Plasmid stability and ecological competence in recombinant cultures

The instability of cell cultures containing plasmid vectors is a major problem in the commercial exploitation of molecular cloning techniques. Plasmid stability is influenced by the nature of the host cell, the type of plasmid and/or environmental conditions. Plasmid encoded properties may confer a selective advantage on the host cell but can be an energy drain due to replication and expression. Stability of recombinant cultures ultimately may be determined by the cost to benefit ratio of plasmid carriage.The relative competition between plasmid containing and plasmid-free or indigenous populations can determine the degree of dominance of recombinant cultures. The use of inocula in biotechnological processes in which dynamic environmental conditions dominate may also result in instabilities resulting from the characteristics of the ecosystem. In such dynamic conditions plasmid stability is just one contribution to culture stability.Strategies to enhance plasmid stability, within such environments, based on manipulation of physiological state of host cells, must consider the responsiveness or plasticity of both cells and populations. The robustness of cells or the responses to stresses or transient environmental conditions can influence the levels of instability detected; for example, instability or mutation in the host genome may lead to enhanced plasmid stability. Competition among subpopulations arising from unstable copy number control may determine the levels of recombinant cells in open versus closed fermenter systems.Thus the ecological competence (ability to survive and compete) of recombinant cells in dynamic or transient environments is fundamental to the understanding of the ultimate dominance or survival of such recombinant cultures and may form the basis of a strategy to enhance or control stability either in fermenter systems or dynamic process environments. The creation of microniches in time and/or space can enhance plasmid stability. Transient operation based on defined environmental stresses or perturbations in fermenter systems or in heterogeneous or dynamic environments found in gel immobilized cultures have resulted in enhanced stability. Spatial organization resulting from immobilization has the additional advantage of regulated cell protection within defined microenvironments and controlled release, depending on the nature of the gel, from these microenvironments or microcosms. This regulation of ecological competence allied to the advantages of microbial cell growth in gel microenvironments combined with the spatial organization (or juxtapositioning of cells, selective agents, nutrients, protectants, etc.) possible through immobilization technology offers new strategies to enhance plasmid and culture stability.     

An integrative model of evolutionary covariance: a symposium on body shape in fishes

A major direction of current and future biological research is to understand how multiple, interacting functional systems coordinate in producing a body that works. This understanding is complicated by the fact that organisms need to work well in multiple environments, with both predictable and unpredictable environmental perturbations. Furthermore, organismal design reflects a history of past environments and not a plan for future environments. How complex, interacting functional systems evolve, then, is a truly grand challenge. In accepting the challenge, an integrative model of evolutionary covariance is developed. The model combines quantitative genetics, functional morphology/physiology, and functional ecology. The model is used to convene scientists ranging from geneticists, to physiologists, to ecologists, to engineers to facilitate the emergence of body shape in fishes as a model system for understanding how complex, interacting functional systems develop and evolve. Body shape of fish is a complex morphology that (1) results from many developmental paths and (2) functions in many different behaviors. Understanding the coordination and evolution of the many paths from genes to body shape, body shape to function, and function to a working fish body in a dynamic environment is now possible given new technologies from genetics to engineering and new theoretical models that integrate the different levels of biological organization (from genes to ecology).     

The systems analysis approach to mechanosensory coding

An important problem in neuroscience is to obtain quantitative knowledge of how information is represented, or encoded, in the signals that nerve cells process and transmit. Sensory receptors have provided important models for the study of neural coding because their inputs can often be relatively easily controlled and measured, while the resultant activity is recorded. A variety of engineering concepts have been successfully applied to physiological sciences, particularly those related to control of dynamic systems. Linear systems analysis was one of the earliest methods used to probe sensory coding, and measurements such as step responses and frequency responses have become standard tools for describing sensory functions. Modern systems analysis has evolved to provide accurate and efficient linear identification of encoding in sensory receptors that use either graded potentials or action potentials. It has also led to nonlinear systems analysis, the creation of parametric nonlinear models, and measures of information coding by sensory neurons. These methods promise to provide important new knowledge about sensory systems in the future, especially when complemented with parallel biophysical and molecular studies of sensory neurons. Mechanoreceptors provided some of the earliest preparations for the investigation of neural coding, and both the linear and nonlinear properties of wide variety of vertebrate and invertebrate mechanoreceptors continue to be explored. This article is part of a special issue on Neuronal Dynamics of Sensory Coding.     

Exploring the role of microRNAs in axolotl regeneration

The axolotl, Ambystoma mexicanum, is used extensively for research in developmental biology, particularly for its ability to regenerate and restore lost organs, including in the nervous system, to full functionality. Regeneration in mammals typically depends on the healing process and scar formation with limited replacement of lost tissue. Other organisms, such as spiny mice (Acomys cahirinus), salamanders, and zebrafish, are able to regenerate some damaged body components. Blastema is a tissue that is formed after tissue injury in such organisms and is composed of progenitor cells or dedifferentiated cells that differentiate into various cell types during regeneration. Thus, identifying the molecules responsible for initiation of blastema formation is an important aspect for understanding regeneration. Introns, a major source of noncoding RNAs (ncRNAs), have characteristic sizes in the axolotl, particularly in genes associated with development. These ncRNAs, particularly microRNAs (miRNAs), exhibit dynamic regulation during regeneration. These miRNAs play an essential role in timing and control of gene expression to order and organize processes necessary for blastema creation. Master keys or molecules that underlie the remarkable regenerative abilities of the axolotl remain to be fully explored and exploited. Further and ongoing research on regeneration promises new knowledge that may allow improved repair and renewal of human tissues.     

Biomimetic approach to tissue engineering

The overall goal of tissue engineering is to create functional tissue grafts that can regenerate or replace our defective or worn out tissues and organs. Examples of grafts that are now in pre-clinical studies or clinical use include engineered skin, cartilage, bone, blood vessels, skeletal muscle, bladder, trachea, and myocardium. Engineered tissues are also finding applications as platforms for pharmacological and physiological studies in vitro. To fully mobilize the cell's biological potential, a new generation of tissue engineering systems is now being developed to more closely recapitulate the native developmental milieu, and mimic the physiologic mechanisms of transport and signaling. We discuss the interactions between regenerative biology and engineering, in the context of (i) creation of functional tissue grafts for regenerative medicine (where biological input is critical), and (ii) studies of stem cells, development and disease (where engineered tissues can serve as advanced 3D models).     

Free Energy Simulations Over Creation/Annihilation Paths for a Flexible Solute

The inclusion of molecular flexibility into free energy simulations over creation/annihilation paths has been analyzed. A new formalism is presented for such simulations with the intramolecular degrees of freedom being active during the simulation and a recently introduced path is reviewed that allows the inclusion of the flexibility using separate simulations.     

The role of GTP-binding proteins in mechanochemical movements of microorganisms and their potential to form filamentous structures

Prokaryotic cells contain proteins which form extended chains or multimers that oscillate between monomers and oligomers of varying length. Hydrolysis of nucleoside triphosphates combined with site-specific disposition of substrates and products to monomers and multimers is the driving force of dynamic instability of these molecules. Polymeric structures are connected in some manner to a variety of signaling systems that adhere to the polymeric matrix, including the GTP-binding protein(s), protein kinases and phosphatases, and other proteins or systems that communicate between the cytoplasmic membrane and the cytosol. Flexible organization allowing regulated dynamic movement is one of the key elements in all living cells. In eukaryotic cells actin and tubulin are the two main components of dynamically controlled spatial system. These proteins are noteworthy for their ability to polymerize, reversibly, into filaments or microtubules in association with hydrolysis of ATP or GTP, respectively. As such, they regulate most of the mechanics of cell movement including cell division, cell differentiation, phagocytosis and other dynamic phenomena. Recent evidence revealed that microbial cells create functional domains at specific sites of the cells and can form cytoplasmic tubules and fibers.     

Programming and engineering biological networks

Synthetic biology aims to build new functions in living organisms. Recent work has addressed the creation of synthetic epigenetic switches in mammalian cells and synthetic intracellular communication. Fundamentally new, and potentially scaleable, modes of gene regulation have been created that enable expansion of the scope of synthetic circuits. Increasingly sophisticated models of gene regulation that include stochastic effects are beginning to predict the behaviour of small synthetic networks. Overall, these advances suggest that a combination of molecular engineering and systems engineering should allow the creation of living matter capable of performing many useful and novel functions.     

Some observations on the geomorphological impact of hippopotamus (Hippopotamus amphibius L.) in the Okavango Delta, Botswana

The Okavango Delta is a large wetland system situated in northern Botswana. The hippopotamus forms an integral part of this dynamic ecosystem, as it exerts a catalytic effect on geomorphological change. In the distal reaches of the wetland, regular movement of hippos to feeding grounds results in the development of incised channels, which are kept clear of vegetation and act as nodes for swamp expansion. Hippos maintain pathways in backswamp areas, which lead to the development of new channel systems during channel avulsion. They create breaches in the vegetation levees which flank channels in the permanent swamps, causing diversion of water and sediment to backswamp areas. Their paths often lead to lakes in the permanent swamps. During channel avulsion, diversion of a channel into lakes may occur via these paths, which can result in lake closure.     

Diversified Control Paths: A Significant Way Disease Genes Perturb the Human Regulatory Network

Background

The complexity of biological systems motivates us to use the underlying networks to provide deep understanding of disease etiology and the human diseases are viewed as perturbations of dynamic properties of networks. Control theory that deals with dynamic systems has been successfully used to capture systems-level knowledge in large amount of quantitative biological interactions. But from the perspective of system control, the ways by which multiple genetic factors jointly perturb a disease phenotype still remain.

Results

In this work, we combine tools from control theory and network science to address the diversified control paths in complex networks. Then the ways by which the disease genes perturb biological systems are identified and quantified by the control paths in a human regulatory network. Furthermore, as an application, prioritization of candidate genes is presented by use of control path analysis and gene ontology annotation for definition of similarities. We use leave-one-out cross-validation to evaluate the ability of finding the gene-disease relationship. Results have shown compatible performance with previous sophisticated works, especially in directed systems.

Conclusions

Our results inspire a deeper understanding of molecular mechanisms that drive pathological processes. Diversified control paths offer a basis for integrated intervention techniques which will ultimately lead to the development of novel therapeutic strategies.     

“GREENCE” technology for <Emphasis Type="Italic">in silico</Emphasis> analysis of gene network dynamics in individual cells of a clonal population

The technology developed for in silico analysis of gene network behavior in a series of successive cell divisions makes it possible to obtain gene expression profiles in the cells of intermediate and final generations and to evaluate the relation among the cells heterogeneous by the functional states of gene subnetworks. Based on the model of a hypothetical gene network, which includes three cyclic digene systems with negative feedbacks, a new property of dynamic epigenes is confirmed, i.e., metastability of some epigenotypes, which earlier was predicted theoretically and found in experiments in vivo. A dynamic epigene is a cyclic system of genes with more than one inherited functional states, or epigenotypes. In a metastable state such a relation among repressors is set in a cell that, as a result of random distribution of the molecules and fluctuations of protein concentrations, subsequent divisions give daughter cells appear determined to alternative epigenotypes. It is shown in computer experiments that even systems in which dynamic epigenes are typical elements can possess this property.     

“GREENCE” technology for in silico analysis of gene network dynamics in individual cells of a clonal population

The technology developed for in silico analysis of gene network behavior in a series of successive cell divisions makes it possible to obtain gene expression profiles in the cells of intermediate and final generations and to evaluate the relation among the cells heterogeneous by the functional states of gene subnetworks. Based on the model of a hypothetical gene network, which includes three cyclic digene systems with negative feedbacks, a new property of dynamic epigenes is confirmed, i.e., metastability of some epigenotypes, which earlier was predicted theoretically and found in experiments in vivo. A dynamic epigene is a cyclic system of genes with more than one inherited functional states, or epigenotypes. In a metastable state such a relation among repressors is set in a cell that, as a result of random distribution of the molecules and fluctuations of protein concentrations, subsequent divisions give daughter cells appear determined to alternative epigenotypes. It is shown in computer experiments that even systems in which dynamic epigenes are typical elements can possess this property.

     

Ecological Therapy for Cancer: Defining Tumors Using an Ecosystem Paradigm Suggests New Opportunities for Novel Cancer Treatments

We propose that there is an opportunity to devise new cancer therapies based on the recognition that tumors have properties of ecological systems. Traditionally, localized treatment has targeted the cancer cells directly by removing them (surgery) or killing them (chemotherapy and radiation). These modes of therapy have not always been effective because many tumors recur after these therapies, either because not all of the cells are killed (local recurrence) or because the cancer cells had already escaped the primary tumor environment (distant recurrence). There has been an increasing recognition that the tumor microenvironment contains host noncancer cells in addition to cancer cells, interacting in a dynamic fashion over time. The cancer cells compete and/or cooperate with nontumor cells, and the cancer cells may compete and/or cooperate with each other. It has been demonstrated that these interactions can alter the genotype and phenotype of the host cells as well as the cancer cells. The interaction of these cancer and host cells to remodel the normal host organ microenvironment may best be conceptualized as an evolving ecosystem. In classic terms, an ecosystem describes the physical and biological components of an environment in relation to each other as a unit. Here, we review some properties of tumor microenvironments and ecological systems and indicate similarities between them. We propose that describing tumors as ecological systems defines new opportunities for novel cancer therapies and use the development of prostate cancer metastases as an example. We refer to this as “ecological therapy” for cancer.     

Heuristic Reusable Dynamic Programming: Efficient Updates of Local Sequence Alignment

Recomputation of the previously evaluated similarity results between biological sequences becomes inevitable when researchers realize errors in their sequenced data or when the researchers have to compare nearly similar sequences, e.g., in a family of proteins. We present an efficient scheme for updating local sequence alignments with an affine gap model. In principle, using the previous matching result between two amino acid sequences, we perform a forward-backward alignment to generate heuristic searching bands which are bounded by a set of suboptimal paths. Given a correctly updated sequence, we initially predict a new score of the alignment path for each contour to select the best candidates among them. Then, we run the Smith-Waterman algorithm in this confined space. Furthermore, our heuristic alignment for an updated sequence shows that it can be further accelerated by using reusable dynamic programming (rDP), our prior work. In this study, we successfully validate "relative node tolerance bound” (RNTB) in the pruned searching space. Furthermore, we improve the computational performance by quantifying the successful RNTB tolerance probability and switch to rDP on perturbation-resilient columns only. In our searching space derived by a threshold value of 90 percent of the optimal alignment score, we find that 98.3 percent of contours contain correctly updated paths. We also find that our method consumes only 25.36 percent of the runtime cost of sparse dynamic programming (sDP) method, and to only 2.55 percent of that of a normal dynamic programming with the Smith-Waterman algorithm.     

Dynamic Management of Maize Landraces in Central Mexico

Conservationists of crop genetic resources have feared that in situ conservation was not viable for agriculture precisely because of changes resulting from introduction of new varieties of existing crops, new crops, and new farm practices. In addition, conservation within farming systems necessarily implies a constantly changing crop population resulting from the processes of crop evolution. Even though in situ conservation of crop genetic resources is now generally understood to be dynamic, there are few examples of how evolution takes place in farmers fields. This study describes several changes in maize landraces in four communities along an altitude transect in Central Mexico (1200 to 2400 masl). While true modern varieties have not been widely adopted in the study region, farmer management results in numerous changes in maize landrace populations. Five types of dynamic management were observed: (1) purposeful hybridization between traditional and modern maize types, (2) possible creation of a new maize landrace by directional selection of the progeny of hybridization between two traditional landraces, (3) displacement of a local landrace by the introduction of a modern variety and a non-local landrace, (4) maintenance of stable populations of a locally dominant landrace, and (5) market-driven selection for a minor variety. We concur that in situ conservation of crops must be conceived as an open process where the objective is not to maintain historic varieties or static genetic conditions. Rather, in situ conservation of crops is totally in the hands of the farmer, although interventions may be designed to influence farmers’ management of agrobiodiversity.     

Building mammalian signalling pathways with RNAi screens

Technological advances in mammalian systems are providing new tools to identify the molecular components of signalling pathways. Foremost among these tools is the ability to knock down gene function through the use of RNA interference (RNAi). The fact that RNAi can be scaled up for use in high-throughput techniques has motivated the creation of genome-wide RNAi reagents. We are now at the brink of being able to harness the power of RNAi for large-scale functional discovery in mammalian cells.     

The role of bacterial metabolism in transformation of non-mutagenic compounds into mutagens. I. Participation of Escherichia coli nitroreductase in creation of mutagens from non-mutagenic new pesticides]

Investigations concerned Escherichia coli nitroreductase in creation of mutagens from non-mutagenic pesticides-derivatives of urea. Three new compounds were studied: N-phenyl-N'-methylurea (IPO 4328), N-methyl,N-(2-hydroxyethyl)-N'phenylurea (IPO 2363), N-(2-hydroxyethyl), N-methyl-N'-(3,4 dichloroethyl) urea, and diurone-3-(3,4 dichlorophenyl)-1,1 dimethylurea. These compounds were incubated in anaerobic conditions with cells of E. coli K-12 (KF) strain and nitrate or nitrite. Using Ames test, mutagenicity of resulting metabolites was investigated. It was found that during incubation of herbicide IPO 4328 with cells of E. coli K-12 (KF) and nitrate, mutagenic product for strain of S. typhimurium TA 1537 is created. Very weak mutagenic metabolite for the same strain was appearing during incubation of herbicide IPO 2363 with cells of E. coli K-12 (KF) in presence of nitrite. Incubation of investigated compounds with E. coli K-12 (KF) cells alone did not result in appearance of mutagenic substances. Thus, role of Escherichia coli in creation of mutagenic compounds from non-mutagenic derivatives of urea consisted of nitrite from nitrate production with participation of nitroreductase, which afterwards in absence of bacteria or action of their enzymes reacted with investigated pesticides.     

simBio: a Java package for the development of detailed cell models

Quantitative dynamic computer models, which integrate a variety of molecular functions into a cell model, provide a powerful tool to create and test working hypotheses. We have developed a new modeling tool, the simBio package (freely available from http://www.sim-bio.org/), which can be used for constructing cell models, such as cardiac cells (the Kyoto model from Matsuoka et al., 2003, 2004a, b, the LRd model from Faber and Rudy, 2000, and the Noble 98 model from Noble et al., 1998), epithelial cells (Strieter et al., 1990) and pancreatic β cells (Magnus and Keizer, 1998). The simBio package is written in Java, uses XML and can solve ordinary differential equations. In an attempt to mimic biological functional structures, a cell model is, in simBio, composed of independent functional modules called Reactors, such as ion channels and the sarcoplasmic reticulum, and dynamic variables called Nodes, such as ion concentrations. The interactions between Reactors and Nodes are described by the graph theory and the resulting graph represents a blueprint of an intricate cellular system. Reactors are prepared in a hierarchical order, in analogy to the biological classification. Each Reactor can be composed or improved independently, and can easily be reused for different models. This way of building models, through the combination of various modules, is enabled through the use of object-oriented programming concepts. Thus, simBio is a straightforward system for the creation of a variety of cell models on a common database of functional modules.