Toward verified biological models

The last several decades have witnessed a vast accumulation of biological data and data analysis. Many of these data sets represent only a small fraction of the system's behavior, making the visualization of full system behavior difficult. A more complete understanding of a biological system is gained when different types of data (and/or conclusions drawn from the data) are integrated into a larger-scale representation or model of the system. Ideally, this type of model is consistent with all available data about the system, and it is then used to generate additional hypotheses to be tested. Computer-based methods intended to formulate models that integrate various events and to test the consistency of these models with respect to the laboratory-based observations on which they are based are potentially very useful. In addition, in contrast to informal models, the consistency of such formal computer-based models with laboratory data can be tested rigorously by methods of formal verification. We combined two formal modeling approaches in computer science that were originally developed for non-biological system design. One is the inter-object approach using the language of live sequence charts (LSCs) with the Play-Engine tool, and the other is the intra-object approach using the language of statecharts and Rhapsody as the tool. Integration is carried out using InterPlay, a simulation engine coordinator. Using these tools, we constructed a combined model comprising three modules. One module represents the early lineage of the somatic gonad of C. elegans in LSCs, while a second more detailed module in statecharts represents an interaction between two cells within this lineage that determine their developmental outcome. Using the advantages of the tools, we created a third module representing a set of key experimental data using LSCs. We tested the combined statechart-LSC model by showing that the simulations were consistent with the set of experimental LSCs. This small-scale modular example demonstrates the potential for using similar approaches for verification by exhaustive testing of models by LSCs. It also shows the advantages of these approaches for modeling biology.     

Discovery of a novel class of 2-ureido thiophene carboxamide checkpoint kinase inhibitors

Checkpoint kinase-1 (Chk1, CHEK1) is a Ser/Thr protein kinase that mediates the cellular response to DNA-damage. A novel class of 2-ureido thiophene carboxamide urea (TCU) Chk1 inhibitors is described. Inhibitors in this chemotype were optimized for cellular potency and selectivity over Cdk1.     

Barbiturate euthanasia solution-induced tissue artifact in nonhuman primates

BACKGROUND: Barbiturate euthanasia solutions are a humane and approved means of euthanasia. Overdosing causes significant tissue damage in a variety of laboratory animals. METHODS: One hundred seventeen non-human primates (NHP) representing 7 species including 12 fetuses euthanized for humane and research reasons by various vascular routes with Euthasol, Sodium Pentobarbital, Fatal Plus, Beuthanasia D, or Euthanasia 5 were evaluated for euthanasia-induced tissue damage. Lungs and livers were histologically graded for hemolysis, vascular damage, edema, and necrosis. Severity of tissue damage was analyzed for differences on the basis of agent, age, sex, dose, and injection route. RESULTS: Severity of tissue damage was directly related to dose and the intracardiac injection route, but did not differ by species, sex, and agent used. CONCLUSIONS: When the recommended dose of agent was used, tissue damage was generally reduced, minimal, or undetectable. Barbiturate-induced artifacts in NHPs are essentially the same as in other laboratory species.     

Patch exploitation in two dimensions: from Daphnia to simulated foragers

We explore the variability that animals display in their movement choices as they forage in a finite-sized food patch with a uniform food distribution, and present a framework for how these choices may be adjusted to optimize foraging efficiency. Inspired by experimental studies of the zooplankton Daphnia, we model foraging animals as “agents” moving in two dimensions in repeated and successive sequences of hops, pauses, and turns. For Daphnia and other species, critical movement parameters such as hop lengths, pause times, and turning angles are typically reported as probability density functions. Similarly, the agents in our simulations choose their movement parameters at random from such distributions. Each distribution is defined by a characteristic width, which we interpret as a “noise width,” available to be tuned for increased foraging efficiency. We investigate the sensitivity of the system by measuring the food gathered by the agents as the turning angle and hop length noise widths are varied. In all cases, we find a maximum in food gathered at some particular value of the noise width in question, suggesting that these results can be considered robust examples of natural stochastic resonance.     

SopD2 is a novel type III secreted effector of Salmonella typhimurium that targets late endocytic compartments upon delivery into host cells

Salmonella typhimurium is a facultative intracellular pathogen that utilizes two type III secretion systems to deliver virulence proteins into host cells. These proteins, termed effectors, alter host cell function to allow invasion into and intracellular survival/replication within a vacuolar compartment. Here we describe SopD2, a novel member of the Salmonella translocated effector (STE) family, which share a conserved N-terminal type III secretion signal. Disruption of the sopD2 gene prolonged the survival of mice infected with a lethal dose of Salmonella typhimurium , demonstrating a significant role for this effector in pathogenesis. Expression of sopD2 was induced inside host cells and was dependent on functional ssrA/B and phoP/Q, two component regulatory systems. HA-tagged SopD2 was delivered into HeLa cells in a SPI-2-dependent manner and associated with both the Salmonella -containing vacuole and with swollen endosomes elsewhere in the cell. Subcellular fractionation confirmed that SopD2 was membrane associated in host cells, while the closely related effector SopD was localized to the cytosol. A SopD2 fusion to GFP associated with small tubular structures and large vesicles containing late endocytic markers, including Rab7. Surprisingly, expression of N-terminal amino acids 1–150 of SopD2 fused to GFP was sufficient to mediate both binding to late endosomes/lysosomes and swelling of these compartments. These findings demonstrate that the N-terminus of SopD2 is a bifunctional domain required for both type III secretion out of Salmonella as well as late endosome/lysosome targeting following translocation into host cells .     

Human uterine mast cells. Isolation, purification, characterization, ultrastructure, and pharmacology.

As part of an ongoing investigation of human mast cell heterogeneity, we have isolated, partially purified, and characterized the uterine mast cell and compared it with mast cells isolated from other organs. The average histamine content of myometrium and leiomyofibroma obtained from hysterectomies was 2.1 +/- 0.3 (mean +/- SEM) microgram/g of tissue (n = 10), and the histamine content of the two tissues did not differ significantly. A mild collagenase, hyaluronidase, and DNase digestion was used to disperse the uterine mast cells, with an average yield of 9.5% (range, 0 to 21%). The average histamine/uterine mast cell was 2.1 +/- 0.2 pg (n = 3), and 61 +/- 7% (n= 3) of the uterine mast cells survived overnight culture. Early purification efforts with Percoll gradients have yielded up to 80% pure uterine mast cells, with an average of 27 +/- 10% (n = 5). Uterine mast cells released histamine in response to the secretogogues anti-IgE and A23187 but did not respond to substance P or to the basophil secretogogues FMLP, C5a, and 12-O-tetradecanoylphorbol-13-acetate. After 1 microgram/ml anti-IgE stimulation, the uterine mast cell appeared to make significant quantities of PGD2 (89 +/- 26 ng/10(6) cells, n = 6) (p less than 0.05), as assayed by RIA. Simultaneously, leukotriene C4 release was 45 +/- 15 ng/10(6) cells, (n = 6) (p less than 0.05), as assayed by RIA. Combined gas-chromatography mass spectroscopy analysis of anti-IgE-stimulated cell supernatants confirmed the production of PGD2. In pharmacologic studies, isobutyl-methylxanthine and isoproterenol blocked anti-IgE-induced histamine release. The uterine mast cell is similar to the lung mast cell in terms of response to secretogogues and release of arachidonic acid metabolites. Ultrastructurally, the uterine mast cell contains scroll granules, crystal granules, combined granules, homogeneously dense granules, and large lipid bodies, many with focal lucencies within them. Particle granules, most frequently present in gut mast cells of mucosal origin, were absent from uterine mast cells. Although certain features are analogous to the ultrastructure of skin or lung mast cells, the combination of structures is distinctive for uterine mast cells.     

Mass spectrometry-based approaches to protein-ligand interactions

One of the greatest current challenges in proteomics is to develop an understanding of cellular communication and regulation processes, most of which involve noncovalent interactions of proteins with various binding partners. Mass spectrometry plays an important role in all aspects of these research efforts. This article provides a survey of mass spectrometry-based approaches for exploring protein-ligand interactions. A wide array of techniques is available, and the choice of method depends on the specific problem at hand. For example, the high-throughput screening of compound libraries for binding to a specific receptor requires different approaches than structural studies on multiprotein complexes. This review is directed to readers wishing to obtain a concise yet comprehensive overview of existing experimental techniques. Specific emphasis is placed on emerging methods that have been developed within the last few years.