Impact of tumor-specific targeting on the biodistribution and efficacy of siRNA nanoparticles measured by multimodality in vivo imaging

Targeted delivery represents a promising approach for the development of safer and more effective therapeutics for oncology applications. Although macromolecules accumulate nonspecifically in tumors through the enhanced permeability and retention (EPR) effect, previous studies using nanoparticles to deliver chemotherapeutics or siRNA demonstrated that attachment of cell-specific targeting ligands to the surface of nanoparticles leads to enhanced potency relative to nontargeted formulations. Here, we use positron emission tomography (PET) and bioluminescent imaging to quantify the in vivo biodistribution and function of nanoparticles formed with cyclodextrin-containing polycations and siRNA. Conjugation of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid to the 5' end of the siRNA molecules allows labeling with 64Cu for PET imaging. Bioluminescent imaging of mice bearing luciferase-expressing Neuro2A s.c. tumors before and after PET imaging enables correlation of functional efficacy with biodistribution data. Although both nontargeted and transferrin-targeted siRNA nanoparticles exhibit similar biodistribution and tumor localization by PET, transferrin-targeted siRNA nanoparticles reduce tumor luciferase activity by {approx}50% relative to nontargeted siRNA nanoparticles 1 d after injection. Compartmental modeling is used to show that the primary advantage of targeted nanoparticles is associated with processes involved in cellular uptake in tumor cells rather than overall tumor localization. Optimization of internalization may therefore be key for the development of effective nanoparticle-based targeted therapeutics

Effect of helium pre- or postconditioning on signal transduction kinases in patients undergoing coronary artery bypass graft surgery

Background: The noble gas helium induces pre- and postconditioning in animals and humans. Volatile anesthetics induce cardioprotection in humans undergoing coronary artery bypass graft (CABG) surgery. We hypothesized that helium induces pre-and postconditioning in CABG-patients, affecting signaling molecules protein kinase C-epsilon (PKC-epsilon), p38 mitogen activated protein kinase (p38 MAPK), extracellular signal-regulated kinase 1/2 (ERK-1/2) and heat shock protein 27 (HSP-27) within cardiac tissue, and reducing postoperative troponin levels. Methods: After ethical approval and informed consent, 125 elective patients undergoing CABG surgery were randomised into this prospective, placebo controlled, investigator blinded, parallel arm single-centre study. Helium preconditioning (3 x 5 min of 70 % helium and 30 % oxygen) was applied before aortic cross clamping; postconditioning (15 min of helium) was applied before release of the aortic cross clamp. Signaling molecules were measured in right atrial appendix specimens. Troponin-T was measured at 4, 12, 24 and 48 h postoperatively. Results: Baseline characteristics of all groups were similar. Helium preconditioning did not significantly alter the primary outcome (molecular levels of kinases PKC-e and HSP-27, ratio of activated p38 MAPK or ERK 1/2). Postoperative troponin T was 11 arbitrary units [5, 31; area-under-the-curve (interquartile range)] for controls, and no statistically significant changes were observed after helium preconditioning [He-pre: 11 (6, 18)], helium postconditioning [He-post: 11 (8, 15)], helium pre-and postconditioning [He-PP: 14 (6, 20)] and after sevoflurane preconditioning [APC: 12 (8, 24), p = 0.13]. No adverse effects related to study treatment were observed in this study. Conclusions: No effect was observed of helium preconditioning, postconditioning or the combination thereof on activation of p38 MAPK, ERK 1/2 or levels of HSP27 and PKC-e in the human heart. Helium pre-and postconditioning did not affect postoperative troponin release in patients undergoing CABG surgery

Light-Controlled Affinity Purification of Protein Complexes Exemplified by the Resting ZAP70 Interactome

Multiprotein complexes control the behavior of cells, such as of lymphocytes of the immune system. Methods to affinity purify protein complexes and to determine their interactome by mass spectrometry are thus widely used. One drawback of these methods is the presence of false positives. In fact, the elution of the protein of interest (POI) is achieved by changing the biochemical properties of the buffer, so that unspecifically bound proteins (the false positives) may also elute. Here, we developed an optogenetics-derived and light-controlled affinity purification method based on the light-regulated reversible protein interaction between phytochrome B (PhyB) and its phytochrome interacting factor 6 (PIF6). We engineered a truncated variant of PIF6 comprising only 22 amino acids that can be genetically fused to the POI as an affinity tag. Thereby the POI can be purified with PhyB-functionalized resin material using 660 nm light for binding and washing, and 740 nm light for elution. Far-red light-induced elution is effective but very mild as the same buffer is used for the wash and elution. As proof-of-concept, we expressed PIF-tagged variants of the tyrosine kinase ZAP70 in ZAP70-deficient Jurkat T cells, purified ZAP70 and associating proteins using our light-controlled system, and identified the interaction partners by quantitative mass spectrometry. Using unstimulated T cells, we were able to detect the known interaction partners, and could filter out all other proteins

Uncoupling bacterial attachment on and detachment from polydimethylsiloxane surfaces through empirical and simulation studies

Bacterial infections related to medical devices can cause severe problems, whose solution requires in-depth understanding of the interactions between bacteria and surfaces. This work investigates the influence of surface physicochemistry on bacterial attachment and detachment under flow through both empirical and simulation studies. We employed polydimethylsiloxane (PDMS) substrates having different degrees of crosslinking as the model material and the extended Derjaguin - Landau - Verwey - Overbeek model as the simulation method. Experimentally, the different PDMS materials led to similar numbers of attached bacteria, which can be rationalized by the identical energy barriers simulated between bacteria and the different materials. However, different numbers of residual bacteria after detachment were observed, which was suggested by simulation that the detachment process is determined by the interfacial physicochemistry rather than the mechanical property of a material. This finding is further supported by analyzing the bacteria detachment from PDMS substrates from which non-crosslinked polymer chains had been removed: similar numbers of residual bacteria were found on the extracted PDMS substrates. The knowledge gained in this work can facilitate the projection of bacterial colonization on a given surface

Diamagnetic response of cylindrical normal metal - superconductor proximity structures with low concentration of scattering centers

We have investigated the diamagnetic response of composite NS proximity wires, consisting of a clean silver or copper coating, in good electrical contact to a superconducting niobium or tantalum core. The samples show strong induced diamagnetism in the normal layer, resulting in a nearly complete Meissner screening at low temperatures. The temperature dependence of the linear diamagnetic susceptibility data is successfully described by the quasiclassical Eilenberger theory including elastic scattering characterised by a mean free path l. Using the mean free path as the only fit parameter we found values of l in the range 0.1-1 of the normal metal layer thickness d_N, which are in rough agreement with the ones obtained from residual resistivity measurements. The fits are satisfactory over the whole temperature range between 5 mK and 7 K for values of d_N varying between 1.6 my m and 30 my m. Although a finite mean free path is necessary to correctly describe the temperature dependence of the linear response diamagnetic susceptibility, the measured breakdown fields in the nonlinear regime follow the temperature and thickness dependence given by the clean limit theory. However, there is a discrepancy in the absolute values. We argue that in order to reach quantitative agreement one needs to take into account the mean free path from the fits of the linear response. [PACS numbers: 74.50.+r, 74.80.-g]Comment: 10 pages, 9 figure

3',5'-Cyclic Adenosine Monophosphate- and Ca2+-Calmodulin-Dependent Endogenous Protein Phosphorylation Activity in Membranes of the Bovine Chromaffin Secretory Vesicles: Identification of Two Phosphorylated Components as Tyrosine Hydroxylase and Protein Kinase Regulatory Subunit Type II

Abstract: Membranes of the secretory vesicles from bovine adrenal medulla were investigated for the presence of the endogenous protein phosphorylation activity. Seven phosphoprotein bands in the molecular weight range of 250,000 to 30,000 were observed by means of the sodium dodecyl sulphate electrophoresis and autoradiography. On the basis of the criteria of molecular weight, selective stimulation of the phosphorylation by cyclic AMP (as compared with cyclic GMP) and immunoprecipitation by specific antibodies, band 5 (molecular weight 60,300) was found to represent the phosphorylated form of the secretory vesicle-bound tyrosine hydroxylase. The electrophoretic mobility, the stimulatory and inhibitory effects of cyclic AMP in presence of Mg2+ and Zn,2+ respectively, and immunoreactivity toward antibodies showed band 6 to contain two forms of the regulatory subunits of the type II cyclic AMP-dependent protein kinase, distinguishable by their molecular weights (56,000 and 52,000, respectively). Phosphorylation of band 7 (molecular weight 29,800) was stimulated about 2 to 3 times by Ca2+ and calmodulin in the concentration range of both agents believed to occur in the secretory tissues under physiological conditions

Precise Prediction for M_W in the MSSM

We present the currently most accurate evaluation of the W boson mass, M_W, in the Minimal Supersymmetric Standard Model (MSSM). The full complex phase dependence at the one-loop level, all available MSSM two-loop corrections as well as the full Standard Model result have been included. We analyse the impact of the different sectors of the MSSM at the one-loop level with a particular emphasis on the effect of the complex phases. We discuss the prediction for M_W based on all known higher-order contributions in representative MSSM scenarios. Furthermore we obtain an estimate of the remaining theoretical uncertainty from unknown higher-order corrections.Comment: 38 pages, 25 figures. Minor corrections, additional reference

Histone 3.3 hotspot mutations in conventional osteosarcomas: a comprehensive clinical and molecular characterization of six H3F3A mutated cases

Background: Histone 3.3 (H3.3) hotspot mutations in bone tumors occur in the vast majority of giant cell tumors of bone (GCTBs; 96%), chondroblastomas (95%) and in a few cases of osteosarcomas. However, clinical presentation, histopathological features, and additional molecular characteristics of H3.3 mutant osteosarcomas are largely unknown. Methods: In this multicentre, retrospective study, a total of 106 conventional high-grade osteosarcomas, across all age groups were re-examined for hotspot mutations in the H3.3 coding genes H3F3A and H3F3B. H3.3 mutant osteosarcomas were re-evaluated in a multidisciplinary manner and analyzed for genome-wide DNA-methylation patterns and DNA copy number aberrations alongside H3.3 wild-type osteosarcomas and H3F3A G34W/L mutant GCTBs. Results: Six osteosarcomas (6/106) carried H3F3A hotspot mutations. No mutations were found in H3F3B. All patients with H3F3A mutant osteosarcoma were older than 30 years with a median age of 65 years. Copy number aberrations that are commonly encountered in high-grade osteosarcomas also occurred in H3F3A mutant osteosarcomas. Unlike a single osteosarcoma with a H3F3A K27M mutation, the DNA methylation profiles of H3F3A G34W/R mutant osteosarcomas were clearly different from H3.3 wild-type osteosarcomas, but more closely related to GCTBs. The most differentially methylated promoters between H3F3A G34W/R mutant and H3.3 wild-type osteosarcomas were in KLLN/PTEN (p < 0.00005) and HIST1H2BB (p < 0.0005). Conclusions: H3.3 mutations in osteosarcomas may occur in H3F3A at mutational hotspots. They are overall rare, but become more frequent in osteosarcoma patients older than 30 years. Osteosarcomas carrying H3F3A G34W/R mutations are associated with epigenetic dysregulation of KLLN/PTEN and HIST1H2BB

Equity as a Prerequisite for Stability of Cooperation on Global Public Good Provision

Analysing cooperative provision of a global public good such as climate protection, we explore the relationship between equitable burden sharing on the one hand and core stability on the other. To assess the size of the burden which a public good contribution entails for a country, we make use of a specific measure based on Moulin (Econometrica 55:963-977, 1987). In particular, we show that a Pareto optimal allocation which is not in the core can always be blocked by a group of countries with the highest Moulin sacrifices. In this sense, it is the 'overburdening' and thus 'unfair' treatment of some countries that provides the reason for core instability. By contrast, a Pareto optimal allocation is in the core if the public good contributions are fairly equally distributed according to their Moulin sacrifices. The potential implications of our theoretical analysis for global climate policy are also discussed