Single-Step Purification of Monomeric l-Selectin via Aptamer Affinity Chromatography

l-selectin is a transmembrane receptor expressed on the surface of white blood cells and responsible for the tethering of leukocytes to vascular endothelial cells. This initial intercellular contact is the first step of the complex leukocyte adhesion cascade that ultimately permits extravasation of leukocytes into the surrounding tissue in case of inflammation. Here we show the binding of a soluble histidine tagged l-selectin to a recently described shortened variant of an l-selectin specific DNA aptamer with surface plasmon resonance. The high specificity of this aptamer in combination with its high binding affinity of ~12 nM, allows for a single-step protein purification from cell culture supernatants. In comparison to the well-established Ni-NTA based technology, aptamer affinity chromatography (AAC) was easier to establish, resulted in a 3.6-fold higher protein yield, and increased protein purity. Moreover, due to target specificity, the DNA aptamer facilitated binding studies directly from cell culture supernatant, a helpful characteristic to quickly monitor successful expression of biological active l-selectin. View Full-Tex

Polymeric near-infrared absorbing dendritic nanogels for efficient in vivo photothermal cancer therapy

In recent years, several near-infrared light absorbing inorganic nanomaterials have been developed for photothermal therapy. However, their biological fate after injection limits their clinical utilization. In this work, we developed a novel polymeric near-infrared light absorbing material based on a biocompatible thermoresponsive nanogel that is semi-interpenetrated with polyaniline, a conjugated polymer with strong near-infrared absorbance. This polymeric nanocomposite generates heat after being irradiated by NIR light, thereby inducing a local hyperthermia that is used for photothermal cancer therapy in vitro and in vivo

Matrix Metalloproteinase-sensitive Multistage Nanogels Promote Drug Transport in 3D Tumor Model

Physiological barriers inside of tumor tissue often result in poor interstitial penetration and heterogeneous intratumoral distribution of nanoparticle-based drug delivery systems (DDS). Novel, matrix metalloproteinase (MMP)-sensitive peptide-crosslinked nanogels (pNGs) as multistage DDS are reported with a beneficial size reduction property to promote the process of deep tissue penetration. Methods: The presented pNGs are based on a dendritic polyglycerol (dPG) scaffold crosslinked by a modified MMP-sensitive fluorogenic peptide. The crosslinker integrates degradability in response to proteases present in the tumor microenvironment. Surfactant-free, inverse nanoprecipitation is employed to prepare the nanogels using strain-promoted click chemistry. The size and crosslinking density of the pNGs are controlled by the functionalization degree of dPG with cyclooctyne groups and by the peptide crosslinker fraction. The intrinsic reporter moiety of the crosslinker was used to study the influence of pNG compositions on the degradation profile. The therapeutic drug Doxorubicin was conjugated through a pH-sensitive linkage to dPG to form a multistage DDS. The penetration behavior of the pNGs was studied using agarose matrix and multicellular tumor spheroids (MCTS). Results: Nanogel sizes were controlled in the range of 150-650 nm with narrow size distributions and varying degrees of crosslinking. The pNGs showed stability in PBS and cell media but were readily degraded in the presence of MMP-7. The crosslinking density influenced the degradation kinetic mediated by MMP-7 or cells. Stable conjugation of DOX at physiological pH and controlled drug release at acidic pH were observed. The digestions of nanogels lead to a size reduction to polymer-drug fragments which efficiently penetrated into agarose gels. Moreover, the degradable multistage pNGs demonstrated deeper penetration into MCTS as compared to their non-degradable counterparts. Thus, degradable pNGs were able to deliver their cargo and efficiently reduce the cell viability in MCTS. Conclusion: The triggered size reduction of the pNGs by enzymatic degradation can facilitate the infiltration of the nanocarrier into dense tissue, and thereby promote the delivery of its cargo

Dendritic polymer imaging systems for the evaluation of conjugate uptake and cleavage

Fluorescent turn-on probes combined with polymers have a broad range of applications, e.g. for intracellular sensing of ions, small molecules, or DNA. In the field of polymer therapeutics, these probes can be applied to extend the in vitro characterization of novel conjugates beyond cytotoxicity and cellular uptake studies. This is particularly true in cases in which polymer conjugates contain drugs attached by cleavable linkers. Better information on the intracellular linker cleavage and drug release would allow a faster evaluation and optimization of novel polymer therapeutic concepts. We therefore developed a fluorescent turn-on probe that enables direct monitoring of pH-mediated cleavage processes over time. This is achieved by exploiting the fluorescence resonance energy transfer (FRET) between two dyes that have been coupled to a dendritic polymer. We demonstrate the use of this probe to evaluate polymer uptake and intracellular release of cargo in a cell based microplate assay that is suitable for high throughput screening

Significantly enhanced proteolytic activity of cyclen complexes by monoalkylation

A simple approach towards efficient artificial proteases based on the cyclen ligand is presented. We thus achieved an increase of the proteolytic activity of two orders of magnitude when compared to the unsubstituted cyclen complex. Amphiphilic Cu(II) and Co(III) complexes cut BSA and myoglobin as model substrates at μM concentrations. MALDI-ToF MS is used to identify the cleavage fragments

Discriminating among Earth composition models using geo-antineutrinos

It has been estimated that the entire Earth generates heat corresponding to about 40 TW (equivalent to 10,000 nuclear power plants) which is considered to originate mainly from the radioactive decay of elements like U, Th and K, deposited in the crust and mantle of the Earth. Radioactivity of these elements produce not only heat but also antineutrinos (called geo-antineutrinos) which can be observed by terrestrial detectors. We investigate the possibility of discriminating among Earth composition models predicting different total radiogenic heat generation, by observing such geo-antineutrinos at Kamioka and Gran Sasso, assuming KamLAND and Borexino (type) detectors, respectively, at these places. By simulating the future geo-antineutrino data as well as reactor antineutrino background contributions, we try to establish to which extent we can discriminate among Earth composition models for given exposures (in units of kt$\cdot$ yr) at these two sites on our planet. We use also information on neutrino mixing parameters coming from solar neutrino data as well as KamLAND reactor antineutrino data, in order to estimate the number of geo-antineutrino induced events.Comment: 24 pages, 10 figures, final version to appear in JHE

Neutrinos and Energetics of the Earth

We estimate terrestrial antineutrino and neutrino fluxes according to different models of Earth composition. We find large variations, corresponding to uncertainties on the estimated $U$, $Th$ and $K$ abundances in the mantle. Information on the mantle composition can be derived from antineutrino flux measurements after subtracting the crust contribution. This requires a good description of the crust composition in the region of the detector site. Measurements of terrestrial antineutrinos will provide a direct insight on the main sources of Earth's heat flow.Comment: 11 pages, RevTeX file. To appear on Phys. Lett. B. Addendum contains comparison with KamLAND result

Post-collisional Tertiary–Quaternary mafic alkalic magmatism in the Carpathian–Pannonian region: a review

Mafic alkalic volcanism was widespread in the Carpathian–Pannonian region (CPR) between 11 and 0.2 Ma. It followed the Miocene continental collision of the Alcapa and Tisia blocks with the European plate, as subduction-related calc-alkaline magmatism was waning. Several groups of mafic alkalic rocks from different regions within the CPR have been distinguished on the basis of ages and/or trace-element compositions. Their trace element and Sr–Nd–Pb isotope systematics are consistent with derivation from complex mantle-source regions, which included both depleted asthenosphere and metasomatized lithosphere. The mixing of DMM-HIMU-EMII mantle components within asthenosphere-derived magmas indicates variable contamination of the shallow asthenosphere and/or thermal boundary layer of the lithosphere by a HIMU-like component prior to and following the introduction of subduction components. Various mantle sources have been identified: Lower lithospheric mantle modified by several ancient asthenospheric enrichments (source A); Young asthenospheric plumes with OIB-like trace element signatures that are either isotopically enriched (source B) or variably depleted (source C); Old upper asthenosphere heterogeneously contaminated by DM-HIMU-EMII-EMI components and slightly influenced by Miocene subduction-related enrichment (source D); Old upper asthenosphere heterogeneously contaminated by DM-HIMU-EMII components and significantly influenced by Miocene subduction-related enrichment (source E). Melt generation was initiated either by: (i) finger-like young asthenospheric plumes rising to and heating up the base of the lithosphere (below the Alcapa block), or (ii) decompressional melting of old asthenosphere upwelling to replace any lower lithosphere or heating and melting former subducted slabs (the Tisia block)

Testing the ureilite projectile hypothesis for the El'gygytgyn impact: determination of siderophile element abundances and Os isotope ratios in ICDP drill core samples and melt rocks

The geochemical nature of the impactites from International Continental Scientific Drilling Project-El'gygytgyn lake drill core 1C is compared with that of impact melt rock fragments collected near the western rim of the structure and literature data. Concentrations of major and trace elements, with special focus on siderophile metals Cr, Co, Ni, and the platinum group elements, and isotope ratios of osmium (Os), were determined to test the hypothesis of an ureilite impactor at El'gygytgyn. Least squares mixing calculations suggest that the upper volcanic succession of rhyolites, dacites, and andesites were the main contributors to the polymict impact breccias. Additions of 2-13.5 vol% of basaltic inclusions recovered from drill core intervals between 391.6 and 423.0 mblf can almost entirely account for the compositional differences observed for the bottom of a reworked fallout deposit at 318.9 mblf, a polymict impact breccia at 471.4 mblf, and three impact melt rock fragments. However, the measured Os isotope ratios and slightly elevated PGE content (up to 0.262 ng g(-1) Ir) of certain impactite samples, for which the CI-normalized logarithmic PGE signature displays a relatively flat (i.e., chondritic) pattern, can only be explained by the incorporation of a small meteoritic contribution. This component is also required to explain the exceptionally high siderophile element contents and corresponding Ni/Cr, Ni/Co, and Cr/Co ratios of impact glass spherules and spherule fragments that were recovered from the reworked fallout deposits and from terrace outcrops of the Enmyvaam River approximately 10 km southeast of the crater center. Mixing calculations support the presence of approximately 0.05 wt% and 0.50-18 wt% of ordinary chondrite (possibly type-LL) in several impactites and in the glassy spherules, respectively. The heterogeneous distribution of the meteoritic component provides clues for emplacement mechanisms of the various impactite units

Effects of Vacancies near Substitutional Implants on Trapping and Desorption of Helium - A Simulation

Trapping of He by vacancies and drainage of He from substitutional implants (Ag and Kr in W) to nearby vacancies are investigated using static lattice calculations. The calculations indicate that drainage of He will occur to vacancies within a radius of 2.5 lattice units from the implant. Furthermore the trapping probability of substitutional and interstitial random walkers on a bcc lattice by substitutional traps or vacancies is calculated. When implantation-produced vacancies are present in the vicinity of the observed trap a shielding effect occurs. Trapping constants are calculated with two random walk models for both the unshielded and the shielded defect. For the latter several configurations were taken. The results show that shielding of a defect by one vacancy at a distance of three lattice units leads already to a reduction of He trapping by that defect of 30% to 40%.