A stochastic model of cellular senescence. II. Concordance with experimental data

This report presents the results of computer simulations of a mathematical model for cell proliferation and senescence. The mathematical model is based on the idea that the in vitro proliferative potential of normal cells is determined by the number of a certain class of self-replicating particles it possesses. In the simulation model these particles are lost by both stochastic and deterministic processes. When all self-replicating particles are lost a cell can divide up to seven times before it irreversibly ceases to replicate. The simulated in vitro lifespans of clones and subclones are shown to be in close agreement with experimental data. We also show that other properties of finitely proliferating cultures can be simulated by this model.     

Conditions for pathogen elimination by immune systems

Summary A continuous harvest effort can lead a population to extinction. How an “unconscious” immune system would perpetrate such an effort in order to eliminate a self-replicating antigen (a pathogen) becomes an intriguing problem if the system responses are functions of the pathogen population: the responses cannot be a continuous effort as the pathogen vanishes. On theoretical grounds, we show some qualities an immune response must have to support pathogen elimination. Then, three specific mechanisms are addressed: a pathogen-independent positive feedback loop among the responding cells of the system (e.g., B-lymphocyte and T-helper); the persistence of antigen bound to presenting cells; and the programmed expansion/contraction of a pool of responding cells. The maintenance of responding cells due to these mechanisms is the essential feature to the effective clearance of self-replicating agents. Thus, evolutionarily, the primary function of a helper lymphocyte would be to amplify a response and the primary function of memory would be the very elimination of pathogens.     

Minimal self-replicating systems

Minimal self-replicating systems typically consist of three components: a product molecule, and two substrate molecules that become joined to form another product molecule. An important characteristic of self-replicating systems is the ability of the product to catalyze the formation of additional product, resulting in autocatalytic behavior. Recent advances in the area of self-replication have led to improved efficiency of autocatalysis, both by increasing the fraction of product molecules that can participate in further rounds of replication, and by improving the efficiency of the catalysts themselves. This review analyzes chemical self-replicating systems that have been developed to date and discusses ongoing challenges in this area of research.     

Cancer therapy using a self-replicating RNA vaccine.

'Naked' nucleic acid vaccines are potentially useful candidates for the treatment of patients with cancer, but their clinical efficacy has yet to be demonstrated. We sought to enhance the immunogenicity of a nucleic acid vaccine by making it 'self-replicating'. We accomplished this by using a gene encoding an RNA replicase polyprotein derived from the Semliki forest virus, in combination with a model antigen. A single intramuscular injection of a self-replicating RNA immunogen elicited antigen-specific antibody and CD8+ T-cell responses at doses as low as 0.1 microg. Pre-immunization with a self-replicating RNA vector protected mice from tumor challenge, and therapeutic immunization prolonged the survival of mice with established tumors. The self-replicating RNA vectors did not mediate the production of substantially more model antigen than a conventional DNA vaccine did in vitro. However, the enhanced efficacy in vivo correlated with a caspase-dependent apoptotic death in transfected cells. This death facilitated the uptake of apoptotic cells by dendritic cells, providing a potential mechanism for enhanced immunogenicity. Naked, non-infectious, self-replicating RNA may be an excellent candidate for the development of new cancer vaccines.     

An example of design optimization for high evolvability: string rewriting grammar

As an example of the optimization of an evolutionary system design, a string rewriting system is studied. A set of rewriting rules that defines the growth of a string is experimentarily optimized in terms of maximizing the 'replicative capacity', that is the occurrence ratio of self-replicating strings. It is shown that the most optimized rule set allows many strings to self-replicate by using a special character able to copy an original string sequentially. Then, using various different rewriting rule sets, the connectivity between self-replicating strings is studied. A set of 'hyperblobs' covering the self-replicating strings is extracted and their connectivity is studied. The experimental results show that a large replicative capacity assures strong connectivity between self-replicating genotypes, making the system highly evolvable.     

Centrioles: duplicating precariously

To assemble a mitotic spindle and accurately segregate chromosomes to progeny, a cell needs to precisely regulate its centrosome number, a feat largely accomplished through the tight control of centriole duplication. Recent work showing that the overexpression of centriolar proteins can lead to the formation of multiple centrioles in the absence of pre-existing centrioles challenges the idea that it is a self-replicating organelle.     

Shrinking genomics

Two bacteria are featured this month, and both are at the lower end of the genome size scale. The first, Mycoplasma gallisepticum, belongs to a group of bacteria that have been studied both as important human and animal pathogens and in the pursuit of understanding the essential functions of a self-replicating minimal cell. The second, Nanoarchaeum equitans, is an obligate symbiont that only grows in co-culture with another archaeon. N. equitans seems to be the coelacanth of the microbial world--it has been assigned to a new phylum and represents a primitive form of prokaroytic life.     

Hepatitis C virus molecular clones: from cDNA to infectious virus particles in cell culture

There has been major progress in our understanding of hepatitis C virus (HCV) molecular virology in recent years. An essential prerequisite for this progress was the availability of functional molecular HCV clones, that serve as a starting point in order to establish cell culture systems. The first of these was the HCV replicon system, which used self-replicating subgenomic viral RNAs. However, these replicons only recapitulated the intracellular life cycle, and did not support production of infectious virus: this became possible with the identification of an HCV isolate that, for unknown reasons, replicates to very high levels in a human hepatoma cell line. Cells containing this genome release virus particles that are infectious in cell culture and in vivo. Without doubt, this system provides new possibilities for molecular studies of the HCV life cycle and the development of novel antiviral concepts.     

Product analysis of RNA generated de novo by Qβ replicase

Q_β replicase synthesizes self-replicating RNA in the absence of exogenous template after a certain lag time (“template-free synthesis”). The products of the template-free RNA synthesis have been investigated by gel electrophoresis and fingerprinting techniques. It has been found that a multitude of self-replicating RNA species appears in the early phases of reaction with variable lengths and sequences. Template-free synthesis in different samples under completely identical conditions yields RNA products with very different and unrelated fingerprints. The early products rapidly undergo an evolution process that alters the phenotypic properties of the self-replicating RNA, and leads to a concomitant increase of replication efficiency. Fingerprints and electrophoretic mobilities of the self-replicating RNA species are altered discontinuously during the evolution process. The evolution process ends with the selection of optimized self-replicating RNA species, whose phenotypes are conserved even after many serial transfers. Some optimized RNA species and midivariant RNA apparently have related sequences, since they contain many identical spots in their fingerprints. The properties of the RNA species produced by template-free synthesis match those of 6 S RNA found in Q_β-infected Escherichia coli cells. The results are in full agreement with the finding of Sumper & Luce (1975), who have presented evidence that Q_β replicase synthesized RNA de novo in the absence of exogenous template.     

Construction of a chemically and conformationally self-replicating system of amyloid-like fibrils

The amyloid-like fibril is considered to be a macromolecular self-assemblage with a highly-ordered quaternary structure, in which numerous beta-stranded polypeptide chains align regularly. Therefore, this kind of fibril has the potential to be engineered into proteinaceous materials, although conformational alteration of proteins from their native form to the amyloid form is a misfolding and undesirable process related to amyloid diseases. In this study, we have attempted to design an artificial system to explore applicability of using the amyloid-like fibril as a construct possessing self-recognition and self-catalytic abilities. A peptide self-replicating system based on the beta-structure of the amyloid-like fibril was designed and constructed. The beta-stranded peptide was self-replicated by the native chemical ligation reaction, and the newly generated peptide was self-assembled into amyloid-like fibrils. Thus, the constructed system was of both chemical and conformational self-replicating fibrils.     

An automaton analogue of unicellularity.

There is presented in outline form an abstract model of a cell in an evolutionary context. The design is based on an elaboration of John Holland's one-dimensional, abstract universe recently posed for the study of the emergence of self-replicating systems. Eight new ingredients constitute the elaboration. They suggest how compartmentation of a set of "molecules" in a one-dimensional universe can be achieved and how a suitable, compartmentalized set of molecules can replicate in a manner analogous to real cell replication, i.e., there is segregation and semi-conservative replication of the genetic material, and there is division of the compartment through the construction of an "inner membrane". The approach to self-replication of a spatially delimited entity exemplified by this design differs from the abstract models of replication of the von Neumann or Laing type. In these the replicating entity constructs a copy external to itself and does not undergo any essential replacement of any of its parts. Also, while in these models the concern is primarily with questions of the "logical" type, our design has in mind features identifiable with both energy and information considerations. Thus, the rules which define the underlying "physics and chemistry" imply that the self-replicating entity is a dissipative structure. A constant flux of energy is required to maintain the system far from thermodynamic equilibrium in order to account for multiple steady states, and hence dynamic structure.     

Mixed-mode oscillations in a self-replicating dimerization mechanism

Recently, self-replicating molecules have been synthesized in the laboratory by Rebek. Given the importance of such molecules, we proposed a simple model of a self-replicating dimer, which works as a template for its own formation. Here we consider a three variable model. For the model, we obtain mixed-mode and chaotic oscillations. Also, we find coexistence between two periodic attractors as well as a periodic and a chaotic attractor.     

最小基因组研究进展

随着许多生物体全基因组测序的完成,兴起了最小基因组的研究,即一个能营独立生活的生物体最少需要多少个基因。已知最小细胞支原体基因组是研究最小基因组的重要内容,还通过比较多种已测序基因组COG分析最小基因组,目前通过转座子插入基因突主为和同源重组删除基因的分析,进行最小基因组研究。     

Mollicutes-wall-less bacteria with internal cytoskeletons

The structure and motility of the Mollicutes (Spiroplasma, Mycoplasma, and Acholeplasma) are briefly reviewed. The data are presented from the perspective of prokaryotic and eukaryotic motors, cytoskeletons, and cell motility. The Mollicutes are eubacteria derived from Clostridia by regressive evolution and genome reduction to produce the smallest and simplest free-living and self-replicating cells. Structurally, the Mollicutes are characterized by a complete lack of a cell wall and the presence of an internal cytoskeleton. Spiroplasma, which are helical cells with a flat, ribbon-like cytoskeleton, are amenable to structural and geometrical analysis. Motility and shape changes can be explained and modeled by the cytoskeleton acting as a linear motor.     

Toward an artificial cell based on gene expression in vesicles

We present a new experimental approach to build an artificial cell using the translation machinery of a cell-free expression system as the hardware and a DNA synthetic genome as the software. This approach, inspired by the self-replicating automata of von Neumann, uses cytoplasmic extracts, encapsulated in phospholipid vesicles, to assemble custom-made genetic circuits to develop the functions of a minimal cell. Although this approach can find applications, especially in biotechnology, the primary goal is to understand how a DNA algorithm can be designed to build an operating system that has some of the properties of life. We provide insights on this cell-free approach as well as new results to transform step by step a long-lived vesicle bioreactor into an artificial cell. We show how the green fluorescent protein can be anchored to the membrane and we give indications of a possible insertion mechanism of integral membrane proteins. With vesicles composed of different phospholipids, the fusion protein alpha-hemolysin-eGFP can be expressed to reveal patterns on the membrane. The specific degradation complex ClpXP from E. coli is introduced to create a sink for the synthesized proteins. Perspectives and subsequent limitations of this approach are discussed.     

Evolutionary dynamics of cellular automata-based self-replicators in hostile environments

In this paper we investigate population dynamics, genealogy and complexity-increase of locally interacting populations of cellular automata-based evolving self-replicating loops (evoloops). We outline experiments indicating that the evolutionary growth in complexity, known to be achievable in principle given the complete genetic accessibility granted by universal construction, may be achievable in practice using much simpler replicating structures. By introducing evoloop populations to hostile environments, we demonstrate that selection pressures toward smaller species can be mediated to enable evolutionary accessibility to larger species, which themselves roam a much more vast portion of genetic state-space. We show that this growth in size results from intrinsically biased genealogy inherent in the rules of the evoloop CA, normally suppressed by selection pressures from direct competition favouring the smallest species. This shows that, in populations of simple self-replicating structures, a limited form of complexity-increase may result from a process which is driven by biased genealogical connectivity--a purely emergent property arising out of bottom-up evolutionary dynamics--and not just by adaptation . Implications of this result are discussed and contrasted with other self-replication studies in Artificial Life and Biology.     

Humoral and cellular immune response to RNA immunization with flavivirus replicons derived from tick-borne encephalitis virus

A new vaccination principle against flaviviruses, based on a tick-borne encephalitis virus (TBEV) self-replicating noninfectious RNA vaccine that produces subviral particles, has recently been introduced (R. M. Kofler, J. H. Aberle, S. W. Aberle, S. L. Allison, F. X. Heinz, and C. W. Mandl, Proc. Natl. Acad. Sci. USA 7:1951-1956, 2004). In this study, we evaluated the potential of the self-replicating RNA vaccine in mice in comparison to those of live, attenuated vaccines and a formalin-inactivated whole-virus vaccine (ImmunInject). For this purpose, mice were immunized using gene gun-mediated application of the RNA vaccine and tested for CD8+ T-cell responses, long-term duration, neutralizing capacity, and isotype profile of specific antibodies and protection against lethal virus challenge. We demonstrate that the self-replicating RNA vaccine induced a broad-based, humoral and cellular (Th1 and CD8+ T-cell response) immune response comparable to that induced by live vaccines and that it protected mice from challenge. Even a single immunization with 1 microg of the replicon induced a long-lasting antibody response, characterized by high neutralizing antibody titers, which were sustained for at least 1 year. Nevertheless, it was possible to boost this response further by a second injection with the RNA vaccine, even in the presence of a concomitant CD8+ T-cell response. In this way it was possible to induce a balanced humoral and cellular immune response, similar to infection-induced immunity but without the safety hazards of infectious agents. The results also demonstrate the value of TBEV replicon RNA for inducing protective long-lasting antiviral responses.     

Method for the isolation of the replication region of a bacterial replicon: construction of a mini-F'kn plasmid.

A purified fragment of deoxyribonucleic acid (DNA) that determines resistance to kanamycin and is incapable of self-replication was used to select a self-replicating fragment from an EcoRI endonuclease digest of the sex factor F'lac. This F'lac fragment, exhibiting a molecular weight of 6 X 10(6), carries the genes essential for maintenance of the F replicon in Escherichia coli cells. The constructed mini-F'km plasmid also retains the incompatibility properties of the parent F'lac plasmid. Large amounts of the kanamycin resistance fragment of a molecular weight of 4.5 X 10(6) with an EcoRI-cleaved, self-replicating derivative of colicinogenic plasmid E1 that has a molecular weight of 2.2 X 10(6), The recombinant plasmid is able to replicate extensively in E. coli in medium containing chloramphenicol, and, therefore, large quantities of this plasmid DNA can be obtained. The substantial difference in size between the two fragments in the recombinant plasmid greatly facilitates their separation by preparative agarose gel electrophoresis.     

Rna catalysis and the origin of life

Until the discovery of catalytic RNAs, first the self-splicing intron inTetrahymena and then the bacterial RNAse P, cellular enzymes had always seemed to be protein in nature. The recognition that RNA can catalytically make and break phosphodiester bonds simplifies some of the assumptions required of a rudimentary self-replicating entity. Available information on the chemistry of RNA-catalyzed reactions is reviewed, with particular attention to self-splicing introns and tRNA processing by RNase P. An explicit model for a self-replicating RNA is described. The model postulates a nucleotide binding/polymerization site in the RNA, and takes advantage of intrinsic fluidity in RNA higher order structure to dissociate parent and progeny complementary strands.