Kinetic and Dynamic Delaunay tetrahedralizations in three dimensions

We describe the implementation of algorithms to construct and maintain three-dimensional dynamic Delaunay triangulations with kinetic vertices using a three-simplex data structure. The code is capable of constructing the geometric dual, the Voronoi or Dirichlet tessellation. Initially, a given list of points is triangulated. Time evolution of the triangulation is not only governed by kinetic vertices but also by a changing number of vertices. We use three-dimensional simplex flip algorithms, a stochastic visibility walk algorithm for point location and in addition, we propose a new simple method of deleting vertices from an existing three-dimensional Delaunay triangulation while maintaining the Delaunay property. The dual Dirichlet tessellation can be used to solve differential equations on an irregular grid, to define partitions in cell tissue simulations, for collision detection etc.Comment: 29 pg (preprint), 12 figures, 1 table Title changed (mainly nomenclature), referee suggestions included, typos corrected, bibliography update

The EM Algorithm and the Rise of Computational Biology

In the past decade computational biology has grown from a cottage industry with a handful of researchers to an attractive interdisciplinary field, catching the attention and imagination of many quantitatively-minded scientists. Of interest to us is the key role played by the EM algorithm during this transformation. We survey the use of the EM algorithm in a few important computational biology problems surrounding the "central dogma"; of molecular biology: from DNA to RNA and then to proteins. Topics of this article include sequence motif discovery, protein sequence alignment, population genetics, evolutionary models and mRNA expression microarray data analysis.Comment: Published in at http://dx.doi.org/10.1214/09-STS312 the Statistical Science (http://www.imstat.org/sts/) by the Institute of Mathematical Statistics (http://www.imstat.org

Filtered Networks of Evolutionary Processors

* Supported by INTAS 00-626 and TIC 2003-09319-c03-03.This paper presents some connectionist models that are widely used to solve NP-problems. Most well known numeric models are Neural Networks that are able to approximate any function or classify any pattern set provided numeric information is injected into the net. Neural Nets usually have a supervised or unsupervised learning stage in order to perform desired response. Concerning symbolic information new research area has been developed, inspired by George Paun, called Membrane Systems. A step forward, in a similar Neural Network architecture, was done to obtain Networks of Evolutionary Processors (NEP). A NEP is a set of processors connected by a graph, each processor only deals with symbolic information using rules. In short, objects in processors can evolve and pass through processors until a stable configuration is reach. This paper just shows some ideas about these two models

The stability and activity of human neuroserpin are modulated by a salt bridge that stabilises the reactive centre loop

Neuroserpin (NS) is an inhibitory protein belonging to the serpin family and involved in several pathologies, including the dementia Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB), a genetic neurodegenerative disease caused by accumulation of NS polymers. Our Molecular Dynamics simulations revealed the formation of a persistent salt bridge between Glu289 on strand s2C and Arg362 on the Reactive Centre Loop (RCL), a region important for the inhibitory activity of NS. Here, we validated this structural feature by simulating the Glu289Ala mutant, where the salt bridge is not present. Further, MD predictions were tested in vitro by purifying recombinant Glu289Ala NS from E. coli. The thermal and chemical stability along with the polymerisation propensity of both Wild Type and Glu289Ala NS were characterised by circular dichroism, emission spectroscopy and non-denaturant gel electrophoresis, respectively. The activity of both variants against the main target protease, tissue-type plasminogen activator (tPA), was assessed by SDS-PAGE and chromogenic kinetic assay. Our results showed that deletion of the salt bridge leads to a moderate but clear reduction of the overall protein stability and activity

Retrotransposon Tto1: functional analysis and engineering for insertional mutagenesis

Retrotransposons are genomic parasites activated by stress conditions that can be seriously detrimental for their host. In this work I demonstrate that Tto1, a typical plant LTR retrotransposon with insertion preference into genes can be turned into a synthetic molecular tool for gene tagging in plants and can be used to predict models for its replication steps. Although retrotransposons have been already used in plant mutagenesis, such application always required establishing protocols for tissue cultures and regeneration in vitro. Here, I show that sequence engineering of Tto1 provides the possibility to obtain transposition in vivo, with a simple screening method based on PCR and with the advantage to skip all in vitro manipulations. An artificial -estradiol inducible promoter has been used to obtain transposition “on demand” in Arabidopsis plants, which generates stable unlinked insertions that follow mendelian segregation in the progeny. Comparing serial deletions of 3’ LTR of the engineered inducible Tto1 (iTto1), I have mapped its two natural terminators and identified the “minimal” R (redundant) region required to achieve the complete reverse transcription of the genomic mRNA into a new cDNA copy. Interestingly, the transcripts ending at the major “early” terminator cannot support reverse transcription, suggesting a mechanism of natural control on the expression. Transcripts with a more extended termination point contain 100 essential nucleotides that define the active nucleus of the R region. This sequence promotes the formation of a stable hairpin structure that “kisses” a complementary identical hairpin on the cDNA and determines the formation of the characteristic cDNA/mRNA heteroduplex. Since the LTR is a repeated sequence the definition of a minimal redundant region has also the important implication to reduce the only possible target for sequence-based gene silencing, which should lead to an increase of the mutagenic efficiency of iTto1. Additional investigations have been carried out in attempt to identify points of improvement of iTto1 performances. By sequence alignment I identified different versions of the integrase that might have influence on insertion efficiency. Furthermore I tested the pOp6/LhGR-N system that will provide higher expression levels in different host plants. The final goal of my work is to extend the application of iTto1 to crop mutagenesis, therefore a big part of my work has been spent to develop Tto1 constructs with activity in barley. Transgenic plants have been obtained, however the constructs still need further experimentation

Molecular recording of mammalian embryogenesis.

Ontogeny describes the emergence of complex multicellular organisms from single totipotent cells. This field is particularly challenging in mammals, owing to the indeterminate relationship between self-renewal and differentiation, variation in progenitor field sizes, and internal gestation in these animals. Here we present a flexible, high-information, multi-channel molecular recorder with a single-cell readout and apply it as an evolving lineage tracer to assemble mouse cell-fate maps from fertilization through gastrulation. By combining lineage information with single-cell RNA sequencing profiles, we recapitulate canonical developmental relationships between different tissue types and reveal the nearly complete transcriptional convergence of endodermal cells of extra-embryonic and embryonic origins. Finally, we apply our cell-fate maps to estimate the number of embryonic progenitor cells and their degree of asymmetric partitioning during specification. Our approach enables massively parallel, high-resolution recording of lineage and other information in mammalian systems, which will facilitate the construction of a quantitative framework for understanding developmental processes

Astrocytic Ion Dynamics: Implications for Potassium Buffering and Liquid Flow

We review modeling of astrocyte ion dynamics with a specific focus on the implications of so-called spatial potassium buffering, where excess potassium in the extracellular space (ECS) is transported away to prevent pathological neural spiking. The recently introduced Kirchoff-Nernst-Planck (KNP) scheme for modeling ion dynamics in astrocytes (and brain tissue in general) is outlined and used to study such spatial buffering. We next describe how the ion dynamics of astrocytes may regulate microscopic liquid flow by osmotic effects and how such microscopic flow can be linked to whole-brain macroscopic flow. We thus include the key elements in a putative multiscale theory with astrocytes linking neural activity on a microscopic scale to macroscopic fluid flow.Comment: 27 pages, 7 figure

Parallel Genetics of Gene Regulatory Sequences in Caenorhabditis elegans

Wie regulatorische Sequenzen die Genexpression steuern, ist von grundlegender Bedeutung für die Erklärung von Phänotypen in Gesundheit und Krankheit. Die Funktion regulatorischer Sequenzen muss letztlich in ihrer genomischen Umgebung und in entwicklungs- oder gewebespezifischen Zusammenhängen verstanden werden. Da dies eine technische Herausforderung ist, wurden bisher nur wenige regulatorische Elemente in vivo charakterisiert. Hier verwenden wir Induktion von Cas9 und multiplexed-sgRNAs, um hunderte von Mutationen in Enhancern/Promotoren und 3′ UTRs von 16 Genen in C. elegans zu erzeugen. Wir quantifizieren die Auswirkungen von Mutationen auf Genexpression und Physiologie durch gezielte RNA- und DNA-Sequenzierung. Bei der Anwendung unseres Ansatzes auf den 3′ UTR von lin-41, bei der wir hunderte von Mutanten erzeugen, stellen wir fest, dass die beiden benachbarten Bindungsstellen für die miRNA let-7 die lin-41-Expression größtenteils unabhängig voneinander regulieren können, mit Hinweisen auf eine mögliche kompensatorische Interaktion. Schließlich verbinden wir regulatorische Genotypen mit phänotypischen Merkmalen für mehrere Gene. Unser Ansatz ermöglicht die parallele Analyse von genregulatorischen Sequenzen direkt in Tieren.How regulatory sequences control gene expression is fundamental for explaining phenotypes in health and disease. The function of regulatory sequences must ultimately be understood within their genomic environment and development- or tissue-specific contexts. Because this is technically challenging, few regulatory elements have been characterized in vivo. Here, we use inducible Cas9 and multiplexed guide RNAs to create hundreds of mutations in enhancers/promoters and 3′ UTRs of 16 genes in C. elegans. We quantify the impact of mutations on expression and physiology by targeted RNA sequencing and DNA sampling. When applying our approach to the lin-41 3′ UTR, generating hundreds of mutants, we find that the two adjacent binding sites for the miRNA let-7 can regulate lin-41 expression largely independently of each other, with indications of a compensatory interaction. Finally, we map regulatory genotypes to phenotypic traits for several genes. Our approach enables parallel analysis of gene regulatory sequences directly in animals

Structural and biochemical characterization of the exopolysaccharide deacetylase Agd3 required for Aspergillus fumigatus biofilm formation

The exopolysaccharide galactosaminogalactan (GAG) is an important virulence factor of the fungal pathogen Aspergillus fumigatus. Deletion of a gene encoding a putative deacetylase, Agd3, leads to defects in GAG deacetylation, biofilm formation, and virulence. Here, we show that Agd3 deacetylates GAG in a metal-dependent manner, and is the founding member of carbohydrate esterase family CE18. The active site is formed by four catalytic motifs that are essential for activity. The structure of Agd3 includes an elongated substrate-binding cleft formed by a carbohydrate binding module (CBM) that is the founding member of CBM family 87. Agd3 homologues are encoded in previously unidentified putative bacterial exopolysaccharide biosynthetic operons and in other fungal genomes. The exopolysaccharide galactosaminogalactan (GAG) is an important virulence factor of the fungal pathogen Aspergillus fumigatus. Here, the authors study an A. fumigatus enzyme that deacetylates GAG in a metal-dependent manner and constitutes a founding member of a new carbohydrate esterase family.Bio-organic Synthesi