A sub-cm micromachined electron microscope

A new approach for fabricating macroscopic (approximately 10x10x10 mm(exp 3)) structures with micron accuracy has been developed. This approach combines the precision of semiconductor processing and fiber optic technologies. A (100) silicon wafer is anisotropically etched to create four orthogonal v-grooves and an aperture on each 10x12 mm die. Precision 308 micron optical fibers are sandwiched between the die to align the v-grooves. The fiber is then anodically bonded to the die above and below it. This procedure is repeated to create thick structures and a stack of 5 or 6 die will be used to create a miniature scanning electron microscope (MSEM). Two die in the structure will have a segmented electrode to deflect the beam and correct for astigmatism. The entire structure is UHV compatible. The performance of an SEM improves as its length is reduced and a sub-cm 2 keV MSEM with a field emission source should have approximately 1 nm resolution. A low voltage high resolution MSEM would be useful for the examination of biological specimens and semiconductors with a minimum of damage. The first MSEM will be tested with existing 6 micron thermionic sources. In the future a micromachined field emission source will be used. The stacking technology presented in this paper can produce an array of MSEMs 1 to 30 mm in length with a 1 mm or larger period. A key question being addressed by this research is the optimum size for a low voltage MSEM which will be determined by the required spatial resolution, field of view, and working distance

Networked buffering: a basic mechanism for distributed robustness in complex adaptive systems

A generic mechanism - networked buffering - is proposed for the generation of robust traits in complex systems. It requires two basic conditions to be satisfied: 1) agents are versatile enough to perform more than one single functional role within a system and 2) agents are degenerate, i.e. there exists partial overlap in the functional capabilities of agents. Given these prerequisites, degenerate systems can readily produce a distributed systemic response to local perturbations. Reciprocally, excess resources related to a single function can indirectly support multiple unrelated functions within a degenerate system. In models of genome:proteome mappings for which localized decision-making and modularity of genetic functions are assumed, we verify that such distributed compensatory effects cause enhanced robustness of system traits. The conditions needed for networked buffering to occur are neither demanding nor rare, supporting the conjecture that degeneracy may fundamentally underpin distributed robustness within several biotic and abiotic systems. For instance, networked buffering offers new insights into systems engineering and planning activities that occur under high uncertainty. It may also help explain recent developments in understanding the origins of resilience within complex ecosystems. \ud \u

Exploring the Contextual Sensitivity of Factors that Determine Cell-to-Cell Variability in Receptor-Mediated Apoptosis

Stochastic fluctuations in gene expression give rise to cell-to-cell variability in protein levels which can potentially cause variability in cellular phenotype. For TRAIL (TNF-related apoptosis-inducing ligand) variability manifests itself as dramatic differences in the time between ligand exposure and the sudden activation of the effector caspases that kill cells. However, the contribution of individual proteins to phenotypic variability has not been explored in detail. In this paper we use feature-based sensitivity analysis as a means to estimate the impact of variation in key apoptosis regulators on variability in the dynamics of cell death. We use Monte Carlo sampling from measured protein concentration distributions in combination with a previously validated ordinary differential equation model of apoptosis to simulate the dynamics of receptor-mediated apoptosis. We find that variation in the concentrations of some proteins matters much more than variation in others and that precisely which proteins matter depends both on the concentrations of other proteins and on whether correlations in protein levels are taken into account. A prediction from simulation that we confirm experimentally is that variability in fate is sensitive to even small increases in the levels of Bcl-2. We also show that sensitivity to Bcl-2 levels is itself sensitive to the levels of interacting proteins. The contextual dependency is implicit in the mathematical formulation of sensitivity, but our data show that it is also important for biologically relevant parameter values. Our work provides a conceptual and practical means to study and understand the impact of cell-to-cell variability in protein expression levels on cell fate using deterministic models and sampling from parameter distributions

Combining Network Modeling and Gene Expression Microarray Analysis to Explore the Dynamics of Th1 and Th2 Cell Regulation

Two T helper (Th) cell subsets, namely Th1 and Th2 cells, play an important role in inflammatory diseases. The two subsets are thought to counter-regulate each other, and alterations in their balance result in different diseases. This paradigm has been challenged by recent clinical and experimental data. Because of the large number of genes involved in regulating Th1 and Th2 cells, assessment of this paradigm by modeling or experiments is difficult. Novel algorithms based on formal methods now permit the analysis of large gene regulatory networks. By combining these algorithms with in silico knockouts and gene expression microarray data from human T cells, we examined if the results were compatible with a counter-regulatory role of Th1 and Th2 cells. We constructed a directed network model of genes regulating Th1 and Th2 cells through text mining and manual curation. We identified four attractors in the network, three of which included genes that corresponded to Th0, Th1 and Th2 cells. The fourth attractor contained a mixture of Th1 and Th2 genes. We found that neither in silico knockouts of the Th1 and Th2 attractor genes nor gene expression microarray data from patients with immunological disorders and healthy subjects supported a counter-regulatory role of Th1 and Th2 cells. By combining network modeling with transcriptomic data analysis and in silico knockouts, we have devised a practical way to help unravel complex regulatory network topology and to increase our understanding of how network actions may differ in health and disease

Stochastic Responses May Allow Genetically Diverse Cell Populations to Optimize Performance with Simpler Signaling Networks

Two theories have emerged for the role that stochasticity plays in biological responses: first, that it degrades biological responses, so the performance of biological signaling machinery could be improved by increasing molecular copy numbers of key proteins; second, that it enhances biological performance, by enabling diversification of population-level responses. Using T cell biology as an example, we demonstrate that these roles for stochastic responses are not sufficient to understand experimental observations of stochastic response in complex biological systems that utilize environmental and genetic diversity to make cooperative responses. We propose a new role for stochastic responses in biology: they enable populations to make complex responses with simpler biochemical signaling machinery than would be required in the absence of stochasticity. Thus, the evolution of stochastic responses may be linked to the evolvability of different signaling machineries.National Institutes of Health (U.S.). Pioneer Awar

An In Silico Modeling Approach to Understanding the Dynamics of Sarcoidosis

BACKGROUND: Sarcoidosis is a polygenic disease with diverse phenotypic presentations characterized by an abnormal antigen-mediated Th1 type immune response. At present, progress towards understanding sarcoidosis disease mechanisms and the development of novel treatments is limited by constraints attendant to conducting human research in a rare disease in the absence of relevant animal models. We sought to develop a computational model to enhance our understanding of the pathological mechanisms of and predict potential treatments of sarcoidosis. METHODOLOGY/RESULTS: Based upon the literature, we developed a computational model of known interactions between essential immune cells (antigen-presenting macrophages, effector and regulatory T cells) and cytokine mediators (IL-2, TNFα, IFNγ) of granulomatous inflammation during sarcoidosis. The dynamics of these interactions are described by a set of ordinary differential equations. The model predicts bistable switching behavior which is consistent with normal (self-limited) and "sarcoidosis-like" (sustained) activation of the inflammatory components of the system following a single antigen challenge. By perturbing the influence of model components using inhibitors of the cytokine mediators, distinct clinically relevant disease phenotypes were represented. Finally, the model was shown to be useful for pre-clinical testing of therapies based upon molecular targets and dose-effect relationships. CONCLUSIONS/SIGNIFICANCE: Our work illustrates a dynamic computer simulation of granulomatous inflammation scenarios that is useful for the investigation of disease mechanisms and for pre-clinical therapeutic testing. In lieu of relevant in vitro or animal surrogates, our model may provide for the screening of potential therapies for specific sarcoidosis disease phenotypes in advance of expensive clinical trials

Attenuated T Cell Responses to a High-Potency Ligand In Vivo

According to this study, the strongest T cell receptor ligands in vitro do not necessarily induce the strongest T cell responses in vivo, suggesting that vaccine designers may need to reconsider their strategies

Phase Transition of a Non-Linear Opinion Dynamics with Noisy Interactions

International audienceIn several real \emph{Multi-Agent Systems} (MAS), it has been observed that only weaker forms of\emph{metastable consensus} are achieved, in which a large majority of agents agree on some opinion while other opinions continue to be supported by a (small) minority of agents. In this work, we take a step towards the investigation of metastable consensus for complex (non-linear) \emph{opinion dynamics} by considering the famous \undecided dynamics in the binary setting, which is known to reach consensus exponentially faster than the \voter dynamics. We propose a simple form of uniform noise in which each message can change to another one with probability $p$ and we prove that the persistence of a \emph{metastable consensus} undergoes a \emph{phase transition} for $p=\frac 16$. In detail, below this threshold, we prove the system reaches with high probability a metastable regime where a large majority of agents keeps supporting the same opinion for polynomial time. Moreover, this opinion turns out to be the initial majority opinion, whenever the initial bias is slightly larger than its standard deviation.On the contrary, above the threshold, we show that the information about the initial majority opinion is ``lost'' within logarithmic time even when the initial bias is maximum.Interestingly, using a simple coupling argument, we show the equivalence between our noisy model above and the model where a subset of agents behave in a \emph{stubborn} way

Biophysics across time and space

Understanding the behaviour of almost any biological object is a fundamentally multiscale problem — a challenge that biophysicists have been increasingly embracing, building on two centuries of biophysical studies at a variety of length scales