Transformation of hematopoietic cell lines to growth-factor independence and induction of a fatal myelo- and lymphoproliferative disease in mice by retrovirally transduced TEL/JAK2 fusion genes

Recent reports have demonstrated fusion of the TEL gene on 12p13 to the JAK2 gene on 9p24 in human leukemias. Three variants have been identified that fuse the TEL pointed (PNT) domain to (i) the JAK2 JH1-kinase domain, (ii) part of and (iii) all of the JH2 pseudokinase domain. We report that all of the human TEL/JAK2 variants, and a human/mouse chimeric hTEL/mJAK2(JH1) fusion gene, transform the interleukin-3 (IL-3)-dependent murine hematopoietic cell line Ba/F3 to IL-3-independent growth. Transformation requires both the TEL PNT domain and JAK2 kinase activity. Furthermore, all TEL/JAK2 variants strongly activated STAT 5 by phosphotyrosine Western blots and by electrophoretic mobility shift assays (EMSA). Mice (n = 40) transplanted with bone marrow infected with the MSCV retrovirus containing either the hTEL/mJAK2(JH1) fusion or its human counterpart developed a fatal mixed myeloproliferative and T-cell lymphoproliferative disorder with a latency of 2-10 weeks. In contrast, mice transplanted with a TEL/JAK2 mutant lacking the TEL PNT domain (n = 10) or a kinase-inactive TEL/JAK2(JH1) mutant (n = 10) did not develop the disease. We conclude that all human TEL/JAK2 fusion variants are oncoproteins in vitro that strongly activate STAT 5, and cause lethal myelo- and lymphoproliferative syndromes in murine bone marrow transplant models of leukemia.     

Functional assay of multidrug resistant cells using JC-1, a carbocyanine fluorescent probe

Multidrug resistance (MDR) is characterized by a decrease in the efficiency of chemotherapeutic agents correlated with the expression and activity of a membrane protein: the permeability-glycoprotein (Pgp 170). Clinically, detection of MDR can be performed by functional tests based on the accumulation of fluorescent compounds such as rhodamine 123. With the aim of improving the sensitivity of such analysis, we have evaluated JC-1, a fluorescent lipophilic carbocyanine dye. Above a critical concentration, JC-1 aggregates in a 'liquid crystal' form. Aggregates display a specific red emission band centered at 597 nm whereas the monomers display a green emission band centered at 540 nm. JC-1 was avidly accumulated in sensitive K562 cells where it displayed both a green cytoplasmic and red mitochondrial fluorescence. In contrast, JC-1 was poorly accumulated in resistant K562 cells, which displayed only a slight green fluorescence. The level of JC-1 accumulation was correlated with the level of Pgp expression detected by MRK16 and UIC2 antibodies on a set of K562 subclones with increasing resistance levels. The specific fluorescence properties of JC-1 allow accurate discrimination between low-level resistant cells and sensitive cells. Chemosensitizers such as verapamil, cyclosporine A or S9788 restored JC-1 accumulation in resistant cells. The fluorescence properties of JC-1 could therefore be used for monitoring the effects of reversing agents.     

Microtribological Studies of Different Nanocomposite TiC/a-C:H Coatings Using a Modified Nanoindentation Setup

The microtribological behavior of different nanocomposite TiC/a-C:H coatings against 100Cr6 (AISI 52100) balls with 250 m radius has been studied using a modified nanoindentation setup and was compared to the results of macroscopic pin-on-disc (POD) experiments. First results reveal significant differences between macroscopic friction coefficients _POD determined using POD tests and microscopic friction coefficients _micro. On the macroscopic scale low friction coefficients can be obtained for high hardness coatings. On the microscopic scale the high hardness samples induce considerable wear on the steel counterbody leading to high microscopic friction coefficients of around 0.3. For samples with lower hardness no wear has been observed and low microscopic friction coefficients (< 0.2) can be acheieved.     

The data-acquisition system for the CMS tracker beam tests

This paper describes the conception and the development of a real-time data-acquisition system for prototype detectors of the Tracker being designed for the compact muon solenoid (CMS) experiment at the Large Hadron Collider of CERN, European Laboratory for Particle Physics, Geneva, Switzerland. The rationale for the development of a dedicated data-acquisition system was the need to perform two fundamental beam tests (the “Milestone Barrel 1” and “Milestone Forward 1”), with large-scale prototypes of the detectors planned as the baseline design. The number of readout channels, the complexity of the readout electronics, and the stringent requirements of the milestone tests mandated that a thorough understanding of the issues related to the physics of the detectors themselves be coupled with the application of leading-edge electronic and software engineering technologies. The implementation described in this paper is based on a distributed architecture. An event builder CPU handles the two main tasks of synchronizing a variable number of front-end processors and formatting the data in preparation for the transfer to a dedicated high-performance storage system, while the front-end processors handle the hardware and the real-time readout. Additional workstations are used to decouple the actual task of transferring the data files and monitoring the detector performance on-line from the readout farm. The system has been successfully operated during the two aforementioned Milestone tests, allowing the CMS Tracker collaboration to pass them, with the simultaneous readout of up to 40000 detector channels. The results of the two Milestones have led to the compilation of the “Tracker Technical Design Report”. Subsequently, the same readout system has been used for a number of other beam tests, and it has formed the basis for the development of further, more advanced data-acquisition systems for the new readout electronic of the CMS Tracker     

Inhibition of the proliferative cycle and apoptotic events in WiDr cells after down-regulation of the calcium-binding protein calretinin using antisense oligodeoxynucleotides

The colon adenocarcinoma cell line WiDr expresses the calcium-binding protein calretinin (CR). In order to deduce possible functions of calretinin in these cells we decreased its concentration by antisense techniques. Treatment of WiDr cells with phosphorothioate antisense oligodeoxynucleotides (AS-ODNs) led to a drop in calretinin expression, as evidenced by immunohistochemical staining of WiDr cells and Western blot analysis of cytosolic cell extracts. The morphology of these epithelial cells changed from polygonal to spherical and they formed dense cell clusters. Cells displaying morphological alterations typical for apoptotic cells were observed after incubation with AS-ODNs, as evidenced by phase-contrast and electron microscopy. The mitotic rate of AS-ODN-treated cells dropped significantly, as demonstrated by mitotic labeling and time-lapse microcinematography. Furthermore, an accumulation of cells in phase G1 and a reduction of [3H]thymidine-labeled cells was observed in antisense-treated cells. The basal level of [Ca2+]i was not influenced by the down-regulation of calretinin. WiDr cells incubated with the nonsense, reverse-sense, or with an oligodeoxynucleotide with a totally unrelated sequence did not show any significant differences when compared to control cells. We conclude that calretinin levels have an impact on the progression of the cell cycle of WiDr cells.     

On the blending of the Landsat and MODIS surface reflectance: predicting daily Landsat surface reflectance

The 16-day revisit cycle of Landsat has long limited its use for studying global biophysical processes, which evolve rapidly during the growing season. In cloudy areas of the Earth, the problem is compounded, and researchers are fortunate to get two to three clear images per year. At the same time, the coarse resolution of sensors such as the Advanced Very High Resolution Radiometer and Moderate Resolution Imaging Spectroradiometer (MODIS) limits the sensors' ability to quantify biophysical processes in heterogeneous landscapes. In this paper, the authors present a new spatial and temporal adaptive reflectance fusion model (STARFM) algorithm to blend Landsat and MODIS surface reflectance. Using this approach, high-frequency temporal information from MODIS and high-resolution spatial information from Landsat can be blended for applications that require high resolution in both time and space. The MODIS daily 500-m surface reflectance and the 16-day repeat cycle Landsat Enhanced Thematic Mapper Plus (ETM+) 30-m surface reflectance are used to produce a synthetic "daily" surface reflectance product at ETM+ spatial resolution. The authors present results both with simulated (model) data and actual Landsat/MODIS acquisitions. In general, the STARFM accurately predicts surface reflectance at an effective resolution close to that of the ETM+. However, the performance depends on the characteristic patch size of the landscape and degrades somewhat when used on extremely heterogeneous fine-grained landscapes.     

Quantitative Topographical Characterization of Thermally Sprayed Coatings by Optical Microscopy

Topography measurements and roughness calculations for different rough surfaces (Rugotest surface comparator and thermally sprayed coatings) are presented. The surfaces are measured with a novel quantitative topography measurement technique based on optical stereomicroscopy and a comparison is made with established scanning stylus and optical profilometers. The results show that for most cases the different methods yield similar results. Stereomicroscopy is therefore a valuable method for topographical investigations in both quality control and research. On the other hand, the method based on optical microscopy demands a careful optimization of the experimental settings like the magnification and the illumination to achieve satisfactory results.