Radiology-Pathology and Surgical Correlation in Astroblastoma.

Astroblastoma is a rare astrocytic glial neoplasm that affects mainly young girls, peaking between 10 and 30 years of age, with low- and high-grade manifestations. Imaging characteristics are well-described, but histopathologic and, more recently, molecular analysis is fundamental to establish the diagnosis, now based on MN1 alterations. We describe a case with typical imaging and histologic features of an MN1-altered astroblastoma.

Co-expression of truncated and full-length tau induces severe neurotoxicity.

Abundant tau inclusions are a defining hallmark of several human neurodegenerative diseases, including Alzheimer's disease. Protein fragmentation is a widely observed event in neurodegenerative proteinopathies. The relevance of tau fragmentation for the neurodegenerative process in tauopathies has yet remained unclear. Here we found that co-expression of truncated and full-length human tau in mice provoked the formation of soluble high-molecular-weight tau, the failure of axonal transport, clumping of mitochondria, disruption of the Golgi apparatus and missorting of synaptic proteins. This was associated with extensive nerve cell dysfunction and severe paralysis by the age of 3 weeks. When the expression of truncated tau was halted, most mice recovered behaviorally and functionally. In contrast, co-expression of full-length tau isoforms did not result in paralysis. Truncated tau thus induces extensive but reversible neurotoxicity in the presence of full-length tau through the formation of nonfilamentous high-molecular-weight tau aggregates, in the absence of tau filaments. Targeting tau fragmentation may provide a novel approach for the treatment of human tauopathies.

A first generation compact microbeam radiation therapy system based on carbon nanotube X-ray technology.

We have developed a compact microbeam radiation therapy device using carbon nanotube cathodes to create a linear array of narrow focal line segments on a tungsten anode and a custom collimator assembly to select a slice of the resulting wedge-shaped radiation pattern. Effective focal line width was measured to be 131 µm, resulting in a microbeam width of ∼300 µm. The instantaneous dose rate was projected to be 2 Gy/s at full-power. Peak to valley dose ratio was measured to be >17 when a 1.4 mm microbeam separation was employed. Finally, multiple microbeams were delivered to a mouse with beam paths verified through histology.

TU-E-217BCD-11: Evaluating the Performance of a Stationary Digital Breast Tomosynthesis System.

PURPOSE: In conventional Digital Breast Tomosynthesis (DBT) systems a single x-ray source moves over a limited angle arc. This leads to motion blurring in the projection images associated with x-ray source motion and total scan times. We have developed a stationary DBT (s-DBT) system which forgoes a rotating source for an array of carbon nanotube (CNT) based x-ray sources. Here we report the results of evaluating the performance and the optimization of image acquisition parameters of the s-DBT system. METHODS: The s-DBT system consists of a linear source array with 31 x-ray generating focal spots distributed over a 30 degree angular span. The source array has been retrofitted onto a Hologic Selenia Dimensions DBT system. An American College of Radiology accreditation phantom was imaged to assess the quality of the reconstruction images in different configurations. A line wire phantom is used to measure the modulation transfer function (MTF). RESULTS: For the standard imaging protocol, the system resolution along the scanning direction is increased from 3.0 cycles/mm in DBT to 4.2 cycles/mm in s-DBT at a magnification factor of 1.08. The MTF did not have a noticeable change between different configurations, whereas in DBT the MTF can be degraded for larger angular spans due to faster x-ray source motion. The overall image quality factor is found to be best for the configuration with a large angular span and intermittent number of projection views. CONCLUSIONS: We demonstrated successful construction and operation of the s-DBT system integrating a CNT x-ray source array with a Hologic DBT system. The spatial resolution of the s-DBT system is demonstrated to be substantially increased over the corresponding DBT system. It was found that a configuration with a large angular span, an intermittent number of projection views, and an even dose distribution resulted in the best overall image quality. Hologic INC has provided the Hologic Selenia Dimensions used in the research. The project is supported by the National Cancer Institute under grant number U54CA119343 and R01CA134598 and the UNC University Cancer Research Fund. Dr. Xin Qian is supported by a fellowship from the Department of Defense under grant number BC087505.

Design and feasibility studies of a stationary digital breast tomosynthesis system.

Studies have shown that digital breast tomosynthesis (DBT) can improve breast cancer diagnosis by reconstructing 3D images. However, DBT scanners based on rotation gantry prolong the imaging time and reduce spatial resolution due to motion comparing with the regular two-view mammography. To obtain three dimension reconstruction images and maintain the high image quality of conventional mammography, we proposed a prototype stationary digital breast tomosynthesis system (s-DBT). The proposed s-DBT system acquires projection images without mechanical movement. The core component of the s-DBT system is a specially designed spatially distributed multi-beam x-ray tube based on the carbon nanotube field emission x-ray technology. The multi-beam x-ray source array enables collection of all projection images from different viewing angles without mechanical motion. Preliminary results show the s-DBT system can achieve a scan time comparable to the regular two-view mammography, and improve the spatial resolution comparing with rotating gantry DBT.

Stationary digital breast tomosynthesis with distributed field emission X-ray tube.

Tomosynthesis requires projection images from different viewing angles. Using a distributed x-ray source this can be achieved without mechanical motion of the source with the potential for faster image acquisition speed. A distributed x-ray tube has been designed and manufactured specifically for breast tomosynthesis. The x-ray tube consists of 31 field emission x-ray sources with an angular range of 30°. The total dose is up to 100mAs with an energy range between 27 and 45 kVp. We discuss the source geometry and results from the characterization of the first prototype. The x-ray tube uses field emission cathodes based on carbon nanotubes (CNT) as electron source. Prior to the manufacturing of the sealed x-ray tube extensive testing on the field emission cathodes has been performed to verify the requirements for commercial tomosynthesis systems in terms of emission current, focal spot size and tube lifetime.

Distributed source x-ray tube technology for tomosynthesis imaging.

Tomosynthesis imaging requires projection images from different viewing angles. Conventional systems use a moving xray source to acquire the individual projections. Using a stationary distributed x-ray source with a number of sources that equals the number of required projections, this can be achieved without any mechanical motion. Advantages are a potentially faster image acquisition speed, higher spatial and temporal resolution and simple system design. We present distributed x-ray sources based on carbon nanotube (CNT) field emission cathodes. The field emission cathodes deliver the electrons required for x-ray production. CNT emitters feature a stable emission at high current density, a cold emission, excellent temporal control of the emitted electrons and good configurability. We discuss the use of stationary sources for two applications: (i) a linear tube for stationary digital breast tomosynthesis (sDBT), and (ii) a square tube for on-board tomosynthesis image-guided radiation therapy (IGRT). Results from high energy distributed sources up to 160kVp are also presented.

Screened radiative corrections from hyperfine-split dielectronic resonances in lithiumlike scandium.

Term energies for dielectronic-recombination Rydberg resonances below 0.07 eV are determined for Sc18+ with absolute accuracies below 0.0002 eV by electron collision spectroscopy in an ion storage ring, using the twin-electron-beam technique and a cryogenic photocathode. The lithiumlike 2s_{1/2}-2p_{3/2} transition energy for Z=21 is determined to 4.6 ppm, less than 1% of the few-body effects on radiative corrections. Features from the hyperfine structure of the 2s state could be resolved in the dielectronic-recombination spectrum.

High-resolution dissociative recombination of cold H3+and first evidence for nuclear spin effects.

The energy-resolved rate coefficient for the dissociative recombination (DR) of H(3)(+) with slow electrons has been measured by the storage-ring method using an ion beam produced from a radiofrequency multipole ion trap, employing buffer-gas cooling at 13 K. The electron energy spread of the merged-beams measurement is reduced to 500 microeV by using a cryogenic GaAs photocathode. This and a previous cold- measurement jointly confirm the capability of ion storage rings, with suitable ion sources, to store and investigate H(3)(+) in the two lowest, (J,G) = (1,1) and (1,0) rotational states prevailing also in cold interstellar matter. The use of para-H(2) in the ion source, expected to enhance para-H(3)(+) in the stored ion beam, is found to increase the DR rate coefficient at meV electron energies.

Mitotic degradation of cyclin A is mediated by multiple and novel destruction signals.

Exit from mitosis requires Cdk1 inactivation, with the most prominent mechanism of Cdk1 inactivation being proteolysis of mitotic cyclins [1]. In higher eukaryotes this involves sequential destruction of A- and B-type cyclins. CycA is destroyed first, and CycA/Cdk1 inactivation is required for the metaphase-to-anaphase transition [2]. The degradation of CycA is delayed in response to DNA damage but is not prevented when the spindle checkpoint is activated [3, 4]. Cyclin destruction is thought to be mediated by a conserved motif, the destruction box (D box). Like B-type cyclins, A-type cyclins contain putative destruction box sequences in their N termini [5]. However, no detailed in vivo analysis of the sequence requirements for CycA destruction has been described so far. Here we tested several mutations in the CycA coding region for destruction in Drosophila embryos. We show that D box sequences are not essential for mitotic destruction of CycA. Destruction is mediated by at least three different elements that act in an overlapping fashion to mediate its mitotic degradation.

The cyclin-dependent kinase inhibitor Roughex is involved in mitotic exit in Drosophila.

BACKGROUND: Exit from mitosis is a tightly regulated event. This process has been studied in greatest detail in budding yeast, where several activities have been identified that cooperate to downregulate activity of the cyclin-dependent kinase (CDK) Cdc28 and force an exit from mitosis. Cdc28 is inactivated through proteolysis of B-type cyclins by the multisubunit ubiquitin ligase termed the anaphase promoting complex/cyclosome (APC/C) and inhibition by the cyclin-dependent kinase inhibitor (CKI) Sic1. In contrast, the only mechanism known to be essential for CDK inactivation during mitosis in higher eukaryotes is cyclin destruction. RESULTS: We now present evidence that the Drosophila CKI Roughex (Rux) contributes to exit from mitosis. Observations of fixed and living embryos show that metaphase is significantly longer in rux mutants than in wild-type embryos. In addition, Rux overexpression is sufficient to drive cells experimentally arrested in metaphase into interphase. Furthermore, rux mutant embryos are impaired in their ability to overcome a transient metaphase arrest induced by expression of a stable cyclin A. Rux has numerous functional similarities with Sic1. While these proteins share no sequence similarity, we show that Sic1 inhibits mitotic Cdk1-cyclin complexes from Drosophila in vitro and in vivo. CONCLUSIONS: Rux inhibits Cdk1-cyclin A kinase activity during metaphase, thereby contributing to exit from mitosis. To our knowledge, this is the first mitotic function ascribed to a CKI in a multicellular organism and indicates the existence of a novel regulatory mechanism for the metaphase to anaphase transition during development.

Cyclins: growing pains for Drosophila.

Tradition holds that cyclin D is required for the initiation of cell division; recent studies in Drosophila, however, suggest that cyclin D has a separate function in governing growth.

Homeodomain position 54 specifies transcriptional versus translational control by Bicoid.

Bicoid (BCD), the anterior determinant of Drosophila, controls embryonic gene expression by transcriptional activation and translational repression. Both functions require the homeodomain (HD), which recognizes DNA motifs at target gene enhancers and a specific sequence interval in the 3' untranslated region of caudal (cad) mRNA. Here we show that the BCD HD is a nucleic acid-binding unit. Its helix III contains an arginine-rich motif (ARM), similar to the RNA-binding domain of the HIV-1 protein REV, needed for both RNA and DNA recognition. Replacement of arginine 54, within this motif, alters the RNA but not the DNA binding properties of the HD. Corresponding BCD mutants fail to repress cad mRNA translation, whereas the transcriptional target genes are still activated.

Rux is a cyclin-dependent kinase inhibitor (CKI) specific for mitotic cyclin-Cdk complexes.

BACKGROUND: Roughex (Rux) is a cell-cycle regulator that contributes to the establishment and maintenance of the G1 state in the fruit fly Drosophila. Genetic data show that Rux inhibits the S-phase function of the cyclin A (CycA)-cyclin-dependent kinase 1 (Cdk1) complex; in addition, it can prevent the mitotic functions of CycA and CycB when overexpressed. Rux has no homology to known Cdk inhibitors (CKIs), and the molecular mechanism of Rux function is not known. RESULTS: Rux interacted with CycA and CycB in coprecipitation experiments. Expression of Rux caused nuclear translocation of CycA and CycB, and inhibited Cdk1 but not Cdk2 kinase activity. Cdk1 inhibition by Rux did not rely on inhibitory phosphorylation, disruption of cyclin-Cdk complex formation or changes in subcellular localization. Rux inhibited Cdk1 kinase in two ways: Rux prevented the activating phosphorylation on Cdk1 and also inhibited activated Cdk1 complexes. Surprisingly, Rux had a stimulating effect on CycA-Cdk1 activity when present in low concentrations. CONCLUSIONS: Rux fulfils all the criteria for a CKI. This is the first description in a multicellular organism of a CKI that specifically inhibits mitotic cyclin-Cdk complexes. This function of Rux is required for the G1 state and male meiosis and could also be involved in mitotic regulation, while the stimulating effect of Rux might assist in any S-phase function of CycA-Cdk1.

Exit from mitosis in Drosophila syncytial embryos requires proteolysis and cyclin degradation, and is associated with localized dephosphorylation.

The cyclin proteolysis that accompanies the exit from mitosis in diverse systems appears to be essential for restoration of interphase. The early syncytial divisions of Drosophila embryos, however, occur without detectable oscillations in the total cyclin level or Cdk1 activity. Nonetheless, we found that injection of an established inhibitor of cyclin proteolysis, a cyclin B amino-terminal peptide, prevents exit from mitosis in syncytial embryos. Similarly, injection of a version of Drosophila cyclin B that is refractory to proteolysis results in mitotic arrest. We infer that proteolysis of cyclins is required for exit from syncytial mitoses. This inference can be reconciled with the failure to observe oscillations in total cyclin levels if only a small pool of cyclins is destroyed in each cycle. We find that antibody detection of histone H3 phosphorylation (PH3) acts as a reporter for Cdk1 activity. A gradient of PH3 along anaphase chromosomes suggests local Cdk1 inactivation near the spindle poles in syncytial embryos. This pattern of Cdk1 inactivation would be consistent with local cyclin destruction at centrosomes or kinetochores. The local loss of PH3 during anaphase is specific to the syncytial divisions and is not observed after cellularization. We suggest that exit from mitosis in syncytial cycles is modified to allow nuclear autonomy within a common cytoplasm.

S-phase function of Drosophila cyclin A and its downregulation in G1 phase.

BACKGROUND: Cyclin E is the normal inducer of S phase in G1 cells of Drosophila embryos. Stable G1 quiescence requires the downregulation both of cyclin E and of other factors that can bypass the normal regulation of cell cycle progression. RESULTS: High-level expression of cyclin A triggered the G1/S transition in wild-type embryos and in mutant embryos lacking cyclin E. Three types of control downregulated this activity of cyclin A. First, cyclin destruction limited the accumulation of cyclin A protein in G1. Second, inhibitory phosphorylation of cdc2, the kinase partner of cyclin A, reduced the S-phase promoting activity of cyclin A in G1. Third, rux, a protein with unknown biochemical function, limited cyclin A function in G1. Overexpression of rux blocked S phase induction by coexpressed cyclin A and promoted the degradation of cyclin A. Rux also prevented a stable cyclin A mutant from inducing S phase, indicating that inhibition does not require cyclin destruction, and drove the nuclear localization of cyclin A. CONCLUSIONS: Cyclin A can drive the G1/S transition, but this function is suppressed by three types of control: cyclin A destruction, inhibitory phosphorylation of cdc2, and inhibition by rux. The partly redundant contributions of these three inhibitory mechanisms safeguard the stability of G1 quiescence until the induction of cyclin E. The action of rux during G1 resembles the action of inhibitors of mitotic kinases present during G1 in yeast, although no obvious sequence similarity exists.

Variation in the number of activated torso receptors correlates with differential gene expression.

Activation of receptor tyrosine kinases triggers many developmental decisions, yet we do not understand how activation of a single receptor can be transduced into different cell responses. The torso pathway in Drosophila provides a model to address this issue since it generates more than one response in the embryo. The torso receptor tyrosine kinase is activated at the embryonic poles under the control of trunk, a protein with similarities to several types of extracellular growth factors. Activation of torso is responsible for the development of a variety of structures, whose appearance can be correlated with activation of at least two different genes along the terminal region. In this study we have analyzed mutations in torso and trunk that express low levels of the respective proteins. We show that different amounts of torso or trunk molecules correlate with the expression of different zygotic genes, implicating changes in the number of activated torso molecules as one of the mechanisms defining differential gene expression. We suggest that variation in the number of activated receptors at the cell surface is a general mechanism that leads to differential gene expression and thus the generation of different cell responses.

The activity of D-raf in torso signal transduction is altered by serine substitution, N-terminal deletion, and membrane targeting.

The Raf family of serine/threonine kinases are essential components in many receptor tyrosine kinase-mediated signal transduction pathways. Here, we analyze the function of D-raf in the Torso (Tor) pathway required to specify cellular fates at the embryonic poles. Using mutant embryos lacking endogenous D-raf protein, we show that D-raf's serine/threonine kinase activity is essential for its role in Tor signal transduction and that human Raf-1 will substitute for D-raf in this pathway. After Tor activation, D-raf becomes hyperphosphorylated. We identified two putative serine phosphorylation sites (S388 and S743) in SF9 cells and demonstrate that S743 or its phosphorylation is essential for D-raf function in embryos. Alanine substitution at S388, N-terminal truncation, or targeted membrane association permits transmission of the Torso signal by D-raf, but these D-raf molecules differ in their rescuing potential and relative biological activity. Membrane-targeted D-raftor4021 showed the highest level of activity, followed by alanine-substituted D-rafS388A and N-terminal-truncated D-raf delta 445. Since the activity profiles for these altered forms of D-raf are distinct, these findings indicate that each structural modification differentially affects the regulation and/or propagation of the Tor signal by these mutant D-raf proteins.