870 research outputs found
Efficient protein depletion by genetically controlled deprotection of a dormant N-degron
Methods that allow for the manipulation of genes or their products have been highly fruitful for biomedical research. Here, we describe a method that allows the control of protein abundance by a genetically encoded regulatory system. We developed a dormant N-degron that can be attached to the N-terminus of a protein of interest. Upon expression of a site-specific protease, the dormant N-degron becomes deprotected. The N-degron then targets itself and the attached protein for rapid proteasomal degradation through the N-end rule pathway. We use an optimized tobacco etch virus (TEV) protease variant combined with selective target binding to achieve complete and rapid deprotection of the N-degron-tagged proteins. This method, termed TEV protease induced protein inactivation (TIPI) of TIPI-degron (TDeg) modified target proteins is fast, reversible, and applicable to a broad range of proteins. TIPI of yeast proteins essential for vegetative growth causes phenotypes that are close to deletion mutants. The features of the TIPI system make it a versatile tool to study protein function in eukaryotes and to create new modules for synthetic or systems biology
Apoptosis at Inflection Point in Liquid Culture of Budding Yeasts
Budding yeasts are highly suitable for aging studies, because the number of bud
scars (stage) proportionally correlates with age. Its maximum stages are known
to reach at 20–30 stages on an isolated agar medium. However, their stage
dynamics in a liquid culture is virtually unknown. We investigate the population
dynamics by counting scars in each cell. Here one cell division produces one new
cell and one bud scar. This simple rule leads to a conservation law: “The
total number of bud scars is equal to the total number of cells.” We find
a large discrepancy: extremely fewer cells with over 5 scars than expected.
Almost all cells with 6 or more scars disappear within a short period of time in
the late log phase (corresponds to the inflection point). This discrepancy is
confirmed directly by the microscopic observations of broken cells. This finding
implies apoptosis in older cells (6 scars or more)
Cycle-centrality in complex networks
Networks are versatile representations of the interactions between entities
in complex systems. Cycles on such networks represent feedback processes which
play a central role in system dynamics. In this work, we introduce a measure of
the importance of any individual cycle, as the fraction of the total
information flow of the network passing through the cycle. This measure is
computationally cheap, numerically well-conditioned, induces a centrality
measure on arbitrary subgraphs and reduces to the eigenvector centrality on
vertices. We demonstrate that this measure accurately reflects the impact of
events on strategic ensembles of economic sectors, notably in the US economy.
As a second example, we show that in the protein-interaction network of the
plant Arabidopsis thaliana, a model based on cycle-centrality better accounts
for pathogen activity than the state-of-art one. This translates into
pathogen-targeted-proteins being concentrated in a small number of triads with
high cycle-centrality. Algorithms for computing the centrality of cycles and
subgraphs are available for download
Predictive diagnostics and personalized medicine for the prevention of chronic degenerative diseases
Progressive increase of mean age and life expectancy in both industrialized and emerging societies parallels an increment of chronic degenerative diseases (CDD) such as cancer, cardiovascular, autoimmune or neurodegenerative diseases among the elderly. CDD are of complex diagnosis, difficult to treat and absorbing an increasing proportion in the health care budgets worldwide. However, recent development in modern medicine especially in genetics, proteomics, and informatics is leading to the discovery of biomarkers associated with different CDD that can be used as indicator of disease’s risk in healthy subjects. Therefore, predictive medicine is merging and medical doctors may for the first time anticipate the deleterious effect of CDD and use markers to identify persons with high risk of developing a given CDD before the clinical manifestation of the diseases. This innovative approach may offer substantial advantages, since the promise of personalized medicine is to preserve individual health in people with high risk by starting early treatment or prevention protocols. The pathway is now open, however the road to an effective personalized medicine is still long, several (diagnostic) predictive instruments for different CDD are under development, some ethical issues have to be solved. Operative proposals for the heath care systems are now needed to verify potential benefits of predictive medicine in the clinical practice. In fact, predictive diagnostics, personalized medicine and personalized therapy have the potential of changing classical approaches of modern medicine to CDD
Error and attack tolerance of complex networks
Many complex systems, such as communication networks, display a surprising
degree of robustness: while key components regularly malfunction, local
failures rarely lead to the loss of the global information-carrying ability of
the network. The stability of these complex systems is often attributed to the
redundant wiring of the functional web defined by the systems' components. In
this paper we demonstrate that error tolerance is not shared by all redundant
systems, but it is displayed only by a class of inhomogeneously wired networks,
called scale-free networks. We find that scale-free networks, describing a
number of systems, such as the World Wide Web, Internet, social networks or a
cell, display an unexpected degree of robustness, the ability of their nodes to
communicate being unaffected by even unrealistically high failure rates.
However, error tolerance comes at a high price: these networks are extremely
vulnerable to attacks, i.e. to the selection and removal of a few nodes that
play the most important role in assuring the network's connectivity.Comment: 14 pages, 4 figures, Late
Evaluating Gene Expression Dynamics Using Pairwise RNA FISH Data
Recently, a novel approach has been developed to study gene expression in single cells with high time resolution using RNA Fluorescent In Situ Hybridization (FISH). The technique allows individual mRNAs to be counted with high accuracy in wild-type cells, but requires cells to be fixed; thus, each cell provides only a “snapshot” of gene expression. Here we show how and when RNA FISH data on pairs of genes can be used to reconstruct real-time dynamics from a collection of such snapshots. Using maximum-likelihood parameter estimation on synthetically generated, noisy FISH data, we show that dynamical programs of gene expression, such as cycles (e.g., the cell cycle) or switches between discrete states, can be accurately reconstructed. In the limit that mRNAs are produced in short-lived bursts, binary thresholding of the FISH data provides a robust way of reconstructing dynamics. In this regime, prior knowledge of the type of dynamics – cycle versus switch – is generally required and additional constraints, e.g., from triplet FISH measurements, may also be needed to fully constrain all parameters. As a demonstration, we apply the thresholding method to RNA FISH data obtained from single, unsynchronized cells of Saccharomyces cerevisiae. Our results support the existence of metabolic cycles and provide an estimate of global gene-expression noise. The approach to FISH data presented here can be applied in general to reconstruct dynamics from snapshots of pairs of correlated quantities including, for example, protein concentrations obtained from immunofluorescence assays
Functional cartography of complex metabolic networks
High-throughput techniques are leading to an explosive growth in the size of
biological databases and creating the opportunity to revolutionize our
understanding of life and disease. Interpretation of these data remains,
however, a major scientific challenge. Here, we propose a methodology that
enables us to extract and display information contained in complex networks.
Specifically, we demonstrate that one can (i) find functional modules in
complex networks, and (ii) classify nodes into universal roles according to
their pattern of intra- and inter-module connections. The method thus yields a
``cartographic representation'' of complex networks. Metabolic networks are
among the most challenging biological networks and, arguably, the ones with
more potential for immediate applicability. We use our method to analyze the
metabolic networks of twelve organisms from three different super-kingdoms. We
find that, typically, 80% of the nodes are only connected to other nodes within
their respective modules, and that nodes with different roles are affected by
different evolutionary constraints and pressures. Remarkably, we find that
low-degree metabolites that connect different modules are more conserved than
hubs whose links are mostly within a single module.Comment: 17 pages, 4 figures. Go to http://amaral.northwestern.edu for the PDF
file of the reprin
Global organization of metabolic fluxes in the bacterium, Escherichia coli
Cellular metabolism, the integrated interconversion of thousands of metabolic
substrates through enzyme-catalyzed biochemical reactions, is the most
investigated complex intercellular web of molecular interactions. While the
topological organization of individual reactions into metabolic networks is
increasingly well understood, the principles governing their global functional
utilization under different growth conditions pose many open questions. We
implement a flux balance analysis of the E. coli MG1655 metabolism, finding
that the network utilization is highly uneven: while most metabolic reactions
have small fluxes, the metabolism's activity is dominated by several reactions
with very high fluxes. E. coli responds to changes in growth conditions by
reorganizing the rates of selected fluxes predominantly within this high flux
backbone. The identified behavior likely represents a universal feature of
metabolic activity in all cells, with potential implications to metabolic
engineering.Comment: 15 pages 4 figure
Feedback control architecture and the bacterial chemotaxis network.
PMCID: PMC3088647This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Bacteria move towards favourable and away from toxic environments by changing their swimming pattern. This response is regulated by the chemotaxis signalling pathway, which has an important feature: it uses feedback to 'reset' (adapt) the bacterial sensing ability, which allows the bacteria to sense a range of background environmental changes. The role of this feedback has been studied extensively in the simple chemotaxis pathway of Escherichia coli. However it has been recently found that the majority of bacteria have multiple chemotaxis homologues of the E. coli proteins, resulting in more complex pathways. In this paper we investigate the configuration and role of feedback in Rhodobacter sphaeroides, a bacterium containing multiple homologues of the chemotaxis proteins found in E. coli. Multiple proteins could produce different possible feedback configurations, each having different chemotactic performance qualities and levels of robustness to variations and uncertainties in biological parameters and to intracellular noise. We develop four models corresponding to different feedback configurations. Using a series of carefully designed experiments we discriminate between these models and invalidate three of them. When these models are examined in terms of robustness to noise and parametric uncertainties, we find that the non-invalidated model is superior to the others. Moreover, it has a 'cascade control' feedback architecture which is used extensively in engineering to improve system performance, including robustness. Given that the majority of bacteria are known to have multiple chemotaxis pathways, in this paper we show that some feedback architectures allow them to have better performance than others. In particular, cascade control may be an important feature in achieving robust functionality in more complex signalling pathways and in improving their performance
Colored Motifs Reveal Computational Building Blocks in the C. elegans Brain
Background: Complex networks can often be decomposed into less complex sub-networks whose structures can give hints about the functional
organization of the network as a whole. However, these structural
motifs can only tell one part of the functional story because in this
analysis each node and edge is treated on an equal footing. In real
networks, two motifs that are topologically identical but whose nodes
perform very different functions will play very different roles in the
network.
Methodology/Principal Findings: Here, we combine structural information
derived from the topology of the neuronal network of the nematode C.
elegans with information about the biological function of these nodes,
thus coloring nodes by function. We discover that particular
colorations of motifs are significantly more abundant in the worm brain
than expected by chance, and have particular computational functions
that emphasize the feed-forward structure of information processing in
the network, while evading feedback loops. Interneurons are strongly
over-represented among the common motifs, supporting the notion that
these motifs process and transduce the information from the sensor
neurons towards the muscles. Some of the most common motifs identified
in the search for significant colored motifs play a crucial role in the
system of neurons controlling the worm's locomotion.
Conclusions/Significance: The analysis of complex networks in terms of
colored motifs combines two independent data sets to generate insight
about these networks that cannot be obtained with either data set
alone. The method is general and should allow a decomposition of any
complex networks into its functional (rather than topological) motifs
as long as both wiring and functional information is available
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