is largely limited to standard short-term (≤48 h) toxicity tests, which lack both NP fate characterization and an understanding of the mechanisms underlying toxicity. Employing slightly longer exposure times (72 to 96 h), we found that suspensions of nanosized (∼100 nm initial mean diameter) titanium dioxide (nTiO2) led to toxicity in Daphnia magna at nominal concentrations of 3.8 (72-h EC50) and 0.73 mg/L (96-h EC50). However, nTiO2 disappeared quickly from the ISO-medium water phase, resulting in toxicity levels as low as 0.24 mg/L (96-h EC50) based on measured concentrations. Moreover, we showed that nTiO2 (∼100 nm) is significantly more toxic than non-nanosized TiO2 (∼200 nm) prepared from the same stock suspension. Most importantly, we hypothesized a mechanistic chain of events for nTiO2 toxicity in D. magna that involves the coating of the organism surface with nTiO2 combined with a molting disruption. Neonate D. magna (≤6 h) exposed to 2 mg/L nTiO2 exhibited a “biological surface coating” that disappeared within 36 h, during which the first molting was successfully managed by 100% of the exposed organisms. Continued exposure up to 96 h led to a renewed formation of the surface coating and significantly reduced the molting rate to 10%, resulting in 90% mortality. Because coating of aquatic organisms by manmade NP might be ubiquitous in nature, this form of physical NP toxicity might result in widespread negative impacts on environmental health

Fate and effects of silver nanoparticles at the aquatic-terrestrial interface: A floodplain mesocosm experiment

The production volume of engineered inorganic nanoparticles (EINP) successively increased over the last years. Once released into the natural environment, these particles may change their size and surface properties in interaction with other substances. This is expected to control their mobility and their impact on biochemical processes. However, the underlying processes are not fully understood yet. Transformation processes and long-term fate of citrate-coated silver nanoparticles (Ag NP) were investigated in an innovative floodplain mesocosm, which was run with river Rhine water and natural soil from an adjacent floodplain for 33 weeks. Flooding events were simulated every three weeks. The Ag NP with a concentration of 5 mg L-1 were continuously introduced into the water for three weeks followed by a three-week period without spiking. Every third week the ecotoxicological impact of Ag NP was determined by means of Gammarus mortality and feeding assays. At the end of the experiment, the total Ag concentrations were measured in profiles of the floodplain soil and the sediment as well as in algae that developed in the mesocosm. The total Ag concentration in the aquatic phase in the main zone as well as in the floodplain fluctuated according to the periodic Ag NP pulse. Further, significant amounts of Ag accumulated in algae (up to 4.7 mg g-1) and exposed leaves (up to 170 μg g-1). However, for the applied experimental conditions we did neither observed mortality nor sublethal effects on Gammarus feeding activity. More than 40 % of the Ag remained in the sediment of the main zone and 7 % were transported during flooding into the floodplain soil. Furthermore, 0.5 % of the Ag was still in the water phase. Most of the particles were immobilized in the top layer of the sediments and soil. Only very little transport in deeper soil layers was observed in the soil columns and sediment. Accumulation in algae, sediment, and soil is alarming for long-term environmental impact assessments and the long lifetime in the aqueous phase suggests long-range transport of Ag NP in rivers

Interaction of cylindrical polymer brushes in dilute and semi-dilute solution

We present a systematic study of flexible cylindrical brush-shaped macromolecules in a good solvent by small-angle neutron scattering (SANS), static light scattering (SLS), and by dynamic light scattering (DLS) in dilute and semi-dilute solution. The SLS and SANS data extrapolated to infinite dilution lead to the shape of the polymer that can be modeled in terms of a worm-like chain with a contour length of 380 nm and a persistence length of 17.5 nm. SANS data taken at higher polymer concentration were evaluated by using the polymer reference interaction site model (PRISM). We find that the persistence length reduce from 17.5 nm at infinite dilution to 5.3 nm at the highest concentration (volume fraction 0.038). This is comparable with the decrease of the persistence length in semi-dilute concentration predicted theoretically for polyelectrolytes. This finding reveals a softening of stiffness of the polymer brushes caused by their mutual interaction

Targeting quiescent leukemic stem cells using second generation autophagy inhibitors

In chronic myeloid leukemia (CML), tyrosine kinase inhibitor (TKI) treatment induces autophagy that promotes survival and TKI-resistance in leukemic stem cells (LSCs). In clinical studies hydroxychloroquine (HCQ), the only clinically approved autophagy inhibitor, does not consistently inhibit autophagy in cancer patients, so more potent autophagy inhibitors are needed. We generated a murine model of CML in which autophagic flux can be measured in bone marrow-located LSCs. In parallel, we use cell division tracing, phenotyping of primary CML cells, and a robust xenotransplantation model of human CML, to investigate the effect of Lys05, a highly potent lysosomotropic agent, and PIK-III, a selective inhibitor of VPS34, on the survival and function of LSCs. We demonstrate that long-term haematopoietic stem cells (LT-HSCs: Lin−Sca-1+c-kit+CD48−CD150+) isolated from leukemic mice have higher basal autophagy levels compared with non-leukemic LT-HSCs and more mature leukemic cells. Additionally, we present that while HCQ is ineffective, Lys05-mediated autophagy inhibition reduces LSCs quiescence and drives myeloid cell expansion. Furthermore, Lys05 and PIK-III reduced the number of primary CML LSCs and target xenografted LSCs when used in combination with TKI treatment, providing a strong rationale for clinical use of second generation autophagy inhibitors as a novel treatment for CML patients with LSC persistence

DARPins detect the formation of hetero-tetramers of p63 and p73 in epithelial tissues and in squamous cell carcinoma

The two p53 homologues p63 and p73 regulate transcriptional programs in epithelial tissues and several cell types in these tissues express both proteins. All members of the p53 family form tetramers in their active state through a dedicated oligomerization domain that structurally assembles as a dimer of dimers. The oligomerization domain of p63 and p73 share a high sequence identity, but the p53 oligomerization domain is more divergent and it lacks a functionally important C-terminal helix present in the other two family members. Based on these structural differences, p53 does not hetero-oligomerize with p63 or p73. In contrast, p63 and p73 form hetero-oligomers of all possible stoichiometries, with the hetero-tetramer built from a p63 dimer and a p73 dimer being thermodynamically more stable than the two homo-tetramers. This predicts that in cells expressing both proteins a p63$_{2}$/p73$_{2}$ hetero-tetramer is formed. So far, the tools to investigate the biological function of this hetero-tetramer have been missing. Here we report the generation and characterization of Designed Ankyrin Repeat Proteins (DARPins) that bind with high affinity and selectivity to the p63$_{2}$/p73$_{2}$ hetero-tetramer. Using these DARPins we were able to confirm experimentally the existence of this hetero-tetramer in epithelial mouse and human tissues and show that its level increases in squamous cell carcinoma

Influence of operating parameters on the biodegradation of steroid estrogens and nonylphenolic compounds during biological wastewater treatment processes

This document is the unedited author's version of a Submitted Work that was subsequently accepted for publication in Environmental Science & Technology, copyright © American Chemical Society after peer review. To access the final edited and published work see http://pubs.acs.org/doi/abs/10.1021/es901612v.This study investigated operational factors influencing the removal of steroid estrogens and nonylphenolic compounds in two sewage treatment works, one a nitrifying/denitrifying activated sludge plant and the other a nitrifying/denitrifying activated sludge plant with phosphorus removal. Removal efficiencies of >90% for steroid estrogens and for longer chain nonylphenol ethoxylates (NP4−12EO) were observed at both works, which had equal sludge ages of 13 days. However, the biological activity in terms of milligrams of estrogen removed per day per tonne of biomass was found to be 50−60% more efficient in the nitrifying/denitrifying activated sludge works compared to the works which additionally incorporated phosphorus removal. A temperature reduction of 6 °C had no impact on the removal of free estrogens, but removal of the conjugated estrone-3-sulfate was reduced by 20%. The apparent biomass sorption (LogKp) values were greater in the nitrifying/denitrifying works than those in the nitrifying/denitrifying works with phosphorus removal for both steroid estrogens and nonylphenolic compounds possibly indicating a different cell surface structure and therefore microbial population. The difference in biological activity (mg tonne−1 d−1) identified in this study, of up to seven times, suggests that there is the potential for enhancing the removal of estrogens and nonylphenols if more detailed knowledge of the factors responsible for these differences can be identified and maximized, thus potentially improving the quality of receiving waters.Public Utilities Board (Singapore), Anglian Water Ltd, Severn Trent Water Ltd, Thames Water Utilities Ltd, United Utilities 393 Plc and Yorkshire Water Services

Multiple Scale Reorganization of Electrostatic Complexes of PolyStyrene Sulfonate and Lysozyme

We report on a SANS investigation into the potential for these structural reorganization of complexes composed of lysozyme and small PSS chains of opposite charge if the physicochemical conditions of the solutions are changed after their formation. Mixtures of solutions of lysozyme and PSS with high matter content and with an introduced charge ratio [-]/[+]intro close to the electrostatic stoichiometry, lead to suspensions that are macroscopically stable. They are composed at local scale of dense globular primary complexes of radius ~ 100 {\AA}; at a higher scale they are organized fractally with a dimension 2.1. We first show that the dilution of the solution of complexes, all other physicochemical parameters remaining constant, induces a macroscopic destabilization of the solutions but does not modify the structure of the complexes at submicronic scales. This suggests that the colloidal stability of the complexes can be explained by the interlocking of the fractal aggregates in a network at high concentration: dilution does not break the local aggregate structure but it does destroy the network. We show, secondly, that the addition of salt does not change the almost frozen inner structure of the cores of the primary complexes, although it does encourage growth of the complexes; these coalesce into larger complexes as salt has partially screened the electrostatic repulsions between two primary complexes. These larger primary complexes remain aggregated with a fractal dimension of 2.1. Thirdly, we show that the addition of PSS chains up to [-]/[+]intro ~ 20, after the formation of the primary complex with a [-]/[+]intro close to 1, only slightly changes the inner structure of the primary complexes. Moreover, in contrast to the synthesis achieved in the one-step mixing procedure where the proteins are unfolded for a range of [-]/[+]intro, the native conformation of the proteins is preserved inside the frozen core

Combined inhibition of Aurora-A and ATR kinase results in regression of MYCN-amplified neuroblastoma

Amplification of MYCN is the driving oncogene in a subset of high-risk neuroblastoma. The MYCN protein and the Aurora-A kinase form a complex during S phase that stabilizes MYCN. Here we show that MYCN activates Aurora-A on chromatin, which phosphorylates histone H3 at serine 10 in S phase, promotes the deposition of histone H3.3 and suppresses R-loop formation. Inhibition of Aurora-A induces transcription-replication conflicts and activates the Ataxia telangiectasia and Rad3 related (ATR) kinase, which limits double-strand break accumulation upon Aurora-A inhibition. Combined inhibition of Aurora-A and ATR induces rampant tumor-specific apoptosis and tumor regression in mouse models of neuroblastoma, leading to permanent eradication in a subset of mice. The therapeutic efficacy is due to both tumor cell-intrinsic and immune cell-mediated mechanisms. We propose that targeting the ability of Aurora-A to resolve transcription-replication conflicts is an effective therapy for MYCN-driven neuroblastoma (141 words)