Cloning of HumanENC-1and Evaluation of Its Expression and Regulation in Nervous System Tumors

We recently identified and characterized a novel murine gene,ENC-1,that is expressed primarily in the nervous system and encodes an actin-binding protein. To gain insight into a potential role forENC-1gene in the processes of cell differentiation and malignant transformation in the human nervous system, we first cloned and characterized the human homologue ofENC-1.The humanENC-1gene appeared to be highly expressed in adult brain and spinal cord, and in a number of cell lines derived from nervous system tumors we detected low steady-state levels ofENC-1mRNA. We used a neuroblastoma differentiation model, the retinoic acid-induced neuronal differentiation of SMS-KCNR cells, to study the regulation of theENC-1gene during neural crest cell differentiation. We found that the expression ofENC-1increased dramatically in the differentiated SMS-KCNR cells as compared to control undifferentiated cells. These results suggest thatENC-1expression plays a role during differentiation of neural crest cells and may be down regulated in neuroblastoma tumors.     

Volterra-Verhulst prey-predator systems with time dependent coefficients: Diffusion type approximation and periodic solutions

In treating the Volterra-Verhulst prey-predator system with time dependent coefficients, we ask how far this deterministic system represents or approximates the dynamics of the population evolving in a realistic environment which is stochastic in nature. We consider a stochastic system withsmall Gaussian noise type fluctuations. It is shown that the higher moments of the deviation of the deterministic system from the stochastic approach zero as the strength δ of the perturbation decays to zero. For any δ>0 and allT>0, ε>0, the sample population paths that stay within ε distance from the deterministic path during [0,T] form a collection of positive probability. In comparing the stationary distributions of the two systems, we show that the weak limits of those of the stochastic system form a subset of those of the deterministic system. This is in analogy with a result of May connected with the stability of the two systems. Plant and rodent populations possess periodic parameters andexhibit periodic behaivor. We establish theoretically this periodicity under periodicity conditions on the coefficients and perturbing random forces. We also establish a central limit property for the prey-predator system.     

Image analysis as a tool for quantitative phycology: a computational approach to cyanobacterial taxa identification

In the following work we discuss the application of image processing and pattern recognition to the field of quantitative phycology. We overview the area of image processing and review previously published literature pertaining to the image analysis of phycological images and, in particular, cyanobacterial image processing. We then discuss the main operations used to process images and quantify data contained within them. To demonstrate the utility of image processing to cyanobacteria classification, we present details of an image analysis system for automatically detecting and classifying several cyanobacterial taxa of Lake Biwa, Japan. Specifically, we initially target the genus Microcystis for detection and classification from among several species of Anabaena. We subsequently extend the system to classify a total of six cyanobacteria species. High-resolution microscope images containing a mix of the above species and other nontargeted objects are analyzed, and any detected objects are removed from the image for further analysis. Following image enhancement, we measure object properties and compare them to a previously compiled database of species characteristics. Classification of an object as belonging to a particular class membership (e.g., “Microcystis,”“A. smithii,”“Other,” etc.) is performed using parametric statistical methods. Leave-one-out classification results suggest a system error rate of approximately 3%. Received: September 6, 1999 / Accepted: February 6, 2000     

Estimating position and velocity of a submerged moving object by the clawed frog Xenopus and by fish—A cybernetic approach

The lateral-line system is a unique facility of aquatic animals to locate predator, prey, or conspecifics. We present a detailed model of how the clawed frog Xenopus, or fish, can localize submerged moving objects in three dimensions by using their lateral-line system. In so doing we develop two models of a slightly different nature. First, we exploit the characteristic properties of the velocity field, such as zeros and maxima or minima, that a moving object generates at the lateral-line organs and that are directly accessible neuronally, in the context of a simplified geometry. In addition, we show that the associated neuronal model is robust with respect to noise. Though we focus on the superficial neuromasts of Xenopus the same arguments apply mutatis mutandis to the canal lateral-line system of fish. Second, we present a full-blown three-dimensional reconstruction of the source on the basis of a maximum likelihood argument.     

Fucci2a: A bicistronic cell cycle reporter that allows Cre mediated tissue specific expression in mice

Markers of cell cycle stage allow estimation of cell cycle dynamics in cell culture and during embryonic development. The Fucci system incorporates genetically encoded probes that highlight G1 and S/G2/M phases of the cell cycle allowing live imaging. However the available mouse models that incorporate Fucci are beset by problems with transgene inactivation, varying expression level, lack of conditional potential and/or the need to maintain separate transgenes—there is no transgenic mouse model that solves all these problems. To address these shortfalls we re-engineered the Fucci system to create 2 bicistronic Fucci variants incorporating both probes fused using the Thosea asigna virus 2A (T2A) self cleaving peptide. We characterize these variants in stable 3T3 cell lines. One of the variants (termed Fucci2a) faithfully recapitulated the nuclear localization and cell cycle stage specific florescence of the original Fucci system. We go on to develop a conditional mouse allele (R26Fucci2aR) carefully designed for high, inducible, ubiquitous expression allowing investigation of cell cycle status in single cell lineages within the developing embryo. We demonstrate the utility of R26Fucci2aR for live imaging by using high resolution confocal microscopy of ex vivo lung, kidney and neural crest development. Using our 3T3 system we describe and validate a method to estimate cell cycle times from relatively short time-lapse sequences that we then apply to our neural crest data. The Fucci2a system and the R26Fucci2aR mouse model are compelling new tools for the investigation of cell cycle dynamics in cell culture and during mouse embryonic development.     

An efficient blue-white screening system for markerless deletions and stable integrations in Streptomyces chromosomes based on the blue pigment indigoidine biosynthetic gene bpsA

We previously developed an efficient deletion system for streptomycetes based on the positive selection of double-crossover events using bpsA, a gene for producing the blue pigment indigoidine. Using this system, we removed interfering secondary metabolite clusters from Streptomyces lividans TK24, resulting in RedStrep strains with dramatically increased heterologous production of mithramycin A (up to 3-g/l culture). This system, however, required a time-consuming step to remove the resistance marker genes. In order to simplify markerless deletions, we prepared a new system based on the plasmid pAMR18A. This plasmid contains a large polylinker with many unique restriction sites flanked by apramycin and kanamycin resistance genes and the bpsA gene for selecting a double-crossover event. The utility of this new markerless deletion system was demonstrated by its deletion of a 21-kb actinorhodin gene cluster from Streptomyces lividans TK24 with 30% efficiency. We used this system to efficiently remove the matA and matB genes in selected RedStrep strains, resulting in biotechnologically improved strains with a highly dispersed growth phenotype involving non-pelleting small and open mycelia. No further increase in mithramycin A production was observed in these new RedStrep strains, however. We also used this system for the markerless insertion of a heterologous mCherry gene, an improved variant of the monomeric red fluorescent protein, under the control of the strong secretory signal sequence of the subtilisin inhibitor protein, into the chromosome of S. lividans TK24. The resulting recombinant strains efficiently secreted mCherry into the growth medium in a yield of 30 mg/l.

     

A microfluidic gradient mixer-flow chamber as a new tool to study biofilm development under defined solute gradients

Understanding the dynamics of biofilm development in response to chemical cues and signals is required toward the development of controllable biofilm-mediated bioprocesses. In this study, we report a new biofilm growth system that integrates a microfluidic gradient mixer with a biofilm growth chamber. The biofilm growth system allows biofilms to grow under defined solute gradients and enables nondestructive monitoring of the biofilm development dynamics in response to the defined gradients. The solute gradients generated in the system were simulated and then validated experimentally. We then demonstrated the applicability of the biofilm growth system in studying biofilm development under defined solute gradients. Specifically, we examined biofilm development of Shewanella oneidensis and Comamonas testosteroni under a defined calcium and nitrate gradient, respectively. Using two C. testosteroni strains (WDL7 and I2), we further demonstrated the applicability of our biofilm growth system to study the development of coculture biofilms under a defined solute gradient. Our results show that the biofilm growth system we have developed here can be a promising tool to reveal the dynamics of biofilm development in response to chemical cues and signals as well as the interorganism interactions in coculture biofilms.     

Toward a Practical Ontology for Socioeconomic Metabolism

The complexity of data and methods in industrial ecology (IE) keeps growing, and the demand for comprehensive and interdisciplinary assessments increases. To keep up with this development, the field needs a data infrastructure that allows researchers to annotate, store, retrieve, combine, and exchange data at low cost, without loss of information, and across disciplines and model frameworks. A consensus‐building debate about how to describe the common object of study, socioeconomic metabolism (SEM), is necessary for the development of practical data structures and databases. We review the definitions of basic concepts to describe SEM in IE and related fields such as integrated assessment modeling. We find that many definitions are not compatible, are implicit, and are sometimes lacking. To resolve the conflicts and inconsistencies within the current definitions, we propose a hierarchical system of terms and definitions, a practical ontology, for describing objects, their properties, and events in SEM. We propose a typology of object properties and use sets to group objects into a hierarchical, mutually exclusive, and collectively exhaustive (H‐MECE) classification. This grouping leads to a general definition of stocks. We show that a MECE representation of events necessarily requires two complementary concepts: processes and flows, for which we propose general definitions based on sets. Using these definitions, we show that the system structure of any interdisciplinary model of SEM can be formulated as a directed graph. We propose guidelines for semantic data annotation and database design, which can help to turn the vision of a powerful data infrastructure for SEM research into reality.     

Mating system variation along a successional gradient in the allogamous and colonizing plant Crepis sancta (Asteraceae)

We analysed mating system in an annual and colonizing plant, Crepis sancta, that occupies different successional stages in the French Mediterranean region. Based on a previous experiment, we hypothesized that low inbreeding depression measured in young successional stages should select for selfing whereas higher inbreeding depression in old stages should select for outcrossing. Nine populations of C. sancta (Asteraceae) from contrasting successional stages were used to analyse (1) Seed set after autonomous and enforced selfing in controlled conditions and (2) outcrossing rates in natural conditions using allozymes (progeny array analysis). We found that C. sancta possesses a pseudo‐self‐incompatibility system and that mating system varies among populations. Allozymes revealed that the population multilocus outcrossing rates vary from 0.77 to 0.99. The lowest outcrossing rates occur in the youngest successional stages and complete outcrossing is found in old stages. The data partially agree with the predictions we made and the results are more generally discussed in the light of factors changing during succession. We did not find any evidence of reproductive assurance in the nine populations, contrary to what is often assumed as a major factor governing mating system evolution in colonizing species. We propose that mating system variation can be interpreted as the result of the balance between the cost of outcrossing and inbreeding depression in a metapopulation context.     

Differentiation of a Miniature Inverted Transposable Element （MITE） System in Asian Rice Cultivars and Its Inference for a Diphyletic Origin of Two Subspecies of Asian Cultivated Rice

In the present study, we report a survey on a Miniature Inverted Transposable Element （MITE） system known as mPing in 102 varieties of Asian cultivated rice （Oryza sativa L.）. We found that mPing populations could be generalized Into two families, mPing-1 and mPing-2, according to their sequence structures. Further analysis showed that these two families of mPing had significant bias in their distribution pattern in two subspecies of rice, namely O. sativa ssp. japonica and indica. 0. sativa japonica has a higher proportion of mPing-1 as a general trait, whereas 0. sativa indica has a higher proportion of roPing-2. We also examined the mPing system In a doubled haploid （DH） cross-breeding population of jingxi 17 （japonica） and zhaiyeqing 8 （indica） varieties and observed that the mPing system was not tightly linked to major subspecies-determining genes. Furthermore, we checked the mPing system in 28 accessions of Asian common wild rice O. rufipogon and found the roPing system in 0. rufipogon. The distribution pattern of the roPing system in O. rufipogon indicated a diphyletlc origin of the Asian cultivated rice O. sativa species. We did not find the mPing system in another 20 Oryza species. These results substantiated a previous hypothesis that O. ruflpogon and O. nivara species were the closest relatives of O. sativa and that the two extant subspecies of O. sativa were evolved independently from corresponding ecotypes of O. ruflpogon.     

A discrete,size-structured model of phytoplankton growth in the chemostat: introduction of inhomogeneous cell division size

 We introduce inhomogeneous, substrate dependent cell division in a time discrete, nonlinear matrix model of size-structured population growth in the chemostat, first introduced by Gage et al. [8] and later analysed by Smith [13]. We show that mass conservation is verified, and conclude that our system admits one non zero globally stable equilibrium, which we express explicitly. Then we run numerical simulations of the system, and compare the predictions of the model to data related to phytoplankton growth, whose obtention we discuss. We end with the identification of several parameters of the system. Received: 9 February 2000 / Revised version: 10 October 2001 / Published online: 23 August 2002 RID="*" ID="*" Present address: Department of Mathematics and Statistics, University of Victoria, B.C., Canada. e-mail: jarino@math.uvic.ca Key words or phrases: Chemostat – Structured population models – Discrete model – Inhomogeneous division size     

Mechanisms of Recombination: Lessons from E. coli

The genetics and biochemistry of genetic recombination in E. coli has been studied for over four decades and provides a useful model system to understand recombination in other organisms. Here we provide an overview of the mechanisms of recombination and how such processes contribute to DNA repair. We describe the E. coli functions that are known to contribute to these mechanisms, step by step, and summarize their biochemical properties in relation to the role these proteins play in vivo. We feature areas of investigation that are newly emerging, as well as work that provides a historical perspective to the field. Finally, we highlight some of the questions that remain unanswered.     

Use of virus-induced gene silencing to characterize genes involved in modulating hypersensitive cell death in maize

Plant disease resistance proteins (R-proteins) detect specific pathogen-derived molecules, triggering a defence response often including a rapid localized cell death at the point of pathogen penetration called the hypersensitive response (HR). The maize Rp1-D21 gene encodes a protein that triggers a spontaneous HR causing spots on leaves in the absence of any pathogen. Previously, we used fine mapping and functional analysis in a Nicotiana benthamiana transient expression system to identify and characterize a number of genes associated with variation in Rp1-D21-induced HR. Here we describe a system for characterizing genes mediating HR, using virus-induced gene silencing (VIGS) in a maize line carrying Rp1-D21. We assess the roles of 12 candidate genes. Three of these genes, SGT1, RAR1, and HSP90, are required for HR induced by a number of R-proteins across several plant–pathogen systems. We confirmed that maize HSP90 was required for full Rp1-D21-induced HR. However, suppression of SGT1 expression unexpectedly increased the severity of Rp1-D21-induced HR while suppression of RAR1 expression had no measurable effect. We confirmed the effects on HR of two genes we had previously validated in the N. benthamiana system, hydroxycinnamoyltransferase and caffeoyl CoA O-methyltransferase. We further showed the suppression the expression of two previously uncharacterized, candidate genes, IQ calmodulin binding protein (IQM3) and vacuolar protein sorting protein 37, suppressed Rp1-D21-induced HR. This approach is an efficient way to characterize the roles of genes modulating the hypersensitive defence response and other dominant lesion phenotypes in maize.     

Comparative genomic analysis of bacteriocin-producing Weissella cibaria 110

Weissella cibaria 110 was isolated from plaa-som, a Thai fermented fish product, and known to produce the weissellicin 110 bacteriocin. We carried out comprehensive comparative genomic analysis of W. cibaria 110 with four other non-bacteriocin-producing W. cibaria strains and identified potential antibiotic-resistant genes. We further identified a type III restriction-modification system, a TA system, and a bacteriocin gene cluster that are unique in W. cibaria 110. Genes related to bacteriocin biosynthesis are organized in clusters and are encoded with minimum genetic machinery consisting of structural cognate immunity genes, including ABC transporter and immunity protein. Finally, we predicted W. cibaria 110 to produce a class IId bacteriocin, weissellicin 110, which is 31 amino acids in length and contains a 21-amino-acid N-terminal leader peptide. This is the first bacteriocin-producing sequencing genome in W. cibaria, and we describe the difference between the bacteriocin-producing and non bacteriocin-producing strains from genome point of view.

     

Improved cadaverine production from mutant Klebsiella oxytoca lysine decarboxylase

Cadaverine has the potential to become an important platform chemical for the production of nylon. Previously, a system that overexpresses the Klebsiella oxytoca lysine decarboxylase in Escherichia coli was engineered. The system was optimized by codon optimization, and tuning the expression level of the gene by testing various promoters and inducer concentrations. Here, we further improved the system by optimizing the sequence located in the region of the ribosome‐binding site in order to enhance translation efficiency. We also identified mutant lysine decarboxylase enzymes that demonstrated enhanced cadaverine‐production ability. Together, these modifications increased cadaverine production in the system by 50%, and the system has a yield of 80% from lysine‐HCl under the conditions we tested. This is the first time that a system to produce cadaverine using the lysine decarboxylase from K. oxytoca performed at a level that is competitive with the traditional systems using the E. coli lysine decarboxylases in both lab‐scale and batch fermentation conditions.     

Cell cycle dynamics in a response/signalling feedback system with a gap

We consider a dynamical model of cell cycles of n cells in a culture in which cells in one specific phase (S for signalling) of the cell cycle produce chemical agents that influence the growth/cell cycle progression of cells in another phase (R for responsive). In the case that the feedback is negative, it is known that subpopulations of cells tend to become clustered in the cell cycle; while for a positive feedback, all the cells tend to become synchronized. In this paper, we suppose that there is a gap between the two phases. The gap can be thought of as modelling the physical reality of a time delay in the production and action of the signalling agents. We completely analyse the dynamics of this system when the cells are arranged into two cell cycle clusters. We also consider the stability of certain important periodic solutions in which clusters of cells have a cyclic arrangement and there are just enough clusters to allow interactions between them. We find that the inclusion of a small gap does not greatly alter the global dynamics of the system; there are still large open sets of parameters for which clustered solutions are stable. Thus, we add to the evidence that clustering can be a robust phenomenon in biological systems. However, the gap does effect the system by enhancing the stability of the stable clustered solutions. We explain this phenomenon in terms of contraction rates (Floquet exponents) in various invariant subspaces of the system. We conclude that in systems for which these models are reasonable, a delay in signalling is advantageous to the emergence of clustering.     

Detecting and avoiding likely false‐positive findings – a practical guide

Recently there has been a growing concern that many published research findings do not hold up in attempts to replicate them. We argue that this problem may originate from a culture of ‘you can publish if you found a significant effect’. This culture creates a systematic bias against the null hypothesis which renders meta‐analyses questionable and may even lead to a situation where hypotheses become difficult to falsify. In order to pinpoint the sources of error and possible solutions, we review current scientific practices with regard to their effect on the probability of drawing a false‐positive conclusion. We explain why the proportion of published false‐positive findings is expected to increase with (i) decreasing sample size, (ii) increasing pursuit of novelty, (iii) various forms of multiple testing and researcher flexibility, and (iv) incorrect P‐values, especially due to unaccounted pseudoreplication, i.e. the non‐independence of data points (clustered data). We provide examples showing how statistical pitfalls and psychological traps lead to conclusions that are biased and unreliable, and we show how these mistakes can be avoided. Ultimately, we hope to contribute to a culture of ‘you can publish if your study is rigorous’. To this end, we highlight promising strategies towards making science more objective. Specifically, we enthusiastically encourage scientists to preregister their studies (including a priori hypotheses and complete analysis plans), to blind observers to treatment groups during data collection and analysis, and unconditionally to report all results. Also, we advocate reallocating some efforts away from seeking novelty and discovery and towards replicating important research findings of one's own and of others for the benefit of the scientific community as a whole. We believe these efforts will be aided by a shift in evaluation criteria away from the current system which values metrics of ‘impact’ almost exclusively and towards a system which explicitly values indices of scientific rigour.     

Der problemlöser robbimat

We first show how to formalize environments in which situations can be changed only by action, and in which a situation should be brought about that is acceptable. This leads us to the concept of a problem constellation that consists of an action system, a reaction system, a judgement system, and an initial situation. We then present a cybernetical system which we call Robbimat. It is capable of testing problem constellations, of modifying them according to the results of the tests, and of judging the final constellation. We designed Robbimat in order to have favourable problem constellations automatically generated. As an example we discuss a simple game. It constitutes a win-loss-environment the structure of which can be deduced from the payoff function. We show how Robbimat analyses such an environment, and utilizing its structure classifies the initial situation.

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Wir danken Herrn Manfred Linder für seine Mitarbeit.