Importance of a C-Terminal Conserved Region of Chk1 for Checkpoint Function

BACKGROUND: The protein kinase Chk1 is an essential component of the DNA damage checkpoint pathway. Chk1 is phosphorylated and activated in the fission yeast Schizosaccharomyces pombe when cells are exposed to agents that damage DNA. Phosphorylation, kinase activation, and nuclear accumulation are events critical to the ability of Chk1 to induce a transient delay in cell cycle progression. The catalytic domain of Chk1 is well-conserved amongst all species, while there are only a few regions of homology within the C-terminus. A potential pseudosubstrate domain exists in the C-terminus of S. pombe Chk1, raising the possibility that the C-terminus acts to inhibit the catalytic domain through interaction of this domain with the substrate binding site. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate this hypothesis, we characterized mutations in the pseudosubstrate region. Mutation of a conserved aspartic acid at position 469 to alanine or glycine compromises Chk1 function when the mutants are integrated as single copies, demonstrating that this domain of Chk1 is critical for function. Our data does not support, however, the hypothesis that the domain acts to inhibit Chk1 function as other mutations in the amino acids predicted to comprise the pseudosubstrate do not result in constitutive activation of the protein. When expressed in multi-copy, Chk1D469A remains non-functional. In contrast, multi-copy Chk1D469G confers cell survival and imposes a checkpoint delay in response to some, though not all forms of DNA damage. CONCLUSIONS/SIGNIFICANCE: Thus, we conclude that this C-terminal region of Chk1 is important for checkpoint function and predict that a limiting factor capable of associating with Chk1D469G, but not Chk1D469A, interacts with Chk1 to elicit checkpoint activation in response to a subset of DNA lesions

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

OPAL: Smaller, Simpler, and Just Plain Luckier

On January 26, 2000 at 7:03PM PST, Stanford University’s first student built satellite, OPAL – the Orbiting Picosatellite Automated Launcher, roared into space on a modified Minuteman II missile. Students from the Space Systems Development Laboratory spent four years designing, fabricating, and testing the OPAL satellite in preparation for the launch. OPAL’s primary mission objectives were to explore a new mothership/daughtership mission architecture for distributed sensing, to characterize an off-the-shelf magnetometer, and to characterize a suite of off-the-shelf accelerometers. Six DARPA sponsored daughterships, also known as picosatellites, were deployed from OPAL. They were built by The Aerospace Corporation, Santa Clara University, and a team of amateur radio operators. The OPAL satellite completely achieved its mission goals and is now in extended mission operations. Long-term characterization of the satellite bus, magnetometer, and accelerometers are underway. OPAL has demonstrated that low cost, albeit high risk, university satellites offer an excellent platform for experimental space testbeds. AMSAT assigned OPAL an amateur satellite number of OO-38. OPAL will soon be accessible to non-Stanford users for educational and research purposes

Fast transparent migration for virtual machines

This paper describes the design and implementation of a system that uses virtual machine technology [1] to provide fast, transparent application migration. This is the first system that can migrate unmodified applications on unmodified mainstream Intel x86-based operating system, including Microsoft Windows, Linux, Novell NetWare and others. Neither the application nor any clients communicating with the application can tell that the application has been migrated. Experimental measurements show that for a variety of workloads, application downtime caused by migration is less than a second.

Pointright: experience with flexible input redirection in interactive workspaces

We describe the design of and experience with PointRight, a peerto-peer pointer and keyboard redirection system that operates in multi-machine, multi-user environments. Point-Right employs a geometric model for redirecting input across screens driven by multiple independent machines and operating systems. It was created for interactive workspaces that include large, shared displays and individual laptops, but is a general tool that supports many different configurations and modes of use. Although previous systems have provided for re-routing pointer and keyboard control, in this paper we present a more general and flexible system, along with an analysis of the types of re-binding that must be handled by any pointer redirection system This paper describes the system, the ways in which it has been used, and the lessons that have been learned from its use over the last two years