A comparison of two cell regulatory models entailing high dimensional attractors representing phenotype

Two models for mammalian cell regulation that invoke the concept of cellular phenotype represented by high dimensional dynamic attractor states are compared. In one model the attractors are derived from an experimentally determined genetic regulatory network (GRN) for the cell type. As the state space architecture within which the attractors are embedded is determined by the binding sites on proteins and the recognition sites on DNA the attractors can be described as “hard-wired” in the genome through the genomic DNA sequence. In the second model attractors arising from the interactions between active gene products (mainly proteins) and independent of the genomic sequence, are descended from a pre-cellular state from which life originated. As this model is based on the cell as an open system the attractor acts as the interface between the cell and its environment. Environmental sources of stress can serve to trigger attractor and therefore phenotypic, transitions without entailing genotypic sequence changes.It is asserted that the evidence from cell and molecular biological research and logic, favours the second model. If correct there are important implications for understanding how environmental factors impact on evolution and may be implicated in hereditary and somatic disease.     

Reverse genetic studies of homologous DNA recombination using the chicken B-lymphocyte line, DT40

DT40 is an avian leucosis virus-transformed chicken B-lymphocyte line which exhibits high ratios of targeted to random integration of transfected DNA constructs. This efficient targeted integration may be related to the ongoing diversification of the variable segment of the immunoglobulin gene through homologous DNA recombination-controlled gene conversion. DT40s are a convenient model system for making gene-targeted mutants. Another advantage is the relative tractability of these cells, which makes it possible to disrupt multiple genes in a single cell and to generate conditionally gene-targeted mutants including temperature-sensitive mutants. There are strong phenotypic similarities between murine and DT40 mutants of various genes involved in DNA recombination. These similarities confirm that the DT40 cell line is a reasonable model for the analysis of vertebrate DNA recombination, despite obvious concerns associated with the use of a transformed cell line, which may have certain cell-line-specific characteristics. Here we describe our studies of homologous DNA recombination in vertebrate somatic cells using reverse genetics in DT40 cells.     

Xenopatients 2.0: Reprogramming the epigenetic landscapes of patient-derived cancer genomes

In the science-fiction thriller film Minority Report, a specialized police department called “PreCrime” apprehends criminals identified in advance based on foreknowledge provided by 3 genetically altered humans called “PreCogs”. We propose that Yamanaka stem cell technology can be similarly used to (epi)genetically reprogram tumor cells obtained directly from cancer patients and create self-evolving personalized translational platforms to foresee the evolutionary trajectory of individual tumors. This strategy yields a large stem cell population and captures the cancer genome of an affected individual, i.e., the PreCog-induced pluripotent stem (iPS) cancer cells, which are immediately available for experimental manipulation, including pharmacological screening for personalized “stemotoxic” cancer drugs. The PreCog-iPS cancer cells will re-differentiate upon orthotopic injection into the corresponding target tissues of immunodeficient mice (i.e., the PreCrime-iPS mouse avatars), and this in vivo model will run through specific cancer stages to directly explore their biological properties for drug screening, diagnosis, and personalized treatment in individual patients. The PreCog/PreCrime-iPS approach can perform sets of comparisons to directly observe changes in the cancer-iPS cell line vs. a normal iPS cell line derived from the same human genetic background. Genome editing of PreCog-iPS cells could create translational platforms to directly investigate the link between genomic expression changes and cellular malignization that is largely free from genetic and epigenetic noise and provide proof-of-principle evidence for cutting-edge “chromosome therapies” aimed against cancer aneuploidy. We might infer the epigenetic marks that correct the tumorigenic nature of the reprogrammed cancer cell population and normalize the malignant phenotype in vivo. Genetically engineered models of conditionally reprogrammable mice to transiently express the Yamanaka stemness factors following the activation of phenotypic copies of specific cancer diseases might crucially evaluate a “reprogramming cure” for cancer. A new era of xenopatients 2.0 generated via nuclear reprogramming of the epigenetic landscapes of patient-derived cancer genomes might revolutionize the current personalized translational platforms in cancer research.     

Gene inactivation as a mechanism for the generation of variability in somatic cells cultivated in vitro

It is proposed that a substantial proportion of cell culture variants result from repression or derepression of genetic information which specifies the synthesis of enzymes. The mechanism responsible for this process may be similar to the inactivation of euchromatic segments in Drosophila resulting from their transposition next to heterochromatin. This model could explain a number of confusing observations made in somatic cell culture systems. (1) The generally high mutation rate for markers in somatic cells. (2) The instability and high reversion rates of a number of genetic markers. (3) Failure of certain drug resistance markers to show the expected decreases in mutation rate with increase in ploidy levels. (4) Anomalous mutation induction kinetics, and lack of specificity of mutagenic agents.Means by which this model can be experimentally examined are proposed. They include: (a) correlation of late labeling patterns and transposition of genetic material with phenotypic changes suspected of having their basis in chromosomal inactivation. (b) Mutagenesis studies using markers which would be expected to show phenotypic variation due to chromosomal inactivation. (c) Studies of gene expression in somatic cell hybrids. (d) Examination of gene products from resistant cell lines suspected of arising from chromosomal inactivation.     

EVOLUTION OF IDENTITY SIGNALS: FREQUENCY‐DEPENDENT BENEFITS OF DISTINCTIVE PHENOTYPES USED FOR INDIVIDUAL RECOGNITION

Identifying broad‐scale evolutionary processes that maintain phenotypic polymorphisms has been a major goal of modern evolutionary biology. There are numerous mechanisms, such as negative frequency‐dependent selection, that may maintain polymorphisms, although it is unknown which mechanisms are prominent in nature. Traits used for individual recognition are strikingly variable and have evolved independently in numerous lineages, providing an excellent model to investigate which factors maintain ecologically relevant phenotypic polymorphisms. Theoretical models suggest that individuals may benefit by advertising their identities with distinctive, recognizable phenotypes. Here, we test the benefits of advertising one's identity with a distinctive phenotype. We manipulated the appearance of Polistes fuscatus paper wasp groups so that three individuals had the same appearance and one individual had a unique, easily recognizable appearance. We found that individuals with distinctive appearances received less aggression than individuals with nondistinctive appearances. Therefore, individuals benefit by advertising their identity with a unique phenotype. Our results provide a potential mechanism through which negative frequency‐dependent selection may maintain the polymorphic identity signals in P. fuscatus. Given that recognition is important for many social interactions, selection for distinctive identity signals may be an underappreciated and widespread mechanism underlying the evolution of phenotypic polymorphisms in social taxa.     

Immortalisation of Primary Cells

Knowledge of the target cells is fundamental to maximise efficiency in attempts at immortalisation of specific cell types. It is also important to optimise the primary cell culture system to promote the survival of the target cell population. Other important factors that may influence the success in obtaining immortalised cells include the toxicity and efficiency of the immortalisation procedure. These can be assessed experimentally and if necessary appropriate techniques can be employed to purify the target cells. When cell lines have been established it is vital to assess them at an early stage for desired scientific and practical features as well as determining their stability and life-span. Furthermore, early characterisation of cell line authenticity (e.g. genetic characters, species of origin) and quality control testing will avoid wasted time and resources should contamination with micro-organisms or another cell line occur. Establishing a programme of immortalisation is a serious undertaking that should only be considered when there are no candidate continuous cell lines available. However, new approaches to modify the biology of cells to give extended life-span, whilst retaining the characteristics of differentiated cells in vivo, will hopefully provide valuable new substrates for in vitro toxicology.     

Using genetic networks and homology to understand the evolution of phenotypic traits

Homology can have different meanings for different kinds of biologists. A phylogenetic view holds that homology, defined by common ancestry, is rigorously identified through phylogenetic analysis. Such homologies are taxic homologies (=synapomorphies). A second interpretation, "biological homology" emphasizes common ancestry through the continuity of genetic information underlying phenotypic traits, and is favored by some developmental geneticists. A third kind of homology, deep homology, was recently defined as "the sharing of the genetic regulatory apparatus used to build morphologically and phylogenetically disparate features." Here we explain the commonality among these three versions of homology. We argue that biological homology, as evidenced by a conserved gene regulatory network giving a trait its "essential identity" (a Character Identity Network or "ChIN") must also be a taxic homology. In cases where a phenotypic trait has been modified over the course of evolution such that homology (taxic) is obscured (e.g. jaws are modified gill arches), a shared underlying ChIN provides evidence of this transformation. Deep homologies, where molecular and cellular components of a phenotypic trait precede the trait itself (are phylogenetically deep relative to the trait), are also taxic homologies, undisguised. Deep homologies inspire particular interest for understanding the evolutionary assembly of phenotypic traits. Mapping these deeply homologous building blocks on a phylogeny reveals the sequential steps leading to the origin of phenotypic novelties. Finally, we discuss how new genomic technologies will revolutionize the comparative genomic study of non-model organisms in a phylogenetic context, necessary to understand the evolution of phenotypic traits.     

Cell line cross-contamination: How aware are mammalian cell culturists of the problem and how to monitor it?

HeLa was the first human cell line established (1952) and became one of the most frequently used lines because of its hardiness and rapid growth rate. During the next two decades, the development of other human cell lines mushroomed. One reason for this became apparent during the 1970s, when it was demonstrated that many of these cell lines had been overgrown and replaced by fast-growing HeLa cells inadvertently introduced into the original cultures. Although the discovery of these "HeLa contaminants" prompted immediate alarm, how aware are cell culturists today of the threat of cell line cross-contamination? To answer this question, we performed a literature search and conducted a survey of 483 mammalian cell culturists to determine how many were using HeLa contaminants without being aware of their true identity and how many were not using available means to ensure correct identity. Survey respondents included scientists, staff, and graduate students in 48 countries. HeLa cells were used by 32% and HeLa contaminants by 9% of survey respondents. Most were also using other cell lines; yet, only about a third of respondents were testing their lines for cell identity. Of all the cell lines used, 35% had been obtained from another laboratory instead of from a repository, thus increasing the risk of false identity. Over 220 publications were found in the PubMed database (1969-2004) in which HeLa contaminants were used as a model for the tissue type of the original cell line. Overall, the results of this study indicate a lack of vigilance in cell acquisition and identity testing. Some researchers are still using HeLa contaminants without apparent awareness of their true identity. The consequences of cell line cross-contamination can be spurious scientific conclusions; its prevention can save time, resources, and scientific reputations.     

Rapid adaptation: a new dimension for evolutionary perspectives in ecology

Although the study of adaptation is central to biology, two types of adaptation are recognized in the biological field: physiological adaptation (accommodation or acclimation; an individual organism’s phenotype is adjusted to its environment) and evolutionary–biological adaptation (adaptation is shaped by natural selection acting on genetic variation). The history of the former concept dates to the late nineteenth and early twentieth centuries, and has more recently been systemized in the twenty-first century. Approaches to the understanding of phenotypic plasticity and learning behavior have only recently been developed, based on cellular–histological and behavioral–neurobiological techniques as well as traditional molecular biology. New developments of the former concepts in phenotypic plasticity are discussed in bacterial persistence, wing di-/polymorphism with transgenerational effects, polyphenism in social insects, and defense traits for predator avoidance, including molecular biology analyses. We also discuss new studies on the concept of genetic accommodation resulting in evolution of phenotypic plasticity through a transgenerational change in the reaction norm based on a threshold model. Learning behavior can also be understood as physiological phenotypic plasticity, associating with the brain–nervous system, and it drives the accelerated evolutionary change in behavioral response (the Baldwin effect) with memory stock. Furthermore, choice behaviors are widely seen in decision-making of animal foragers. Incorporating flexible phenotypic plasticity and learning behavior into modeling can drastically change dynamical behavior of the system. Unification of biological sciences will be facilitated and integrated, such as behavioral ecology and behavioral neurobiology in the area of learning, and evolutionary ecology and molecular developmental biology in the theme of phenotypic plasticity.     

Cell Fate Decisions in Malignant Hematopoiesis: Leukemia Phenotype Is Determined by Distinct Functional Domains of the MN1 Oncogene

Extensive molecular profiling of leukemias and preleukemic diseases has revealed that distinct clinical entities, like acute myeloid (AML) and T-lymphoblastic leukemia (T-ALL), share similar pathogenetic mutations. It is not well understood how the cell of origin, accompanying mutations, extracellular signals or structural differences in a mutated gene determine the phenotypic identity of leukemias. We dissected the functional aspects of different protein regions of the MN1 oncogene and their effect on the leukemic phenotype, building on the ability of MN1 to induce leukemia without accompanying mutations. We found that the most C-terminal region of MN1 was required to block myeloid differentiation at an early stage, and deletion of an extended C-terminal region resulted in loss of myeloid identity and cell differentiation along the T-cell lineage in vivo. Megakaryocytic/erythroid lineage differentiation was blocked by the N-terminal region. In addition, the N-terminus was required for proliferation and leukemogenesis in vitro and in vivo through upregulation of HoxA9, HoxA10 and Meis2. Our results provide evidence that a single oncogene can modulate cellular identity of leukemic cells based on its active gene regions. It is therefore likely that different mutations in the same oncogene may impact cell fate decisions and phenotypic appearance of malignant diseases.     

SNP panel identification assay (SPIA): a genetic-based assay for the identification of cell lines

Translational research hinges on the ability to make observations in model systems and to implement those findings into clinical applications, such as the development of diagnostic tools or targeted therapeutics. Tumor cell lines are commonly used to model carcinogenesis. The same tumor cell line can be simultaneously studied in multiple research laboratories throughout the world, theoretically generating results that are directly comparable. One important assumption in this paradigm is that researchers are working with the same cells. However, recent work using high throughput genomic analyses questions the accuracy of this assumption. Observations by our group and others suggest that experiments reported in the scientific literature may contain pre-analytic errors due to inaccurate identities of the cell lines employed. To address this problem, we developed a simple approach that enables an accurate determination of cell line identity by genotyping 34 single nucleotide polymorphisms (SNPs). Here, we describe the empirical development of a SNP panel identification assay (SPIA) compatible with routine use in the laboratory setting to ensure the identity of tumor cell lines and human tumor samples throughout the course of long term research use.     

Megakaryocyte cell sorting from diosgenin-differentiated human erythroleukemia cells by sedimentation field-flow fractionation

Anticancer differentiation therapy could be one strategy to stop cancer cell proliferation. Human erythroleukemia (HEL) cell line, incubated with 10 microM diosgenin, underwent megakaryocytic differentiation. Thus, the association diosgenin/HEL could be used as a model of chemically induced cellular differentiation and anticancer treatment. The goal of this work was to determine the capacity of sedimentation field-flow fractionation (SdFFF) to sort megakaryocytic differentiated cells. SdFFF cell sorting was associated with cellular characterization methods to calibrate specific elution profiles. As demonstrated by cell size measurement methods, cellular morphology, ploidy, and phenotype, we obtained an enriched, sterile, viable, and functional fraction of megakaryocytic cells. Thus, SdFFF is proposed as a routine method to prepare differentiated cells that will be further used to better understand the megakaryocytic differentiation process.     

论DNA C-值与植物入侵性的关系

外来植物的入侵已引起世界普遍关注,强调并迅速提高对外来植物的预警能力是目前首当其冲的任务,由此,如何预测植物的入侵能力,也就成为入侵生态学的一个核心问题。20世纪90年代以来,关于植物入侵争论的焦点集中于入侵植物本身的生物学特点或入侵生境特点,然而,争议多于结论,至今未能找出有效预测外来植物入侵性的答案。着重从DNAC-值与植物入侵性关系这一角度进行论述。自20世纪30年代以来,染色体数目、大小、倍性在细胞水平的变化被认为可能与植物入侵性相关,因为染色体数目、大小变化是物种在细胞水平上的一种表型变异形式,而细胞水平累积的效应有可能决定着植物整体水平上对环境的适应能力,从而决定植物的分布范围,最终与入侵性相关。但是,这些领域的研究也没有得到一致的结论。近年来,人们将注意力转移至被子植物DNAC-值变化在植物环境适应中的生物学意义。现有资料表明,DNAC-值与细胞大小、体积、重量、发育速率等细胞水平上的表型特征存在正相关关系,这些与核型相关的DNAC-值的影响效应,可扩展到多细胞植物有机体的发育速率,在植物生活史的各个阶段起作用,其中就影响到两个受时间因子限制同时又与植物分布相关联的特征——最短世代时间及生活周期类型,而许多入侵成功植物即表现为世代时间短等特点,对于入侵性植物,其不可避免会受生长时间及分布环境的限制,如能保证其在这两方面占有优势便能入侵成功。已有研究结果表明,某些外来入侵种比同属其它种类具有较低的核DNA含量,由此,提出通过研究植物DNAC值,就有可能预测植物入侵能力的强弱,低DNAC-值的植物具有更强的适应环境的能力,即与入侵性大小呈负相关,这为发现新的植物入侵性预测指标提供了思路。     

DEMOGRAPHIC MECHANISMS DETERMINING THE DYNAMICS OF THE RELATIVE ABUNDANCE OF PHASES IN BIPHASIC LIFE CYCLES1

Studies investigating the demographic traits that drive the patterns of phase dominance (the ploidy ratio) in isomorphic biphasic life cycles have not found an integrative solution. Either fertility or survival has been suggested independently as the main driver. Here, we provide a global theoretical framework on how demographic mechanisms determine the ploidy ratio, unifying previous numerical and observational attempts at this question. The analytical solutions of both the ploidy ratio and its elasticities to model parameters of a stage/size‐structured model patterned after the life cycle of a marine alga were derived and analyzed. A complex interaction among vital rates determines the patterns of phase dominance of biphasic life cycles. Three co‐occurring processes—growth, fertility, and looping—may dominate the dynamics of the population, determining both its growth rate and ploidy ratio. Our analyses show that in species where fertility is low, the ploidy ratio is highly elastic to looping transitions (survival, breakage, and clonal growth). Consequently, the subtle morphological, ecophysiological, and biochemistry phase differences that have been reported in isomorphic life cycles as not explaining the observed ploidy ratios, may, in fact, explain them if they translate into slight phase differences in looping transitions. In species where fertility is low, the looping dissimilarities between phases cannot be too high favoring simultaneously one phase, as the population structure would be completely dominated by that phase. In the case of ecological similarity between phases (equal looping and growth rates between phases), a ploidy ratio different from one can only be set by strong phase differences in fertility.     

Environmental epigenetic transgenerational inheritance and somatic epigenetic mitotic stability

The majority of environmental factors can not modify DNA sequence, but can influence the epigenome. The mitotic stability of the epigenome and ability of environmental epigenetics to influence phenotypic variation and disease, suggests environmental epigenetics will have a critical role in disease etiology and biological areas such as evolutionary biology. The current review presents the molecular basis of how environment can promote stable epigenomes and modified phenotypes, and distinguishes the difference between epigenetic transgenerational inheritance through the germ line versus somatic cell mitotic stability.     

Insight into the basis of root growth in Arabidopsis thaliana provided by a simple mathematical model

Plant organ growth changes under genetic and environmental influences can be observed as altered cell proliferation and volume growth. The two aspects are mutually dependent and intricately related. For comprehensive growth analysis, it is necessary to specify the relationship quantitatively. Here, we develop a simple mathematical model for this purpose. Our model assumes that the biological activity of a given organ is proportional to the cell number of the organ and is allocated into three aspects: cell proliferation, volume growth, and organ maintenance. We analyzed the growth of primary roots of Arabidopsis thaliana (L.) Heynh. in one tetraploid and four diploid strains using this model. The analysis determined various growth parameters, such as specific cost coefficients of cell proliferation and volume growth for each strain. The results provide insight into the basis of interstrain variations and ploidy effects in root growth.     

Dynamic genome plasticity during unisexual reproduction in the human fungal pathogen Cryptococcus deneoformans

Genome copy number variation occurs during each mitotic and meiotic cycle and it is crucial for organisms to maintain their natural ploidy. Defects in ploidy transitions can lead to chromosome instability, which is a hallmark of cancer. Ploidy in the haploid human fungal pathogen Cryptococcus neoformans is exquisitely orchestrated and ranges from haploid to polyploid during sexual development and under various environmental and host conditions. However, the mechanisms controlling these ploidy transitions are largely unknown. During C. deneoformans (formerly C. neoformans var. neoformans, serotype D) unisexual reproduction, ploidy increases prior to the onset of meiosis, can be independent from cell-cell fusion and nuclear fusion, and likely occurs through an endoreplication pathway. To elucidate the molecular mechanisms underlying this ploidy transition, we identified twenty cell cycle-regulating genes encoding cyclins, cyclin-dependent kinases (CDK), and CDK regulators. We characterized four cyclin genes and two CDK regulator genes that were differentially expressed during unisexual reproduction and contributed to diploidization. To detect ploidy transition events, we generated a ploidy reporter, called NURAT, which can detect copy number increases via double selection for nourseothricin-resistant, uracil-prototrophic cells. Utilizing this ploidy reporter, we showed that ploidy transition from haploid to diploid can be detected during the early phases of unisexual reproduction. Interestingly, selection for the NURAT reporter revealed several instances of segmental aneuploidy of multiple chromosomes, which conferred azole resistance in some isolates. These findings provide further evidence of ploidy plasticity in fungi with significant biological and public health implications.