IGF1R Signaling in Ewing Sarcoma Is Shaped by Clathrin-/Caveolin-Dependent Endocytosis

Receptor endocytosis is critical for cell signaling. IGF1R mediates an autocrine loop that is de-regulated in Ewing Sarcoma (ES) cells. Here we study the impact of IGF1R internalization, mediated by clathrin and caveolin-1 (CAV1), in ES signaling. We used clathrin and CAV1-siRNA to interfere in clathrin- and caveolin-dependent endocytosis. Chlorpromazine (CPMZ) and methyl-beta-cyclo-dextrin (MCD) were also used in order to inhibit clathrin- and caveolin-dependent endocytosis, respectively. We analyzed IGF1R internalization and co-localization with clathrin and CAV1 upon ligand binding, as well as the status of the IGF1R pathway, cellular proliferation, and the apoptosis of interfered and inhibited ES cells. We performed a high-throughput tyrosine kinase phosphorylation assay to analyze the effects of combining the IGF1R tyrosine kinase inhibitor AEW541 (AEW) with CPMZ or MCD on the intracellular phospho-proteome. We observed that IGF1R is internalized upon ligand binding in ES cells and that this process is dependent on clathrin or CAV1. The blockage of receptor internalization inhibited AKT and MAPK phosphorylation, reducing the proliferative rate of ES cells and increasing the levels of apoptosis. Combination of AEW with CPMZ or MCD largely enhanced these effects. CAV1 and clathrin endocytosis controls IGF1R internalization and signaling and has a profound impact on ES IGF1R-promoted survival signaling. We propose the combination of tyrosine-kinase inhibitors with endocytosis inhibitors as a new therapeutic approach to achieve a stronger degree of receptor inhibition in this, or other neoplasms dependent on IGF1R signaling

Trapping in irradiated p-on-n silicon sensors at fluences anticipated at the HL-LHC outer tracker

The degradation of signal in silicon sensors is studied under conditions expected at the CERN High-Luminosity LHC. 200 $\mu$m thick n-type silicon sensors are irradiated with protons of different energies to fluences of up to $3 \cdot 10^{15}$ neq/cm$^2$. Pulsed red laser light with a wavelength of 672 nm is used to generate electron-hole pairs in the sensors. The induced signals are used to determine the charge collection efficiencies separately for electrons and holes drifting through the sensor. The effective trapping rates are extracted by comparing the results to simulation. The electric field is simulated using Synopsys device simulation assuming two effective defects. The generation and drift of charge carriers are simulated in an independent simulation based on PixelAV. The effective trapping rates are determined from the measured charge collection efficiencies and the simulated and measured time-resolved current pulses are compared. The effective trapping rates determined for both electrons and holes are about 50% smaller than those obtained using standard extrapolations of studies at low fluences and suggests an improved tracker performance over initial expectations

Urine recirculation improves hemodynamics and enhances function in normothermic kidney perfusion

Background: The study compares urine recirculation (URC) to urine replacement (UR) with Ringer's lactate in a porcine normothermic kidney machine perfusion (NMP) model using a preclinical prototype device. Methods:Kidney pairs were recovered uninjured (as live-donor nephrectomy) and perfused consecutively. Pig kidneys (n = 10) were allocated to either NMP with URC (n = 5) or NMP with volume replacement (n = 5). Cold ischemia time was either 2 or 27 hours for the first or second perfusion (URC or UR) of a kidney pair. An autologous blood-based perfusate, leukocyte-filtered, was used and NMP performed up to 24 hours. Perfusion parameters, biochemistry/metabolic parameters were monitored and samples collected. Results:Physiological mean arterial pressures and flows were achieved in both groups but were sustainable only with URC. Significantly higher arterial flow was observed with URC (326.7 ± 1.8 versus 242.5 ± 14.3 mL/min, P = 0.001). Perfusate sodium levels were lower with URC, 129.6 ± 0.7 versus 170.3±2.7 mmol/L, P < 0.001). Stable physiological pH levels were only observed with URC. Perfusate lactate levels were lower with URC (2.2 ± 0.1 versus 7.2 ± 0.5 mmol/L, P < 0.001). Furthermore, the hourly rate of urine output was lower with URC and closer to physiological levels (150 versus 548 mL/h, P = 0.008). Normothermic kidney perfusion with URC was associated with longer achievable durations of perfusion: the objective in all experiments was a 24-hour perfusion, but this was not achieved in every case. The mean perfusions were 17.3 ± 9.2 hours with URC versus 5.3 ± 1.3 hours NMP with UR; P = 0.02. There appeared to be no differences in baseline tubular condition with and without URC. Conclusions:URC facilitates long-term kidney NMP in a porcine model. Perfusate homeostasis and stability of renal arterial flow throughout the perfusion period was only achievable with URC, independent of cold ischemia time duration

Studies on the curing behaviour and mechanical properties of styrene/methyl methacrylate grafted deproteinized natural rubber latex

The graft copolymerization of styrene/methyl methacrylate (MMA) onto deproteinized natural rubber (DPNR) latex was carried out using ammonium peroxy disulfate (N2H8O8S2) as the initiator. The presence of the grafted polystyrene (PS) and polymethyl methacrylate (PMMA) on the rubber backbone was confirmed by FTIR spectroscopy. The effects of monomer concentrations on curing characteristics and mechanical properties were studied. It was found that the cure time and scorch time were increased with increasing monomer concentration whereas the torque max-min value was slightly decreased. It was also noted that the increase in the monomer concentration resulted in stiffer rubber with increased modulus and reduced elongation at break