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PDF file - 42K, Quizartinib inhibition of MV4-11 tumor grwoth in vivo

PDF file - 279K, Immunoblots of MV4-11, MOLM14, and SEM-K2 with quizartinib DR

PDF file - 116K, Elevation of pro-apoptotic BimEL mirrors loss of anti-apoptotic Mcl-1

PDF file - 46K, Durability of quizartinib inhibition translates to loss of viability after brief exposure

PDF file - 54K, DMSO and DR to quizartinib, percent apoptotic cells is percent of cells with

PDF file - 74K, CEP-32496 Supplemental Data.

PDF file - 54K, Inhibition of FLT3 phosphorylation and cell viability by FLT3 inhibitors

Statechart notations with ‘run to completion’ semantics, are popular with engineers for designing controllers that respond to events in the environment with a sequence of state transitions. However, they lack formal refinement and rigorous verification methods. Open image in new window , on the other hand, is based on refinement from an initial abstraction and is designed to make formal verification by automatic theorem provers feasible. We introduce a notion of refinement into a ‘run to completion’ statechart modelling notation, and leverage Open image in new window tool support for theorem proving. We describe the difficulties in translating ‘run to completion’ semantics into Open image in new window refinements and suggest a solution. We outline how safety and liveness properties could be verified.

Benefits of local recovery (restarting only a failed process or task) have been previously demonstrated in parallel solvers. Local recovery has a reduced impact on application performance due to masking of failure delays (for message-passing codes) or dynamic load balancing (for asynchronous many-task codes). In this paper, we implement MPI-process-local checkpointing and recovery of data (as an extension of the Fenix library) in combination with an existing method for local detection of silent errors in partial-differential-equation solvers, to show a path for incorporating lightweight silent-error resilience. In addition, we demonstrate how asynchrony introduced by maximizing computation-communication overlap can halt the propagation of delays. For a prototype stencil solver (including an iterative-solver-like variant) with injected memory bit flips, results show greatly reduced overhead under weak scaling compared to global recovery, and high failure-masking efficiency. The approach is expected to be generalizable to other MPI-based solvers.

The aggregation of attractive colloids has been extensively studied from both theoretical and experimental perspectives as the fraction of solid particles is changed, and the range, type, and strength of attractive or repulsive forces between particles varies. The resulting gels, consisting of disordered assemblies of attractive colloidal particles, have also been investigated with regards to percolation, phase separation, and the mechanical characteristics of the resulting fractal networks. Despite tremendous progress in our understanding of the gelation process, and the exploration of different routes for arresting the dynamics of attractive colloids, the complex interplay between convective transport processes and many-body effects in such systems has limited our ability to drive the system toward a specific configuration. Here, we study a model attractive colloidal system over a wide range of particle characteristics and flow conditions undergoing aggregation far from equilibrium. The complex multiscale dynamics of the system can be understood using a time-rate-transformation diagram adapted from understanding of materials processing in block copolymers, supercooled liquids, and much stiffer glassy metals to direct targeted assembly of attractive colloidal particles. Keywords: Colloidal gels, Directed assembly, Rheology, Attractive colloids, Targeted-design of colloids

Although popular in industry, state-chart notations with ‘run to completion’ semantics lack formal refinement and rigorous verification methods. State-chart models are typically used to design complex control systems that respond to environmental triggers with a sequential process. The model is usually constructed at a concrete level and verified and validated using animation techniques relying on human judgement. Event-B, on the other hand, is based on refinement from an initial abstraction and is designed to make formal verification by automatic theorem provers feasible. We introduce a notion of refinement into a ‘run to completion’ statechart modelling notation, and leverage Event-B ’s tool support for theorem proving. We describe the difficulties in translating ‘run to completion’ semantics into Event-B refinements and suggest a solution. We illustrate our approach and show how critical (e.g. safety) invariant properties can be verified by proof despite the reactive nature of the system. We also show how behavioural aspects of the system can be verified by testing the expected reactions using a temporal logic model checking approach.

We discuss techniques for efficient local detection of silent data corruption in parallel scientific computations, leveraging physical quantities such as momentum and energy that may be conserved by discretized PDEs. The conserved quantities are analogous to “algorithm-based fault tolerance” checksums for linear algebra but, due to their physical foundation, are applicable to both linear and nonlinear equations and have efficient local updates based on fluxes between subdomains. These physics-based checksums enable precise intermittent detection of errors and recovery by rollback to a checkpoint, with very low overhead when errors are rare. We present applications to both explicit hyperbolic and iterative elliptic (unstructured finite-element) solvers with injected memory bit flips.

Colloids with short range attractions self-assemble into sample-spanning structures, whose dynamic nature results in a thermokinematic memory of the deformation history, also referred to as "thixotropy." Here, we study the origins of the thixotropic effect in these time- and rate-dependent materials by investigating hysteresis across different length scales: from particle-level local measurements of coordination number (microscale), to the appearance of density and velocity fluctuations (mesoscale), and up to the shear stress response to an imposed deformation (macroscale). The characteristic time constants at each scale become progressively shorter, and hysteretic effects become more significant as we increase the strength of the interparticle attraction. There are also strong correlations between the thixotropic effects we observe at each scale.

We present a general technique to solve Partial Differential Equations, called robust stencils, which make them tolerant to soft faults, i.e. bit flips arising in memory or CPU calculations. We show how it can be applied to a two-dimensional Lax-Wendroff solver. The resulting 2D robust stencils are derived using an orthogonal application of their 1D counterparts. Combinations of 3 to 5 base stencils can then be created. We describe how these are then implemented in a parallel advection solver. Various robust stencil combinations are explored, representing tradeoff between performance and robustness. The results indicate that the 3-stencil robust combinations are slightly faster on large parallel workloads than Triple Modular Redundancy (TMR). They also have one third of the memory footprint. We expect the improvement to be significant if suitable optimizations are performed. Because faults are avoided each time new points are computed, the proposed stencils are also comparably robust to faults as TMR for a large range of error rates. The technique can be generalized to 3D (or higher dimensions) with similar benefits.

This work outlines an equation-based formulation of a digital control program and transducer interacting with a continuous physical process, and an approach using the Coq theorem prover for verifying the performance of the combined hybrid system. Considering thermal dynamics with linear dissipation for simplicity, we focus on a generalizable, physically consistent description of the interaction of the real-valued temperature and the digital program acting as a thermostat. Of interest in this work is the discovery and formal proof of bounds on the temperature, the degree of variation, and other performance characteristics. Our approach explicitly addresses the need to mathematically represent the decision problem inherent in an analog-to-digital converter, which for rare values can take an arbitrarily long time to produce a digital answer (the so-called Buridan's Principle); this constraint ineluctably manifests itself in the verification of thermostat performance. Furthermore, the temporal causality constraints in the thermal physics must be made explicit to obtain a consistent model for analysis. We discuss the significance of these findings toward the verification of digital control for more complex physical variables and fields.

Modern digital hardware and software designs are increasingly complex but are themselves only idealizations of a real system that is instantiated in, and interacts with, an analog physical environment. Insights from physics, formal methods, and complex systems theory can aid in extending reliability and security measures from pure digital computation (itself a challenging problem) to the broader cyber-physical and out-of-nominal arena. Example applications to design and analysis of high-consequence controllers and extreme-scale scientific computing illustrate the interplay of physics and computation. In particular, we discuss the limitations of digital models in an analog world, the modeling and verification of out-of-nominal logic, and the resilience of computational physics simulation. A common theme is that robustness to failures and attacks is fostered by cyber-physical system designs that are constrained to possess inherent stability or smoothness. This chapter contains excerpts from previous publications by the authors.

Verifying that hardware design implementations adhere to specifications is a time intensive and sometimes intractable problem due to the massive size of the system's state space. Formal methods techniques can be used to prove certain tractable specification properties; however, they are expensive, and often require subject matter experts to develop and solve. Nonetheless, hardware verification is a critical process to ensure security and safety properties are met, and encapsulates problems associated with trust and reliability. For complex designs where coverage of the entire state space is unattainable, prioritizing regions most vulnerable to security or reliability threats would allow efficient allocation of valuable verification resources. Stackelberg security games model interactions between a defender, whose goal is to assign resources to protect a set of targets, and an attacker, who aims to inflict maximum damage on the targets after first observing the defender's strategy. In equilibrium, the defender has an optimal security deployment strategy, given the attacker's best response. We apply this Stackelberg security framework to synthesized hardware implementations using the design's network structure and logic to inform defender valuations and verification costs. The defender's strategy in equilibrium is thus interpreted as a prioritization of the allocation of verification resources in the presence of an adversary. We demonstrate this technique on several open-source synthesized hardware designs.

Fms-like tyrosine kinase 3 (FLT3) is implicated in the pathogenesis of acute myeloid leukemia (AML). FLT3-activating internal tandem duplication (ITD) mutations are found in approximately 30% of patients with AML and are associated with poor outcome in this patient population. Quizartinib (AC220) has previously been shown to be a potent and selective FLT3 inhibitor. In the current study, we expand on previous observations by showing that quizartinib potently inhibits the phosphorylation of FLT3 and downstream signaling molecules independent of FLT3 genotype, yet induces loss of viability only in cells expressing constitutively activated FLT3. We further show that transient exposure to quizartinib, whether in vitro or in vivo, leads to prolonged inhibition of FLT3 signaling, induction of apoptosis, and drastic reductions in tumor volume and pharmacodynamic endpoints. In vitro experiments suggest that these prolonged effects are mediated by slow binding kinetics that provide for durable inhibition of the kinase following drug removal/clearance. Together these data suggest quizartinib, with its unique combination of selectivity and potent/sustained inhibition of FLT3, may provide a safe and effective treatment against FLT3-driven leukemia. Mol Cancer Ther; 12(4); 438–47. ©2013 AACR.

We identify the sequence of microstructural changes that characterize the evolution of an attractive particulate gel under flow and discuss their implications on macroscopic rheology. Dissipative Particle Dynamics (DPD) is used to monitor shear-driven evolution of a fabric tensor constructed from the ensemble spatial configuration of individual attractive constituents within the gel. By decomposing this tensor into isotropic and non-isotropic components we show that the average coordination number correlates directly with the flow curve of the shear stress vs. shear rate, consistent with theoretical predictions for attractive systems. We show that the evolution in non-isotropic local particle rearrangements are primarily responsible for stress overshoots (strain-hardening) at the inception of steady shear flow and also lead, at larger times and longer scales, to microstructural localization phenomena such as shear banding flow-induced structure formation in the vorticity direction., 15 pages, 4 figures

We present algorithmic techniques for parallel PDE solvers that leverage numerical smoothness properties of physics simulation to detect and correct silent data corruption within local computations. We initially model such silent hardware errors (which are of concern for extreme scale) via injected DRAM bit flips. Our mitigation approach generalizes previously developed "robust stencils" and uses modified linear algebra operations that spatially interpolate to replace large outlier values. Prototype implementations for 1D hyperbolic and 3D elliptic solvers, tested on up to 2048 cores, show that this error mitigation enables tolerating orders of magnitude higher bit-flip rates. The runtime overhead of the approach generally decreases with greater solver scale and complexity, becoming no more than a few percent in some cases. A key advantage is that silent data corruption can be handled transparently with data in cache, reducing the cost of false-positive detections compared to rollback approaches.

Traditional parallel programming techniques will suffer rapid deterioration of performance scaling with growing platform size, as the work of coping with increasingly frequent failures dominates over useful computation. To address this challenge, we introduce and simulate a novel software architecture that combines a task dependency graph with a substitution graph. The role of the dependency graph is to limit communication and checkpointing and enhance fault tolerance by allowing graph neighbors to exchange data, while the substitution graph promotes fault oblivious computing by allowing a failed task to be substituted on-the-fly by another task, incurring a quantifiable error. We present optimization formulations for trading off substitution errors and other factors such as available system capacity and low-overlap task partitioning among processors, and demonstrate that these can be approximately solved in real time after some simplifications. Simulation studies of our proposed approach indicate that a substitution network adds considerable resilience and simple enhancements can limit the aggregate substitution errors.

SummaryInteractions between kinases and small molecule inhibitors can be activation state dependent. A detailed understanding of inhibitor binding therefore requires characterizing interactions across multiple activation states. We have systematically explored the effects of ABL1 activation loop phosphorylation and PDGFR family autoinhibitory juxtamembrane domain docking on inhibitor binding affinity. For a diverse compound set, the affinity patterns correctly classify inhibitors as having type I or type II binding modes, and we show that juxtamembrane domain docking can have dramatic negative effects on inhibitor affinity. The results have allowed us to associate ligand-induced conformational changes observed in cocrystal structures with specific energetic costs. The approach we describe enables investigation of the complex relationship between kinase activation state and compound binding affinity and should facilitate strategic inhibitor design.

Activating mutations in the receptor tyrosine kinase FLT3 are present in up to approximately 30% of acute myeloid leukemia (AML) patients, implicating FLT3 as a driver of the disease and therefore as a target for therapy. We report the characterization of AC220, a second-generation FLT3 inhibitor, and a comparison of AC220 with the first-generation FLT3 inhibitors CEP-701, MLN-518, PKC-412, sorafenib, and sunitinib. AC220 exhibits low nanomolar potency in biochemical and cellular assays and exceptional kinase selectivity, and in animal models is efficacious at doses as low as 1 mg/kg given orally once daily. The data reveal that the combination of excellent potency, selectivity, and pharmacokinetic properties is unique to AC220, which therefore is the first drug candidate with a profile that matches the characteristics desirable for a clinical FLT3 inhibitor.

In high-performance scientific software development, the emphasis is often on short time to first solution. Even when the development of new components mostly reuses existing components or libraries and only small amounts of new code must be created, dealing with the component glue code and software build processes to obtain complete applications is still tedious and error-prone. Component-based software meant to reduce complexity at the application level increases complexity to the extent that the user must learn and remember the interfaces and conventions of the component model itself. To address these needs, we introduce Bocca, the first tool to enable application developers to perform rapid component prototyping while maintaining robust software-engineering practices suitable to HPC environments. Bocca provides project management and a comprehensive build environment for creating and managing applications composed of Common Component Architecture components. Of critical importance for high-performance computing (HPC) applications, Bocca is designed to operate in a language-agnostic way, simultaneously handling components written in any of the languages commonly used in scientific applications: C, C++, Fortran, Python and Java. Bocca automates the tasks related to the component glue code, freeing the user to focus on the scientific aspects of the application. Bocca embraces the philosophy pioneered by Ruby on Rails for web applications: start with something that works, and evolve it to the user's purpose.

Digital systems in an out-of-nominal environment (e.g., one causing hardware bit flips) may not be expected to function correctly in all respects but may be required to fail safely. We present an approach for understanding and verifying a system’s out-of-nominal behavior as an abstraction of nominal behavior that preserves designated critical safety requirements. Because abstraction and refinement are already widely used for improved tractability in formal design and proof techniques, this additional way of viewing an abstraction can potentially verify a system’s out-of-nominal safety with little additional work. We illustrate the approach with a simple model of a turnstile controller with possible logic faults (formalized in the temporal logic of actions and NuSMV), noting how design choices can be guided by the desired out-of-nominal abstraction. Principles of robustness in complex systems (specifically, Boolean networks) are found to be compatible with the formal abstraction approach. This work indicates a direction for broader use of formal methods in safety-critical systems.

Current programming languages and programming models make it easy to create software and hardware systems that fulfill an intended function but also leave such systems open to unintended function and vulnerabilities. Software engineering and code hygiene may make systems incrementally safer, but do not produce the wholesale change necessary for secure systems from the outset. Yet there exists an approach with impressive results: We cite recent examples showing that formal methods, coupled with formally informed digital design, have produced objectively more robust code even beyond the properties directly proven. Though discovery of zero-day vulnerabilities is almost always a surprise and powerful tools like semantic fuzzers can cover a larger search space of vulnerabilities than a developer can conceive of, formal models seem to produce robustness of a higher qualitative order than traditionally developed digital systems. Because the claim is necessarily a qualitative one, we illustrate similar results with an idealized programming language in the form of Boolean networks where we have control of parameters related to stability and adaptability. We argue that verifiability with formal methods is an instance of broader design constraints that promote robustness. We draw analogies to real-world programming models and languages that can be mathematically reasoned about in contrast to ones that are essentially undecidable.

The Common Component Architecture (CCA) provides a means for software developers to manage the complexity of large-scale scientific simulations and to move toward a plug-and-play environment for high-performance coputing. In the scientific computing context, component models also promote collaboration using independently developed software, thereby allowing particular individals or groups to focus on the aspects of greatest interest to them. The CCA supports parallel and distributed coputing as well as local high-performance connections between components in a language-independent manner. The design places minimal requirements on components and thus facilitates the integration of existing code into the CCA environment. The CCA model imposes minimal ovehead to minimize the impact on application performance. The focus on high performance distinguishes the CCA from most other component models. The CCA is being applied within an increasing range of disciplines, including cobustion research, global climate simulation, and computtional chemistry.

Proteins in the Bcl-2 family are central regulators of programmed cell death, and members that inhibit apoptosis, such as Bcl-X(L) and Bcl-2, are overexpressed in many cancers and contribute to tumour initiation, progression and resistance to therapy. Bcl-X(L) expression correlates with chemo-resistance of tumour cell lines, and reductions in Bcl-2 increase sensitivity to anticancer drugs and enhance in vivo survival. The development of inhibitors of these proteins as potential anti-cancer therapeutics has been previously explored, but obtaining potent small-molecule inhibitors has proved difficult owing to the necessity of targeting a protein-protein interaction. Here, using nuclear magnetic resonance (NMR)-based screening, parallel synthesis and structure-based design, we have discovered ABT-737, a small-molecule inhibitor of the anti-apoptotic proteins Bcl-2, Bcl-X(L) and Bcl-w, with an affinity two to three orders of magnitude more potent than previously reported compounds. Mechanistic studies reveal that ABT-737 does not directly initiate the apoptotic process, but enhances the effects of death signals, displaying synergistic cytotoxicity with chemotherapeutics and radiation. ABT-737 exhibits single-agent-mechanism-based killing of cells from lymphoma and small-cell lung carcinoma lines, as well as primary patient-derived cells, and in animal models, ABT-737 improves survival, causes regression of established tumours, and produces cures in a high percentage of the mice.

ARC, an apoptosis suppressor limited to terminally differentiated cells, is induced in human breast cancer and confers chemo- and radiation-resistance

Recent advances in both computational hardware and multidisciplinary science have given rise to an unprecedented level of complexity in scientific simulation software. This paper describes an ongoing grass roots effort aimed at addressing complexity in high-performance computing through the use of Component-Based Software Engineering (CBSE). Highlights of the benefits and accomplishments of the Common Component Architecture (CCA) Forum and SciDAC ISIC are given, followed by an illustrative example of how the CCA has been applied to drive scientific discovery in quantum chemistry. Thrusts for future research are also described briefly.

Regulators of apoptosis are thought to work in concert, but the molecular interactions of this process are not understood. Here, we show that in response to cell death stimulation, survivin, a member of the inhibitor of apoptosis (IAP) gene family, associates with another IAP protein, XIAP, via conserved baculovirus IAP repeats. Formation of a survivin-XIAP complex promotes increased XIAP stability against ubiquitination/proteasomal destruction and synergistic inhibition of apoptosis, which is abolished in XIAP(-/-) cells. Therefore, orchestration of an IAP-IAP complex regulates apoptosis.

This study compares the predictions of several “suspension temperature” models of particle migration to laser Doppler velocimetry measurements in a concentrated suspension of noncolloidal spheres. We compare the shear rate, concentration, and suspension temperature profiles in narrow-gap Couette flow. The models predict the observed macroscopic shear rate and concentration profiles well at moderate bulk particle concentration but diverge from one another and from the data at high concentrations. In addition, the predictions of the models compare poorly with suspension temperature measurements. Most of the models greatly underpredict the magnitude of the scalar temperature, capturing instead only the magnitude of the smaller two diagonal components of the temperature tensor. Also, the models do not predict the observed variation of the suspension temperature with particle concentration. Our investigation shows that both neglect of suspension temperature anisotropy and qualitative choices of model coefficie...

High-resolution laser Doppler velocimetry and flow-induced birefringence measurements were used to determine velocity and stress fields and to calculate a transient planar elongation viscosity profile for viscoelastic flow on the shear-free centerplane of a planar contraction geometry. A novel, two-component Boger fluid was developed that enabled Weissenberg numbers, We, as high as 2.9 to be attained and allowed the stress-optical coefficients of the fluid components to be measured. The experimental apparatus was designed to subject the fluid traveling on the centerplane to Hencky strains, e, as great as 3.5. Despite the high We and e, the flow induced only a weakly nonlinear viscoelastic stress response in the fluid. A spectral decomposition of predictions of linear and nonlinear constitutive equations was used to demonstrate that the weakly nonlinear response was a result of the nonhomogeneous form of the centerplane strain-rate profile in conjunction with the finite response time of the individual relaxation modes. The degree of nonlinear stress response in this experiment cannot be increased by increasing the contraction ratio to increase the total Hencky strain experienced by a fluid element traveling along the centerplane, increasing the flow rate to increase the strain rate at a given point along the centerplane, or introducing a taper to the contraction geometry to change the strain rate profile. Thus, the experimental configuration of flow through a contraction appears to be of limited value in evaluating the accuracy of nonlinear constitutive models in predicting the stress response of a viscoelastic polymer solution to a generalized elongational flow.

Survivin is an anti-apoptotic protein that is overexpressed in most human cancers. We show that survivin forms complexes with a cellular protein, hepatitis B X-interacting protein (HBXIP), which was originally recognized for its association with the X protein of hepatitis B virus (HBX). Survivin–HBXIP complexes, but neither survivin nor HBXIP individually, bind pro-caspase-9, preventing its recruitment to Apaf1, and thereby selectively suppressing apoptosis initiated via the mitochondria/cytochrome c pathway. Viral HBX protein also interacts with the survivin– HBXIP complex and suppresses caspase activation in a survivin-dependent manner. Thus, HBXIP functions as a cofactor for survivin, and serves as a link between the cellular apoptosis machinery and a viral pathogen involved in hepatocellular carcinogenesis.

Heart failure is a common, lethal condition whose pathogenesis is poorly understood. Recent studies have identified low levels of myocyte apoptosis (80-250 myocytes per 10(5) nuclei) in failing human hearts. It remains unclear, however, whether this cell death is a coincidental finding, a protective process, or a causal component in pathogenesis. Using transgenic mice that express a conditionally active caspase exclusively in the myocardium, we demonstrate that very low levels of myocyte apoptosis (23 myocytes per 10(5) nuclei, compared with 1.5 myocytes per 10(5) nuclei in controls) are sufficient to cause a lethal, dilated cardiomyopathy. Interestingly, these levels are four- to tenfold lower than those observed in failing human hearts. Conversely, inhibition of cardiac myocyte death in this murine model largely prevents the development of cardiac dilation and contractile dysfunction, the hallmarks of heart failure. To our knowledge, these data provide the first direct evidence that myocyte apoptosis may be a causal mechanism of heart failure, and they suggest that inhibition of this cell death process may constitute the basis for novel therapies.

▪ Abstract Recent advances in the computational modeling of molecular conformational and orientational effects in the flow of viscoelastic fluids are described. These advances involve the coupling of molecular models for the underlying microstructure of macromolecules with the macroscopic equations of change. The kinetic theory for polymeric liquids is described along with the most useful micromechanical models for computing the fluid flow of polymeric liquids. Three levels of description are covered for the computation of molecular orientation effects: methods for molecular models for which closed-form, continuum-like evolution equations for average quantities describing molecular conformations can be obtained, hybrid methods that involve coupling direct solution of the Fokker-Planck equation describing the distribution function for molecular orientations with the equations of change, and hybrid methods that couple stochastic simulations of individual molecule trajectories with the macroscopic equations of change. Illustrative results for rheometric flows (flows with homogeneous, fixed kinematics) and complex flows are given.

Recent statistical constitutive models of suspensions of neutrally buoyant, noncolloidal, solid spheres in Newtonian fluids suggest that the particles migrate in response to gradients in “suspension temperature,” defined as the average kinetic energy contained in the particle velocity fluctuations. These models have not yet been compared systematically with experimental data. In addition the “temperature'’ models assume isotropic particle velocity fluctuations, since the suspension temperature is given as a scalar. However, highly anisotropic particle velocity fluctuations have been observed experimentally, which suggests that a suspension temperature tensor is more realistic. We use laser Doppler velocimetry to measure particle velocity fluctuations arising from interparticle collisions in a concentrated suspension under nearly homogeneous shear flow in a narrow-gap concentric cylinder Couette device. We compare the relative sizes of the fluctuating velocity components and determine the variation of each...

Formal methods have come into wide use because of their effectiveness in verifying %22safety and security%22 requirements of digital systems; a set of requirements for which testing is mostly ineffective. Formal methods are routinely used in the design and verification of high-consequence digital systems in industry. This report outlines our work in assessing the capabilities of commercial and open source formal tools and the ways in which they can be leveraged in digital design workflows.

We present a characterization of short-term stability of Kauffman's $NK$ (random) Boolean networks under arbitrary distributions of transfer functions. Given such a Boolean network where each transfer function is drawn from the same distribution, we present a formula that determines whether short-term chaos (damage spreading) will happen. Our main technical tool which enables the formal proof of this formula is the Fourier analysis of Boolean functions, which describes such functions as multilinear polynomials over the inputs. Numerical simulations on mixtures of threshold functions and nested canalyzing functions demonstrate the formula's correctness.

A wide variety of bead-spring kinetic theory models have been proposed to explain the stress growth and hysteretic behavior of dilute polymer solutions in uniaxial extension. We analyze the Kramers chain, a fine-scale model for polymer dynamics, in order to assess the validity of the coarser-grained bead-spring models in these deformations. Whereas the spring force is a simple function of the dumbbell length for the FENE spring, we find that the relationship between the ensemble-averaged end-to-end force and the extension for a Kramers chain depends on the kinematic history to which it has been subjected. In a quiescent fluid, the Kramers chain force–extension relationship is identical to the FENE force law. However, during start up of elongational flow, the ensemble-averaged end-to-end force for a given (end-to-end) length of the molecule increases with strain until steady state is reached. If the extensional flow is suddenly stopped, the Kramers chain force–extension relationship relaxes back to the FEN...

Septic shock is a leading cause of mortality in intensive care units. No new interventions in the last 20 years have made a substantial impact on the outcome of patients with septic shock. Identification of inhibitable pathways that mediate death in shock is an important goal. Two novel caspase inhibitors, (2-indolyl)-carbonyl-Ala-Asp-fluoromethylketone (IDN 1529) and (1-methyl-3-methyl-2-indolyl)-carbonyl-Val-Asp-fluoromethylketone (IDN 1965), were studied in a murine model of endotoxic shock. IDN 1529 prolonged survival when given before or up to 3 hr after high-dose LPS (p < 0.01) and increased by 2.2-fold the number of animals surviving longterm after a lower dose of LPS (p < 0.01). Despite its similar chemical structure, IDN 1965 lacked these protective effects. Both compounds inhibited caspases 1, 2, 3, 6, 8, and 9, and both afforded comparable reduction in Fas- and LPS-induced caspase 3-like activity and apoptosis. Paradoxically, administration of IDN 1529 but not IDN 1965 led to an increase in the LPS-induced elevation of serum cytokines related directly (IL-1β, IL-18) or indirectly (IL-1α, IL-1Ra) to the action of caspase 1. A process that appears to be distinct from both apoptosis and the release of inflammatory cytokines is a late-acting requirement for lethality in endotoxic shock. Inhibition of this process can rescue mice even when therapy is initiated after LPS has made the mice severely ill.

The mechanism by which membrane-bound Bcl-2 inhibits the activation of cytoplasmic procaspases is unknown. Here we characterize an intracellular, membrane-associated form of procaspase-3 whose activation is controlled by Bcl-2. Heavy membranes isolated from control cells contained a spontaneously activatable caspase-3 zymogen. In contrast, in Bcl-2 overexpressing cells, although the caspase-3 zymogen was still associated with heavy membranes, its spontaneous activation was blocked. However, Bcl-2 expression had little effect on the levels of cytoplasmic caspase activity in unstimulated cells. Furthermore, the membrane-associated caspase-3 differed from cytosolic caspase-3 in its responsiveness to activation by exogenous cytochrome c. Our results demonstrate that intracellular membranes can generate active caspase-3 by a Bcl-2–inhibitable mechanism, and that control of caspase activation in membranes is distinct from that observed in the cytoplasm. These data suggest that Bcl-2 may control cytoplasmic events in part by blocking the activation of membrane-associated procaspases.

Bcl-xL, an antiapoptotic member of the Bcl-2 family, inhibits programmed cell death in a broad variety of cell types. Recent reports have demonstrated that cytochrome c is released from mitochondria during apoptosis and have suggested that this release may be a critical step in the activation of proapoptotic caspases and subsequent cell death. Furthermore, it has been demonstrated that Bcl-2 can prevent the release of cytochrome c from mitochondria in cells triggered to undergo apoptosis. This has led to the hypothesis that the antiapoptotic effects of Bcl-2 family members are due specifically to their ability to prevent cytochrome c release thus preventing subsequent cytochrome c-dependent caspase activation. In the present report, we use microinjection techniques to investigate the relationship between cytochrome c release, induction of apoptosis, and Bcl-xL activity in intact cells. We demonstrate that microinjection of cytochrome c into the cytosol of human kidney 293 cells results in a dose-dependent induction of apoptosis. In contrast, MCF7 breast carcinoma cells (stably transfected to express the Fas antigen CD95, and denoted MCF7F) that lack detectable levels of caspase 3 (CPP32), are totally resistant to microinjection of cytochrome c. However, transfection of MCF7F cells with an expression plasmid coding for pro-caspase 3, but not other pro-caspases, restores cytochrome c sensitivity. Although MCF7F cells are insensitive to cytochrome c microinjection, they rapidly undergo apoptosis in a caspase-dependent manner in response to either tumor necrosis factor or anti-Fas plus cycloheximide, and these deaths are strongly inhibited by Bcl-xL expression. Furthermore, microinjection of cytochrome c does not overcome these antiapoptotic effects of Bcl-xL. Our results support the concept that the release of cytochrome c into the cytoplasm can promote the apoptotic process in cells expressing pro-caspase 3 but that cytochrome c release is not sufficient to induce death in all cells. Importantly, the ability of Bcl-xL to inhibit cell death in the cytochrome c-insensitive MCF7F cells cannot be due solely to inhibition of cytochrome c release from mitochondria.

Fas- and tumor necrosis factor receptor 1 (TNFR1)-induced apoptosis is mediated by the interaction of FADD with caspase-8. Here, we report that the bovine herpesvirus 4 (BHV4) BORFE2 gene encodes a protein that inhibits Fas- and TNFR1-induced apoptosis and contains death effector domains (DEDs). Using the yeast two-hybrid system, we found that the BORFE2 protein interacts with the prodomain of caspase-8. Furthermore, we show that BHV4 BORFE2 is a member of a family of DED-containing proteins that includes other gamma-2 herpesviruses, such as Kaposi's sarcoma-associated herpesvirus and herpesvirus saimiri.

We identified and cloned a novel human protein that contains FADD/Mort1 death effector domain homology regions, designated FLAME-1. FLAME-1, although most similar in structure to Mch4 and Mch5, does not possess caspase activity but can interact specifically with FADD, Mch4, and Mch5. Interestingly, FLAME-1 is recruited to the Fas receptor complex and can abrogate Fas/TNFR-induced apoptosis upon expression in FasL/tumor necrosis factor-sensitive MCF-7 cells, possibly by acting as a dominant-negative inhibitor. These findings identify a novel endogenous control point that regulates Fas/TNFR1-mediated apoptosis.