Nonlinear signaling on biological networks: The role of stochasticity and spectral clustering.

Signal transduction within biological cells is governed by networks of interacting proteins. Communication between these proteins is mediated by signaling molecules which bind to receptors and induce stochastic transitions between different conformational states. Signaling is typically a cooperative process which requires the occurrence of multiple binding events so that reaction rates have a nonlinear dependence on the amount of signaling molecule. It is this nonlinearity that endows biological signaling networks with robust switchlike properties which are critical to their biological function. In this study we investigate how the properties of these signaling systems depend on the network architecture. Our main result is that these nonlinear networks exhibit bistability where the network activity can switch between states that correspond to a low and high activity level. We show that this bistable regime emerges at a critical coupling strength that is determined by the spectral structure of the network. In particular, the set of nodes that correspond to large components of the leading eigenvector of the adjacency matrix determines the onset of bistability. Above this transition the eigenvectors of the adjacency matrix determine a hierarchy of clusters, defined by its spectral properties, which are activated sequentially with increasing network activity. We argue further that the onset of bistability occurs either continuously or discontinuously depending upon whether the leading eigenvector is localized or delocalized. Finally, we show that at low network coupling stochastic transitions to the active branch are also driven by the set of nodes that contribute more strongly to the leading eigenvector. However, at high coupling, transitions are insensitive to network structure since the network can be activated by stochastic transitions of a few nodes. Thus this work identifies important features of biological signaling networks that may underlie their biological function.

Assessing family history of chronic disease in primary care: Prevalence, documentation, and appropriate screening.

OBJECTIVE: To assess the proportion of primary care patients who report a family history (FH) of type 2 diabetes, coronary artery disease, breast cancer, or colorectal cancer (CRC); assess concordance of FH information derived from the electronic medical record (EMR) compared with patient-completed health questionnaires; and assess whether appropriate screening was informed by risk based solely on FH. DESIGN: Data from the BETTER (Building on Existing Tools to Improve Chronic Disease Prevention and Screening in Primary Care) trial were used. Patients were mailed questionnaires. Baseline FH and screening data were obtained for enrolled patients from the EMR and health questionnaires. SETTING: Ontario and Alberta. PARTICIPANTS: Randomly selected patients from 8 family practices. MAIN OUTCOME MEASURES: Agreement on FH between the EMR and questionnaire was determined; logistic regression was used to assess significant predictors of screening. RESULTS: In total, 775 of 789 (98%) patients completed the health questionnaire. The mean age of participants was 52.5 years and 72% were female. A minimum of 12% of patients (range 12% to 36%) had a reported FH of 1 of 4 chronic diseases. Among patients with positive FH, the following proportions of patients had that FH recorded in the EMR compared with the questionnaire: diabetes, 24% in the EMR versus 36% on the questionnaire, κ = 0.466; coronary artery disease, 35% in the EMR versus 22% on the questionnaire, κ = 0.225; breast cancer, 21% in the EMR versus 22% on the questionnaire, κ = 0.241; and CRC, 12% in the EMR versus 14% on the questionnaire, κ = 0.510. There was moderate agreement for diabetes and CRC. The presence of FH was a significant predictor of CRC screening (odds ratio 1.9, 95% CI 1.1 to 3.1). CONCLUSION: A moderate prevalence of FH was found for 4 conditions for which screening recommendations vary with risk based on FH. Having patients self-complete an FH was thought to be feasible; however, questions about FH accuracy and completeness from both self-report and EMR remain. Work is needed to determine how to facilitate the adoption of FH tools into practice as well as strategies linking familial risk to appropriate screening.Trial registration number ISRCTN07170460 (ISRCTN Registry).

N(2)O(3) enhances the nitrosative potential of IFNgamma-primed macrophages in response to Salmonella.

We show here that the nitric oxide (NO)-detoxifying Hmp flavohemoprotein increases by 3-fold the transcription of the Salmonella pathogenicity island 2 (SPI2) in macrophages expressing a functional inducible NO synthase (iNOS). However, Hmp does not prevent NO-related repression of SPI2 transcription in IFNgamma-primed phagocytes, despite preserving intracellular transcription of sdhA sdhB subunits of Salmonella succinate dehydrogenase within both control and IFNgamma-primed phagocytes. To shed light into the seemingly paradoxical role that Hmp plays in protecting intracellular SPI2 expression in various populations of macrophages, N(2)O(3) was quantified as an indicator of the nitrosative potential of Salmonella-infected phagocytes in different states of activation. Hmp was found to prevent the formation of 300nM N(2)O(3)/h/bacteria in IFNgamma-primed macrophages, accounting for about a 60% reduction of the nitrosative power of activated phagocytes. Utilization of the vacuolar ATPase inhibitor bafilomycin indicates that a fourth of the approximately 200nM N(2)O(3)/h sustained by IFNgamma-primed macrophages is generated in endosomal compartments via condensation of HNO(2). In sharp contrast, control macrophages infected with wild-type Salmonella produce as little N(2)O(3) as iNOS-deficient controls. Collectively, these findings indicate that the NO-metabolizing activity of Salmonella Hmp is functional in both control and IFNgamma-primed macrophages. Nonetheless, a substantial amount of the NO generated by IFNgamma-primed macrophages gives rise to N(2)O(3), a species that not only enhances the nitrosative potential of activated phagocytes but also avoids detoxification by Salmonella Hmp.

Constitutive acid sphingomyelinase enhances early and late macrophage killing of Salmonella enterica serovar Typhimurium.

We have identified acid sphingomyelinase (ASM) as an important player in the early and late anti-Salmonella activity of macrophages. A functional ASM participated in the killing activity of macrophages against wild-type Salmonella enterica serovar Typhimurium. The role of ASM in early macrophage killing of Salmonella appears to be linked to an active NADPH phagocyte oxidase enzymatic complex, since the flavoprotein inhibitor diphenyleneiodonium not only blocked a productive respiratory burst but also abrogated the survival advantage of Salmonella in macrophages lacking ASM. Lack of ASM activity also increased the intracellular survival of an isogenic DeltaspiC::FRT Salmonella strain deficient in a translocator and effector of the Salmonella pathogenicity island 2 (SPI2) type III secretion system, suggesting that the antimicrobial activity associated with ASM is manifested regardless of the SPI2 status of the bacteria. Constitutively expressed ASM is responsible for the role that this lipid-metabolizing hydrolase plays in the innate host defense of macrophages against Salmonella. Accordingly, the ASM activity and intracellular concentration and composition of ceramide, gangliosides, and neutral sphingolipids did not increase upon Salmonella infection. Salmonella triggered, nonetheless, a significant increase in the secreted fraction of ASM. Collectively, these findings have elucidated a novel role for constitutive ASM in the anti-Salmonella activity of murine macrophages.

Localized reactive oxygen and nitrogen intermediates inhibit escape of Listeria monocytogenes from vacuoles in activated macrophages.

Listeria monocytogenes (Lm) evades being killed after phagocytosis by macrophages by escaping from vacuoles into cytoplasm. Activated macrophages are listericidal, in part because they can retain Lm in vacuoles. This study examined the contribution of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) to the inhibition of Lm escape from vacuoles. Lm escaped from vacuoles of nonactivated macrophages within 30 min of infection. Macrophages activated with IFN-gamma, LPS, IL-6, and a neutralizing Ab against IL-10 retained Lm within the vacuoles, and inhibitors of ROI and RNI blocked inhibition of vacuolar escape to varying degrees. Measurements of Lm escape in macrophages from gp91(phox-/-) and NO synthase 2(-/-) mice showed that vacuolar retention required ROI and was augmented by RNI. Live cell imaging with the fluorogenic probe dihydro-2',4,5,6,7,7'-hexafluorofluorescein coupled to BSA (DHFF-BSA) indicated that oxidative chemistries were generated rapidly and were localized to Lm vacuoles. Chemistries that oxidized DHFF-BSA were similar to those that retained Lm in phagosomes. Fluorescent conversion of DHFF-BSA occurred more efficiently in smaller vacuoles, indicating that higher concentrations of ROI or RNI were generated in more confining volumes. Thus, activated macrophages retained Lm within phagosomes by the localization of ROI and RNI to vacuoles, and by their combined actions in a small space

Development of genetic circuitry exhibiting toggle switch or oscillatory behavior in Escherichia coli.

Analysis of the system design principles of signaling systems requires model systems where all components and regulatory interactions are known. Components of the Lac and Ntr systems were used to construct genetic circuits that display toggle switch or oscillatory behavior. Both devices contain an "activator module" consisting of a modified glnA promoter with lac operators, driving the expression of the activator, NRI. Since NRI activates the glnA promoter, this creates an autoactivated circuit repressible by LacI. The oscillator contains a "repressor module" consisting of the NRI-activated glnK promoter driving LacI expression. This circuitry produced synchronous damped oscillations in turbidostat cultures, with periods much longer than the cell cycle. For the toggle switch, LacI was provided constitutively; the level of active repressor was controlled by using a lacY mutant and varying the concentration of IPTG. This circuitry provided nearly discontinuous expression of activator.

Calcium spikes in activated macrophages during Fcgamma receptor-mediated phagocytosis.

Rises in intracellular-free calcium ([Ca(2+)](i)) have been variously associated with Fcgamma receptor (FcR)-mediated phagocytosis in macrophages. We show here that activation of murine bone marrow-derived macrophages increases calcium spiking after FcR ligation. Ratiometric fluorescence microscopy was used to measure [Ca(2+)](i) during phagocytosis of immunoglobulin G (IgG)-opsonized erythrocytes. Whereas 13% of nonactivated macrophages increased [Ca(2+)](i) in the form of one or more spikes, 56% of those activated with lipopolysaccharides (LPS; 18 h at 100 ng/ml) and interferon-gamma (IFN-gamma; 100 U/ml) and 73% of macrophages activated with LPS, IFN-gamma, interleukin (IL)-6 (5 ng/ml), and anti-IL-10 IgG (5 micro g/ml) spiked calcium during phagocytosis. Calcium spikes were inhibited by thapsigargin (Tg), indicating that they originated from endoplasmic reticulum. The fact that activated macrophages showed a more dramatic response suggested that calcium spikes during phagocytosis mediate or regulate biochemical mechanisms for microbicidal activities. However, lowering [Ca(2+)](i) with ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid or inhibiting calcium spikes with Tg did not inhibit phagosome-lysosome fusion or the generation of reactive oxygen or nitrogen species. Thus, the increased calcium spiking in activated macrophages was not directly associated with the mechanism of phagocytosis or the increased antimicrobial activities of activated macrophages.

pH-dependent regulation of lysosomal calcium in macrophages.

Calcium measurements in acidic vacuolar compartments of living cells are few, primarily because calibration of fluorescent probes for calcium requires knowledge of pH and the pH-dependence of the probe calcium-binding affinities. Here we report pH-corrected measurements of free calcium concentrations in lysosomes of mouse macrophages, using both ratiometric and time-resolved fluorescence microscopy of probes for pH and calcium. Average free calcium concentration in macrophage lysosomes was 4-6x10(-4) M, less than half of the extracellular calcium concentration, but much higher than cytosolic calcium levels. Incubating cells in varying extracellular calcium concentrations did not alter lysosomal pH, and had only a modest effect on lysosomal calcium concentrations, indicating that endocytosis of extracellular fluid provided a small but measurable contribution to lysosomal calcium concentrations. By contrast, increases in lysosomal pH, mediated by either bafilomycin A(1) or ammonium chloride, decreased lysosomal calcium concentrations by several orders of magnitude. Re-acidification of the lysosomes allowed rapid recovery of lysosomal calcium concentrations to higher concentrations. pH-dependent reductions of lysosomal calcium concentrations appeared to result from calcium movement out of lysosomes into cytoplasm, since increases in cytosolic calcium levels could be detected upon lysosome alkalinization. These studies indicate that lysosomal calcium concentration is high and is maintained in part by the proton gradient across lysosomal membranes. Moreover, lysosomes could provide an intracellular source for physiological increases in cytosolic calcium levels.