Decoupling the Lookaside Buffer From IPv7 in Voice-over-IP

The deployment of semaphores is an unproven riddle. After years of extensive research into the lookaside buffer, we verify the improvement of courseware, which embodies the significant principles of noisy software engineering. RoralSizel, our new system for the exploration of virtual machines, is the solution to all of these issues [1]

Hypoxia leads to significant changes in alternative splicing and elevated expression of CLK splice factor kinases in PC3 prostate cancer cells

© 2018 The Author(s). Background: Mounting evidence suggests that one of the ways that cells adapt to hypoxia is through alternative splicing. The aim of this study was firstly to examine the effect of hypoxia on the alternative splicing of cancer associated genes using the prostate cancer cell line PC3 as a model. Secondly, the effect of hypoxia on the expression of several regulators of splicing was examined. Methods: PC3 cells were grown in 1% oxygen in a hypoxic chamber for 48 h, RNA extracted and sent for high throughput PCR analysis at the RNomics platform at the University of Sherbrooke, Canada. Genes whose exon inclusion rate PSI (ψ) changed significantly were identified, and their altered exon inclusion rates verified by RT-PCR in three cell lines. The expression of splice factors and splice factor kinases in response to hypoxia was examined by qPCR and western blotting. The splice factor kinase CLK1 was inhibited with the benzothiazole TG003. Results: In PC3 cells the exon inclusion rate PSI (ψ) was seen to change by >25% in 12 cancer-associated genes; MBP, APAF1, PUF60, SYNE2, CDC42BPA, FGFR10P, BTN2A2, UTRN, RAP1GDS1, PTPN13, TTC23 and CASP9 (caspase 9). The expression of the splice factors SRSF1, SRSF2, SRSF3, SAM68, HuR, hnRNPA1, and of the splice factor kinases SRPK1 and CLK1 increased significantly in hypoxia. We also observed that the splice factor kinase CLK3, but not CLK2 and CLK4, was also induced in hypoxic DU145 prostate, HT29 colon and MCF7 breast cancer cell lines. Lastly, we show that the inhibition of CLK1 in PC3 cells with the benzothiazole TG003 increased expression of the anti-apoptotic isoform caspase 9b. Conclusions: Significant changes in alternative splicing of cancer associated genes occur in prostate cancer cells in hypoxic conditions. The expression of several splice factors and splice factor kinases increases during hypoxia, in particular the Cdc-like splice factor kinases CLK1 and CLK3. We suggest that in hypoxia the elevated expression of these regulators of splicing helps cells adapt through alternative splicing of key cancer-associated genes. We suggest that the CLK splice factor kinases could be targeted in cancers in which hypoxia contributes to resistance to therapy

The Linked Data Benchmark Council (LDBC): Driving competition and collaboration in the graph data management space

Graph data management is instrumental for several use cases such as recommendation, root cause analysis, financial fraud detection, and enterprise knowledge representation. Efficiently supporting these use cases yields a number of unique requirements, including the need for a concise query language and graph-aware query optimization techniques. The goal of the Linked Data Benchmark Council (LDBC) is to design a set of standard benchmarks that capture representative categories of graph data management problems, making the performance of systems comparable and facilitating competition among vendors. LDBC also conducts research on graph schemas and graph query languages. This paper introduces the LDBC organization and its work over the last decade

Reduced P53 levels ameliorate neuromuscular junction loss without affecting motor neuron pathology in a mouse model of spinal muscular atrophy

Spinal Muscular Atrophy (SMA) is a childhood motor neuron disease caused by mutations or deletions within the SMN1 gene. At endstages of disease there is profound loss of motor neurons, loss of axons within ventral roots and defects at the neuromuscular junctions (NMJ), as evidenced by pathological features such as pre-synaptic loss and swelling and post-synaptic shrinkage. Although these motor unit defects have been widely described, the time course and interdependancy of these aspects of motor unit degeneration are unclear. Recent reports have also revealed an early upregulation of transcripts associated with the P53 signalling pathway. The relationship between the upregulation of these transcripts and pathology within the motor unit is also unclear. In this study, we exploit the prolonged disease timecourse and defined pre-symptomatic period in the Smn mouse model to perform a temporal analysis of the different elements of motor unit pathology. We demonstrate that NMJ loss occurs prior to cell body loss, and coincides with the onset of symptoms. The onset of NMJ pathology also coincides with an increase in P53-related transcripts at the cell body. Finally, using a tamoxifen inducible P53 knockout, we demonstrate that post-natal reduction in P53 levels can reduce NMJ loss, but does not affect other aspects of NMJ pathology, motor neuron loss or the phenotype of the Smn mouse model. Together this work provides a detailed temporal description of pathology within motor units of an SMA mouse model, and demonstrates that NMJ loss is a P53-dependant process. This work supports the role for P53 as an effector of synaptic and axonal degeneration in a die-back neuropathy

Stress-induced alternative splice forms of MDM2 and MDMX modulate the p53-pathway in distinct ways.

MDM2 and MDMX are the chief negative regulators of the tumor-suppressor protein p53 and are essential for maintaining homeostasis within the cell. In response to genotoxic stress and also in several cancer types, MDM2 and MDMX are alternatively spliced. The splice variants MDM2-ALT1 and MDMX-ALT2 lack the p53-binding domain and are incapable of negatively regulating p53. However, they retain the RING domain that facilitates dimerization of the full-length MDM proteins. Concordantly, MDM2-ALT1 has been shown to lead to the stabilization of p53 through its interaction with and inactivation of full-length MDM2. The impact of MDM2-ALT1 expression on the p53 pathway and the nature of its interaction with MDMX remain unclear. Also, the role of the architecturally similar MDMX-ALT2 and its influence of the MDM2-MDMX-p53 axis are yet to be elucidated. We show here that MDM2-ALT1 is capable of binding full-length MDMX as well as full-length MDM2. Additionally, we demonstrate that MDMX-ALT2 is able to dimerize with both full-length MDMX and MDM2 and that the expression of MDM2-ALT1 and MDMX-ALT2 leads to the upregulation of p53 protein, and also of its downstream target p21. Moreover, MDM2-ALT1 expression causes cell cycle arrest in the G1 phase in a p53 and p21 dependent manner, which is consistent with the increased levels of p21. Finally we present evidence that MDM2-ALT1 and MDMX-ALT2 expression can activate subtly distinct subsets of p53-transcriptional targets implying that these splice variants can modulate the p53 tumor suppressor pathway in unique ways. In summary, our study shows that the stress-inducible alternative splice forms MDM2-ALT1 and MDMX-ALT2 are important modifiers of the p53 pathway and present a potential mechanism to tailor the p53-mediated cellular stress response

MDM2-ALT1 and MDMX-ALT2 expression causes upregulation of p53 and its downstream target p21.

<p><b>A.</b> The over-expression (O/E cell lysates) of the myc-tagged LacZ, MDM2-ALT1 or MDMX-ALT2 was confirmed using anti-myc tag antibody in the MCF7 cells that were transfected with the corresponding expression constructs (top panel). The level of p53 protein was examined in these samples using the anti-p53 antibody and an upregulation of p53 protein was observed upon MDM2-ALT1 or MDMX-ALT2 over-expression compared to LacZ expressing cells although the increase is more modest in MDMX-ALT2 over-expression (lanes 2 and 3 compared to lane 1). The positive control, UVC (50 J/m²) irradiated MCF7 cells show a strong upregulation of p53 protein levels in response to the stress when compared to untreated cells (lanes 4 and 5). β-actin was used as loading control. A minimum of three independent experiments was performed and representative gel images are shown. <b>B. </b><i>p21</i> expression at the mRNA level was examined using quantitative real-time PCR and <i>GAPDH</i> levels were used as the endogenous control. The ratio of <i>p21</i> to <i>GAPDH</i> is represented graphically and the error bars represent standard deviations from at least 3 independent experiments. MCF7 cells over-expressing MDM2-ALT1 (2Alt1) show statistically significant increase in <i>p21</i> transcript levels compared to LacZ expressing cells (p<0.01). The cells expressing MDMX-ALT2 (XAlt2) did not show statistically significant changes in <i>p21</i> expression at the mRNA level. <b>C.</b> The levels of p21 protein in the MCF7 cells transfected with myc-tagged LacZ, MDM2-ALT1 or MDMX-ALT2 was examined using anti-p21 antibody. Both MDM2-ALT1 and MDMX-ALT2 over-expression lead to upregulation of p21 protein levels compared to LacZ over-expression (compare lanes 2 and 3 with lane 1). A minimum of three independent experiments was performed and consistent results observed. Representative images are shown here. Additionally, UVC-irradiated MCF7 cells were used as positive control and show an upregulation of p21 compared to untreated cells (lanes 4 and 5).</p

MDM2-ALT1 and MDMX-ALT2 interact with full-length MDM2 and MDMX.

<p>A. Full-length MDM2 is encoded by exons 3 to 12 of the <i>MDM2</i> gene and consists of the N-terminal p53-binding domain, the nuclear localization (NLS) and export signals (NES), the central ARF binding and Zinc finger domains and the C-terminal RING domain. <i>MDM2-ALT1</i> comprises only exons 3 and 12 spliced together and the protein lacks the p53-binding domain. However, it retains the RING domain. <b>B.</b> Full-length MDMX, a close family member of MDM2, also comprises an N-terminal p53-binding domain, a central Zinc finger domain and a C-terminal RING domain and is encoded by exons 2 to 11 of the <i>MDMX</i> gene. <i>MDMX-ALT2</i> consists of exons 2,3,10 and 11 and the protein is architecturally similar to MDM2-ALT1 in that it lacks the p53-binding domain but retains the RING domain. <b>C.</b> Myc-tagged constructs of LacZ, MDM2-ALT1 or MDMX-ALT2 were transfected into MCF7 cells. Immunoprecipitation of the myc-tagged proteins revealed the specific binding of full-length MDM2 to MDM2-ALT1 and MDMX-ALT2 and not to negative control protein myc-LacZ (compare lanes 2 and 3 to lane 1). Experiments were repeated a minimum of three times and consistent results were observed. Representative gel images are presented in the figure. <b>D.</b> Myc-tagged MDM2-ALT1 and MDMX-ALT2 co-immunoprecipitate with full-length MDMX while the negative control protein myc-LacZ does not interact with MDMX (compare lanes 2 and 3 to lane 1). These results were observed in two independent trials and representative images are shown.</p