An Evolutionary/Biochemical Connection Between Promoter- and Primer-Dependent Polymerases Revealed by Selective Evolution of Ligands by Exponential Enrichment (SELEX).

PubMed

Fenstermacher, Katherine J; Achuthan, Vasudevan; Schneider, Thomas D; DeStefano, Jeffrey J

2018-01-16

DNA polymerases (DNAPs) recognize 3' recessed termini on duplex DNA and carry out nucleotide catalysis. Unlike promoter-specific RNA polymerases (RNAPs), no sequence specificity is required for binding or initiation of catalysis. Despite this, previous results indicate that viral reverse transcriptases bind much more tightly to DNA primers that mimic the polypurine tract. In the current report, primer sequences that bind with high affinity to Taq and Klenow polymerases were identified using a modified Selective Evolution of Ligands by Exponential Enrichment (SELEX) approach. Two Taq -specific primers that bound ∼10 (Taq1) and over 100 (Taq2) times more stably than controls to Taq were identified. Taq1 contained 8 nucleotides (5' -CACTAAAG-3') that matched the phage T3 RNAP "core" promoter. Both primers dramatically outcompeted primers with similar binding thermodynamics in PCR reactions. Similarly, exonuclease minus Klenow polymerase also selected a high affinity primer that contained a related core promoter sequence from phage T7 RNAP (5' -ACTATAG-3'). For both Taq and Klenow, even small modifications to the sequence resulted in large losses in binding affinity suggesting that binding was highly sequence-specific. The results are discussed in the context of possible effects on multi-primer (multiplex) PCR assays, molecular information theory, and the evolution of RNAPs and DNAPs. Importance This work further demonstrates that primer-dependent DNA polymerases can have strong sequence biases leading to dramatically tighter binding to specific sequences. These may be related to biological function, or be a consequences of the structural architecture of the enzyme. New sequence specificity for Taq and Klenow polymerases were uncovered and among them were sequences that contained the core promoter elements from T3 and T7 phage RNA polymerase promoters. This suggests the intriguing possibility that phage RNA polymerases exploited intrinsic binding affinities of ancestral DNA polymerases to develop their promotors. Conversely, DNA polymerases could have evolved from related RNA polymerases and retained the intrinsic binding preference despite there being no clear function for such a preference in DNA biology. Copyright © 2018 American Society for Microbiology.

Structural and functional characterization of a calcium-activated cation channel from Tsukamurella paurometabola

NASA Astrophysics Data System (ADS)

Dhakshnamoorthy, Balasundaresan; Rohaim, Ahmed; Rui, Huan; Blachowicz, Lydia; Roux, Benoît

2016-09-01

The selectivity filter is an essential functional element of K+ channels that is highly conserved both in terms of its primary sequence and its three-dimensional structure. Here, we investigate the properties of an ion channel from the Gram-positive bacterium Tsukamurella paurometabola with a selectivity filter formed by an uncommon proline-rich sequence. Electrophysiological recordings show that it is a non-selective cation channel and that its activity depends on Ca2+ concentration. In the crystal structure, the selectivity filter adopts a novel conformation with Ca2+ ions bound within the filter near the pore helix where they are coordinated by backbone oxygen atoms, a recurrent motif found in multiple proteins. The binding of Ca2+ ion in the selectivity filter controls the widening of the pore as shown in crystal structures and in molecular dynamics simulations. The structural, functional and computational data provide a characterization of this calcium-gated cationic channel.

The GS (genetic selection) Principle.

PubMed

Abel, David L

2009-01-01

The GS (Genetic Selection) Principle states that biological selection must occur at the nucleotide-sequencing molecular-genetic level of 3'5' phosphodiester bond formation. After-the-fact differential survival and reproduction of already-living phenotypic organisms (ordinary natural selection) does not explain polynucleotide prescription and coding. All life depends upon literal genetic algorithms. Even epigenetic and "genomic" factors such as regulation by DNA methylation, histone proteins and microRNAs are ultimately instructed by prior linear digital programming. Biological control requires selection of particular configurable switch-settings to achieve potential function. This occurs largely at the level of nucleotide selection, prior to the realization of any integrated biofunction. Each selection of a nucleotide corresponds to the setting of two formal binary logic gates. The setting of these switches only later determines folding and binding function through minimum-free-energy sinks. These sinks are determined by the primary structure of both the protein itself and the independently prescribed sequencing of chaperones. The GS Principle distinguishes selection of existing function (natural selection) from selection for potential function (formal selection at decision nodes, logic gates and configurable switch-settings).

Does TATA matter? A structural exploration of the selectivity determinants in its complexes with TATA box-binding protein.

PubMed Central

Pastor, N; Pardo, L; Weinstein, H

1997-01-01

The binding of the TATA box-binding protein (TBP) to a TATA sequence in DNA is essential for eukaryotic basal transcription. TBP binds in the minor groove of DNA, causing a large distortion of the DNA helix. Given the apparent stereochemical equivalence of AT and TA basepairs in the minor groove, DNA deformability must play a significant role in binding site selection, because not all AT-rich sequences are bound effectively by TBP. To gain insight into the precise role that the properties of the TATA sequence have in determining the specificity of the DNA substrates of TBP, the solution structure and dynamics of seven DNA dodecamers have been studied by using molecular dynamics simulations. The analysis of the structural properties of basepair steps in these TATA sequences suggests a reason for the preference for alternating pyrimidine-purine (YR) sequences, but indicates that these properties cannot be the sole determinant of the sequence specificity of TBP. Rather, recognition depends on the interplay between the inherent deformability of the DNA and steric complementarity at the molecular interface. Images FIGURE 2 PMID:9251783

Continuous aesthetic judgment of image sequences.

PubMed

Khaw, Mel W; Freedberg, David

2018-05-18

Perceptual judgments are said to be reference-dependent as they change on the basis of recent experiences. Here we quantify sequence effects within two types of aesthetic judgments: (i) individual ratings of single images (during self-paced trials) and (ii) continuous ratings of image sequences. As in the case of known contrast effects, trial-by-trial aesthetic responses are negatively correlated with judgments made toward the preceding image. During continuous judgment, a different type of bias is observed. The onset of change within a sequence introduces a persistent increase in ratings (relative to when the same images are judged in isolation). Furthermore, subjects indicate adjustment patterns and choices that selectively favor sequences that are rich in change. Sequence effects in aesthetic judgments thus differ greatly depending on the continuity and arrangement of presented stimuli. The effects highlighted here are important in understanding sustained aesthetic responses over time, such as those elicited during choreographic and musical arrangements. In contrast, standard measurements of aesthetic responses (over trials) may represent a series of distinct aesthetic experiences (e.g., viewing artworks in a museum). Copyright © 2018 Elsevier B.V. All rights reserved.

Selection of an Aptamer Antidote to the Anticoagulant Drug Bivalirudin

PubMed Central

Martin, Jennifer A.; Parekh, Parag; Kim, Youngmi; Morey, Timothy E.; Sefah, Kwame; Gravenstein, Nikolaus; Dennis, Donn M.; Tan, Weihong

2013-01-01

Adverse drug reactions, including severe patient bleeding, may occur following the administration of anticoagulant drugs. Bivalirudin is a synthetic anticoagulant drug sometimes employed as a substitute for heparin, a commonly used anticoagulant that can cause a condition called heparin-induced thrombocytopenia (HIT). Although bivalrudin has the advantage of not causing HIT, a major concern is lack of an antidote for this drug. In contrast, medical professionals can quickly reverse the effects of heparin using protamine. This report details the selection of an aptamer to bivalirudin that functions as an antidote in buffer. This was accomplished by immobilizing the drug on a monolithic column to partition binding sequences from nonbinding sequences using a low-pressure chromatography system and salt gradient elution. The elution profile of binding sequences was compared to that of a blank column (no drug), and fractions with a chromatographic difference were analyzed via real-time PCR (polymerase chain reaction) and used for further selection. Sequences were identified by 454 sequencing and demonstrated low micromolar dissociation constants through fluorescence anisotropy after only two rounds of selection. One aptamer, JPB5, displayed a dose-dependent reduction of the clotting time in buffer, with a 20 µM aptamer achieving a nearly complete antidote effect. This work is expected to result in a superior safety profile for bivalirudin, resulting in enhanced patient care. PMID:23483901

Fitness in time-dependent environments includes a geometric phase contribution

PubMed Central

Tănase-Nicola, Sorin; Nemenman, Ilya

2012-01-01

Phenotypic evolution implies sequential rise in frequency of new genomic sequences. The speed of the rise depends, in part, on the relative fitness (selection coefficient) of the mutant versus the ancestor. Using a simple population dynamics model, we show that the relative fitness in dynamical environments is not equal to the geometric average of the fitness over individual environments. Instead, it includes a term that explicitly depends on the sequence of the environments. For slowly varying environments, this term depends only on the oriented area enclosed by the trajectory taken by the system in the environment state space. It is closely related to the well-studied geometric phases in classical and quantum physical systems. We discuss possible biological implications of these observations, focusing on evolution of novel metabolic or stress-resistant functions. PMID:22112653

An algorithm for a single machine scheduling problem with sequence dependent setup times and scheduling windows

NASA Technical Reports Server (NTRS)

Moore, J. E.

1975-01-01

An enumeration algorithm is presented for solving a scheduling problem similar to the single machine job shop problem with sequence dependent setup times. The scheduling problem differs from the job shop problem in two ways. First, its objective is to select an optimum subset of the available tasks to be performed during a fixed period of time. Secondly, each task scheduled is constrained to occur within its particular scheduling window. The algorithm is currently being used to develop typical observational timelines for a telescope that will be operated in earth orbit. Computational times associated with timeline development are presented.

Synchronized excitability in a network enables generation of internal neuronal sequences

PubMed Central

Wang, Yingxue; Roth, Zachary; Pastalkova, Eva

2016-01-01

Hippocampal place field sequences are supported by sensory cues and network internal mechanisms. In contrast, sharp-wave (SPW) sequences, theta sequences, and episode field sequences are internally generated. The relationship of these sequences to memory is unclear. SPW sequences have been shown to support learning and have been assumed to also support episodic memory. Conversely, we demonstrate these SPW sequences were present in trained rats even after episodic memory was impaired and after other internal sequences – episode field and theta sequences – were eliminated. SPW sequences did not support memory despite continuing to ‘replay’ all task-related sequences – place- field and episode field sequences. Sequence replay occurred selectively during synchronous increases of population excitability -- SPWs. Similarly, theta sequences depended on the presence of repeated synchronized waves of excitability – theta oscillations. Thus, we suggest that either intermittent or rhythmic synchronized changes of excitability trigger sequential firing of neurons, which in turn supports learning and/or memory. DOI: http://dx.doi.org/10.7554/eLife.20697.001 PMID:27677848

Evolutionary dynamics on networks of selectively neutral genotypes: effects of topology and sequence stability.

PubMed

Aguirre, Jacobo; Buldú, Javier M; Manrubia, Susanna C

2009-12-01

Networks of selectively neutral genotypes underlie the evolution of populations of replicators in constant environments. Previous theoretical analysis predicted that such populations will evolve toward highly connected regions of the genome space. We first study the evolution of populations of replicators on simple networks and quantify how the transient time to equilibrium depends on the initial distribution of sequences on the neutral network, on the topological properties of the latter, and on the mutation rate. Second, network neutrality is broken through the introduction of an energy for each sequence. This allows to study the competition between two features (neutrality and energetic stability) relevant for survival and subjected to different selective pressures. In cases where the two features are negatively correlated, the population experiences sudden migrations in the genome space for values of the relevant parameters that we calculate. The numerical study of larger networks indicates that the qualitative behavior to be expected in more realistic cases is already seen in representative examples of small networks.

Evolutionary dynamics on networks of selectively neutral genotypes: Effects of topology and sequence stability

NASA Astrophysics Data System (ADS)

Aguirre, Jacobo; Buldú, Javier M.; Manrubia, Susanna C.

2009-12-01

Networks of selectively neutral genotypes underlie the evolution of populations of replicators in constant environments. Previous theoretical analysis predicted that such populations will evolve toward highly connected regions of the genome space. We first study the evolution of populations of replicators on simple networks and quantify how the transient time to equilibrium depends on the initial distribution of sequences on the neutral network, on the topological properties of the latter, and on the mutation rate. Second, network neutrality is broken through the introduction of an energy for each sequence. This allows to study the competition between two features (neutrality and energetic stability) relevant for survival and subjected to different selective pressures. In cases where the two features are negatively correlated, the population experiences sudden migrations in the genome space for values of the relevant parameters that we calculate. The numerical study of larger networks indicates that the qualitative behavior to be expected in more realistic cases is already seen in representative examples of small networks.

SNPGenie: estimating evolutionary parameters to detect natural selection using pooled next-generation sequencing data.

PubMed

Nelson, Chase W; Moncla, Louise H; Hughes, Austin L

2015-11-15

New applications of next-generation sequencing technologies use pools of DNA from multiple individuals to estimate population genetic parameters. However, no publicly available tools exist to analyse single-nucleotide polymorphism (SNP) calling results directly for evolutionary parameters important in detecting natural selection, including nucleotide diversity and gene diversity. We have developed SNPGenie to fill this gap. The user submits a FASTA reference sequence(s), a Gene Transfer Format (.GTF) file with CDS information and a SNP report(s) in an increasing selection of formats. The program estimates nucleotide diversity, distance from the reference and gene diversity. Sites are flagged for multiple overlapping reading frames, and are categorized by polymorphism type: nonsynonymous, synonymous, or ambiguous. The results allow single nucleotide, single codon, sliding window, whole gene and whole genome/population analyses that aid in the detection of positive and purifying natural selection in the source population. SNPGenie version 1.2 is a Perl program with no additional dependencies. It is free, open-source, and available for download at https://github.com/hugheslab/snpgenie. nelsoncw@email.sc.edu or austin@biol.sc.edu Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Fungal diversity in grape must and wine fermentation assessed by massive sequencing, quantitative PCR and DGGE

PubMed Central

Wang, Chunxiao; García-Fernández, David; Mas, Albert; Esteve-Zarzoso, Braulio

2015-01-01

The diversity of fungi in grape must and during wine fermentation was investigated in this study by culture-dependent and culture-independent techniques. Carignan and Grenache grapes were harvested from three vineyards in the Priorat region (Spain) in 2012, and nine samples were selected from the grape must after crushing and during wine fermentation. From culture-dependent techniques, 362 isolates were randomly selected and identified by 5.8S-ITS-RFLP and 26S-D1/D2 sequencing. Meanwhile, genomic DNA was extracted directly from the nine samples and analyzed by qPCR, DGGE and massive sequencing. The results indicated that grape must after crushing harbored a high species richness of fungi with Aspergillus tubingensis, Aureobasidium pullulans, or Starmerella bacillaris as the dominant species. As fermentation proceeded, the species richness decreased, and yeasts such as Hanseniaspora uvarum, Starmerella bacillaris and Saccharomyces cerevisiae successively occupied the must samples. The “terroir” characteristics of the fungus population are more related to the location of the vineyard than to grape variety. Sulfur dioxide treatment caused a low effect on yeast diversity by similarity analysis. Because of the existence of large population of fungi on grape berries, massive sequencing was more appropriate to understand the fungal community in grape must after crushing than the other techniques used in this study. Suitable target sequences and databases were necessary for accurate evaluation of the community and the identification of species by the 454 pyrosequencing of amplicons. PMID:26557110

Peroxisomal Pex11 is a pore-forming protein homologous to TRPM channels.

PubMed

Mindthoff, Sabrina; Grunau, Silke; Steinfort, Laura L; Girzalsky, Wolfgang; Hiltunen, J Kalervo; Erdmann, Ralf; Antonenkov, Vasily D

2016-02-01

More than 30 proteins (Pex proteins) are known to participate in the biogenesis of peroxisomes-ubiquitous oxidative organelles involved in lipid and ROS metabolism. The Pex11 family of homologous proteins is responsible for division and proliferation of peroxisomes. We show that yeast Pex11 is a pore-forming protein sharing sequence similarity with TRPM cation-selective channels. The Pex11 channel with a conductance of Λ=4.1 nS in 1.0M KCl is moderately cation-selective (PK(+)/PCl(-)=1.85) and resistant to voltage-dependent closing. The estimated size of the channel's pore (r~0.6 nm) supports the notion that Pex11 conducts solutes with molecular mass below 300-400 Da. We localized the channel's selectivity determining sequence. Overexpression of Pex11 resulted in acceleration of fatty acids β-oxidation in intact cells but not in the corresponding lysates. The β-oxidation was affected in cells by expression of the Pex11 protein carrying point mutations in the selectivity determining sequence. These data suggest that the Pex11-dependent transmembrane traffic of metabolites may be a rate-limiting step in the β-oxidation of fatty acids. This conclusion was corroborated by analysis of the rate of β-oxidation in yeast strains expressing Pex11 with mutations mimicking constitutively phosphorylated (S165D, S167D) or unphosphorylated (S165A, S167A) protein. The results suggest that phosphorylation of Pex11 is a mechanism that can control the peroxisomal β-oxidation rate. Our results disclose an unexpected function of Pex11 as a non-selective channel responsible for transfer of metabolites across peroxisomal membrane. The data indicate that peroxins may be involved in peroxisomal metabolic processes in addition to their role in peroxisome biogenesis. Copyright © 2015 Elsevier B.V. All rights reserved.

A coarse-grained biophysical model of sequence evolution and the population size dependence of the speciation rate

PubMed Central

Khatri, Bhavin S.; Goldstein, Richard A.

2015-01-01

Speciation is fundamental to understanding the huge diversity of life on Earth. Although still controversial, empirical evidence suggests that the rate of speciation is larger for smaller populations. Here, we explore a biophysical model of speciation by developing a simple coarse-grained theory of transcription factor-DNA binding and how their co-evolution in two geographically isolated lineages leads to incompatibilities. To develop a tractable analytical theory, we derive a Smoluchowski equation for the dynamics of binding energy evolution that accounts for the fact that natural selection acts on phenotypes, but variation arises from mutations in sequences; the Smoluchowski equation includes selection due to both gradients in fitness and gradients in sequence entropy, which is the logarithm of the number of sequences that correspond to a particular binding energy. This simple consideration predicts that smaller populations develop incompatibilities more quickly in the weak mutation regime; this trend arises as sequence entropy poises smaller populations closer to incompatible regions of phenotype space. These results suggest a generic coarse-grained approach to evolutionary stochastic dynamics, allowing realistic modelling at the phenotypic level. PMID:25936759

Limits of neutral drift: lessons from the in vitro evolution of two ribozymes.

PubMed

Petrie, Katherine L; Joyce, Gerald F

2014-10-01

The relative contributions of adaptive selection and neutral drift to genetic change are unknown but likely depend on the inherent abundance of functional genotypes in sequence space and how accessible those genotypes are to one another. To better understand the relative roles of selection and drift in evolution, local fitness landscapes for two different RNA ligase ribozymes were examined using a continuous in vitro evolution system under conditions that foster the capacity for neutral drift to mediate genetic change. The exploration of sequence space was accelerated by increasing the mutation rate using mutagenic nucleotide analogs. Drift was encouraged by carrying out evolution within millions of separate compartments to exploit the founder effect. Deep sequencing of individuals from the evolved populations revealed that the distribution of genotypes did not escape the starting local fitness peak, remaining clustered around the sequence used to initiate evolution. This is consistent with a fitness landscape where high-fitness genotypes are sparse and well isolated, and suggests, at least in this context, that neutral drift alone is not a primary driver of genetic change. Neutral drift does, however, provide a repository of genetic variation upon which adaptive selection can act.

A chain-retrieval model for voluntary task switching.

PubMed

Vandierendonck, André; Demanet, Jelle; Liefooghe, Baptist; Verbruggen, Frederick

2012-09-01

To account for the findings obtained in voluntary task switching, this article describes and tests the chain-retrieval model. This model postulates that voluntary task selection involves retrieval of task information from long-term memory, which is then used to guide task selection and task execution. The model assumes that the retrieved information consists of acquired sequences (or chains) of tasks, that selection may be biased towards chains containing more task repetitions and that bottom-up triggered repetitions may overrule the intended task. To test this model, four experiments are reported. In Studies 1 and 2, sequences of task choices and the corresponding transition sequences (task repetitions or switches) were analyzed with the help of dependency statistics. The free parameters of the chain-retrieval model were estimated on the observed task sequences and these estimates were used to predict autocorrelations of tasks and transitions. In Studies 3 and 4, sequences of hand choices and their transitions were analyzed similarly. In all studies, the chain-retrieval model yielded better fits and predictions than statistical models of event choice. In applications to voluntary task switching (Studies 1 and 2), all three parameters of the model were needed to account for the data. When no task switching was required (Studies 3 and 4), the chain-retrieval model could account for the data with one or two parameters clamped to a neutral value. Implications for our understanding of voluntary task selection and broader theoretical implications are discussed. Copyright © 2012 Elsevier Inc. All rights reserved.

Biased selection of propagation-related TUPs from phage display peptide libraries.

PubMed

Zade, Hesam Motaleb; Keshavarz, Reihaneh; Shekarabi, Hosna Sadat Zahed; Bakhshinejad, Babak

2017-08-01

Phage display is rapidly advancing as a screening strategy in drug discovery and drug delivery. Phage-encoded combinatorial peptide libraries can be screened through the affinity selection procedure of biopanning to find pharmaceutically relevant cell-specific ligands. However, the unwanted enrichment of target-unrelated peptides (TUPs) with no true affinity for the target presents an important barrier to the successful screening of phage display libraries. Propagation-related TUPs (Pr-TUPs) are an emerging but less-studied category of phage display-derived false-positive hits that are displayed on the surface of clones with faster propagation rates. Despite long regarded as an unbiased selection system, accumulating evidence suggests that biopanning may create biological bias toward selection of phage clones with certain displayed peptides. This bias can be dependent on or independent of the displayed sequence and may act as a major driving force for the isolation of fast-growing clones. Sequence-dependent bias is reflected by censorship or over-representation of some amino acids in the displayed peptide and sequence-independent bias is derived from either point mutations or rare recombination events occurring in the phage genome. It is of utmost interest to clean biopanning data by identifying and removing Pr-TUPs. Experimental and bioinformatic approaches can be exploited for Pr-TUP discovery. With no doubt, obtaining deeper insight into how Pr-TUPs emerge during biopanning and how they could be detected provides a basis for using cell-targeting peptides isolated from phage display screening in the development of disease-specific diagnostic and therapeutic platforms.

Detecting consistent patterns of directional adaptation using differential selection codon models.

PubMed

Parto, Sahar; Lartillot, Nicolas

2017-06-23

Phylogenetic codon models are often used to characterize the selective regimes acting on protein-coding sequences. Recent methodological developments have led to models explicitly accounting for the interplay between mutation and selection, by modeling the amino acid fitness landscape along the sequence. However, thus far, most of these models have assumed that the fitness landscape is constant over time. Fluctuations of the fitness landscape may often be random or depend on complex and unknown factors. However, some organisms may be subject to systematic changes in selective pressure, resulting in reproducible molecular adaptations across independent lineages subject to similar conditions. Here, we introduce a codon-based differential selection model, which aims to detect and quantify the fine-grained consistent patterns of adaptation at the protein-coding level, as a function of external conditions experienced by the organism under investigation. The model parameterizes the global mutational pressure, as well as the site- and condition-specific amino acid selective preferences. This phylogenetic model is implemented in a Bayesian MCMC framework. After validation with simulations, we applied our method to a dataset of HIV sequences from patients with known HLA genetic background. Our differential selection model detects and characterizes differentially selected coding positions specifically associated with two different HLA alleles. Our differential selection model is able to identify consistent molecular adaptations as a function of repeated changes in the environment of the organism. These models can be applied to many other problems, ranging from viral adaptation to evolution of life-history strategies in plants or animals.

Direct and selective immobilization of proteins by means of an inorganic material-binding peptide: discussion on functionalization in the elongation to material-binding peptide.

PubMed

Yokoo, Nozomi; Togashi, Takanari; Umetsu, Mitsuo; Tsumoto, Kouhei; Hattori, Takamitsu; Nakanishi, Takeshi; Ohara, Satoshi; Takami, Seiichi; Naka, Takashi; Abe, Hiroya; Kumagai, Izumi; Adschiri, Tadafumi

2010-01-14

Using an artificial peptide library, we have identified a peptide with affinity for ZnO materials that could be used to selectively accumulate ZnO particles on polypropylene-gold plates. In this study, we fused recombinant green fluorescent protein (GFP) with this ZnO-binding peptide (ZnOBP) and then selectively immobilized the fused protein on ZnO particles. We determined an appropriate condition for selective immobilization of recombinant GFP, and the ZnO-binding function of ZnOBP-fused GFP was examined by elongating the ZnOBP tag from a single amino acid to the intact sequence. The fusion of ZnOBP with GFP enabled specific adsorption of GFP on ZnO substrates in an appropriate solution, and thermodynamic studies showed a predominantly enthalpy-dependent electrostatic interaction between ZnOBP and the ZnO surface. The ZnOBP's binding affinity for the ZnO surface increased first in terms of material selectivity and then in terms of high affinity as the GFP-fused peptide was elongated from a single amino acid to intact ZnOBP. We concluded that the enthalpy-dependent interaction between ZnOBP and ZnO was influenced by the presence of not only charged amino acids but also their surrounding residues in the ZnOBP sequence.

Variation in Campylobacter Mulilocus Sequence Typing Subtypes Detected on Three Different Plating Media

USDA-ARS?s Scientific Manuscript database

Introduction: There are multiple selective plating media available for detection and enumeration of naturally occurring Campylobacter. Campylobacter produce colonies with differing morphology and characteristics depending on the plating medium used. It is unclear if choice of plating medium can a...

An organismic critique of molecular darwinism.

PubMed

Wicken, J S

1985-12-21

The molecular darwinian approach to the emergence of life treats the competition between RNA sequences for nucleotide resources as the primordial selective process in prebiotic evolution, which prescribes possible pathways for the subsequent elaboration of organizational relationships. Since success in this competition is determined by the "phenotypic" properties of RNA strands in the absence of organizational context, the genesis of biotic organization is dependent upon the establishment of co-operative, hypercyclic interactions between competing RNA sequences. The thesis of this paper is that hypercycle theory is based on unwarranted assumptions about the conditions of prebiotic evolution, and that the implications of these assumptions run counter to both empirical evidence and to the rational by which natural selection operates in evolution generally. An organismic alternative to hypercycle theory is suggested, based on the catalytic microsphere and the thermodynamics of selection.

Bit error rate tester using fast parallel generation of linear recurring sequences

DOEpatents

Pierson, Lyndon G.; Witzke, Edward L.; Maestas, Joseph H.

2003-05-06

A fast method for generating linear recurring sequences by parallel linear recurring sequence generators (LRSGs) with a feedback circuit optimized to balance minimum propagation delay against maximal sequence period. Parallel generation of linear recurring sequences requires decimating the sequence (creating small contiguous sections of the sequence in each LRSG). A companion matrix form is selected depending on whether the LFSR is right-shifting or left-shifting. The companion matrix is completed by selecting a primitive irreducible polynomial with 1's most closely grouped in a corner of the companion matrix. A decimation matrix is created by raising the companion matrix to the (n*k).sup.th power, where k is the number of parallel LRSGs and n is the number of bits to be generated at a time by each LRSG. Companion matrices with 1's closely grouped in a corner will yield sparse decimation matrices. A feedback circuit comprised of XOR logic gates implements the decimation matrix in hardware. Sparse decimation matrices can be implemented with minimum number of XOR gates, and therefore a minimum propagation delay through the feedback circuit. The LRSG of the invention is particularly well suited to use as a bit error rate tester on high speed communication lines because it permits the receiver to synchronize to the transmitted pattern within 2n bits.

Correlation of Local Effects of DNA Sequence and Position of Beta-Alanine Inserts with Polyamide-DNA Complex Binding Affinities and Kinetics

PubMed Central

Wang, Shuo; Nanjunda, Rupesh; Aston, Karl; Bashkin, James K.; Wilson, W. David

2012-01-01

In order to better understand the effects of β-alanine (β) substitution and the number of heterocycles on DNA binding affinity and selectivity, the interactions of an eight-ring hairpin polyamide (PA) and two β derivatives as well as a six-heterocycle analog have been investigated with their cognate DNA sequence, 5′-TGGCTT-3′. Binding selectivity and the effects of β have been investigated with the cognate and five mutant DNAs. A set of powerful and complementary methods have been employed for both energetic and structural evaluations: UV-melting, biosensor-surface plasmon resonance, isothermal titration calorimetry, circular dichroism and a DNA ligation ladder global structure assay. The reduced number of heterocycles in the six-ring PA weakens the binding affinity; however, the smaller PA aggregates significantly less than the larger PAs, and allows us to obtain the binding thermodynamics. The PA-DNA binding enthalpy is large and negative with a large negative ΔCp, and is the primary driving component of the Gibbs free energy. The complete SPR binding results clearly show that β substitutions can substantially weaken the binding affinity of hairpin PAs in a position-dependent manner. More importantly, the changes in PA binding to the mutant DNAs further confirm the position-dependent effects on PA-DNA interaction affinity. Comparison of mutant DNA sequences also shows a different effect in recognition of T•A versus A•T base pairs. The effects of DNA mutations on binding of a single PA as well as the effects of the position of β substitution on binding tell a clear and very important story about sequence dependent binding of PAs to DNA. PMID:23167504

The right inferior frontal gyrus processes nested non-local dependencies in music.

PubMed

Cheung, Vincent K M; Meyer, Lars; Friederici, Angela D; Koelsch, Stefan

2018-02-28

Complex auditory sequences known as music have often been described as hierarchically structured. This permits the existence of non-local dependencies, which relate elements of a sequence beyond their temporal sequential order. Previous studies in music have reported differential activity in the inferior frontal gyrus (IFG) when comparing regular and irregular chord-transitions based on theories in Western tonal harmony. However, it is unclear if the observed activity reflects the interpretation of hierarchical structure as the effects are confounded by local irregularity. Using functional magnetic resonance imaging (fMRI), we found that violations to non-local dependencies in nested sequences of three-tone musical motifs in musicians elicited increased activity in the right IFG. This is in contrast to similar studies in language which typically report the left IFG in processing grammatical syntax. Effects of increasing auditory working demands are moreover reflected by distributed activity in frontal and parietal regions. Our study therefore demonstrates the role of the right IFG in processing non-local dependencies in music, and suggests that hierarchical processing in different cognitive domains relies on similar mechanisms that are subserved by domain-selective neuronal subpopulations.

An electrostatic selection mechanism controls sequential kinase signaling downstream of the T cell receptor

PubMed Central

Shah, Neel H; Wang, Qi; Yan, Qingrong; Karandur, Deepti; Kadlecek, Theresa A; Fallahee, Ian R; Russ, William P; Ranganathan, Rama; Weiss, Arthur; Kuriyan, John

2016-01-01

The sequence of events that initiates T cell signaling is dictated by the specificities and order of activation of the tyrosine kinases that signal downstream of the T cell receptor. Using a platform that combines exhaustive point-mutagenesis of peptide substrates, bacterial surface-display, cell sorting, and deep sequencing, we have defined the specificities of the first two kinases in this pathway, Lck and ZAP-70, for the T cell receptor ζ chain and the scaffold proteins LAT and SLP-76. We find that ZAP-70 selects its substrates by utilizing an electrostatic mechanism that excludes substrates with positively-charged residues and favors LAT and SLP-76 phosphosites that are surrounded by negatively-charged residues. This mechanism prevents ZAP-70 from phosphorylating its own activation loop, thereby enforcing its strict dependence on Lck for activation. The sequence features in ZAP-70, LAT, and SLP-76 that underlie electrostatic selectivity likely contribute to the specific response of T cells to foreign antigens. DOI: http://dx.doi.org/10.7554/eLife.20105.001 PMID:27700984

Computational design of enzyme-ligand binding using a combined energy function and deterministic sequence optimization algorithm.

PubMed

Tian, Ye; Huang, Xiaoqiang; Zhu, Yushan

2015-08-01

Enzyme amino-acid sequences at ligand-binding interfaces are evolutionarily optimized for reactions, and the natural conformation of an enzyme-ligand complex must have a low free energy relative to alternative conformations in native-like or non-native sequences. Based on this assumption, a combined energy function was developed for enzyme design and then evaluated by recapitulating native enzyme sequences at ligand-binding interfaces for 10 enzyme-ligand complexes. In this energy function, the electrostatic interaction between polar or charged atoms at buried interfaces is described by an explicitly orientation-dependent hydrogen-bonding potential and a pairwise-decomposable generalized Born model based on the general side chain in the protein design framework. The energy function is augmented with a pairwise surface-area based hydrophobic contribution for nonpolar atom burial. Using this function, on average, 78% of the amino acids at ligand-binding sites were predicted correctly in the minimum-energy sequences, whereas 84% were predicted correctly in the most-similar sequences, which were selected from the top 20 sequences for each enzyme-ligand complex. Hydrogen bonds at the enzyme-ligand binding interfaces in the 10 complexes were usually recovered with the correct geometries. The binding energies calculated using the combined energy function helped to discriminate the active sequences from a pool of alternative sequences that were generated by repeatedly solving a series of mixed-integer linear programming problems for sequence selection with increasing integer cuts.

Catch a tiger snake by its tail: Differential toxicity, co-factor dependence and antivenom efficacy in a procoagulant clade of Australian venomous snakes.

PubMed

Lister, Callum; Arbuckle, Kevin; Jackson, Timothy N W; Debono, Jordan; Zdenek, Christina N; Dashevsky, Daniel; Dunstan, Nathan; Allen, Luke; Hay, Chris; Bush, Brian; Gillett, Amber; Fry, Bryan G

2017-11-01

A paradigm of venom research is adaptive evolution of toxins as part of a predator-prey chemical arms race. This study examined differential co-factor dependence, variations relative to dietary preference, and the impact upon relative neutralisation by antivenom of the procoagulant toxins in the venoms of a clade of Australian snakes. All genera were characterised by venoms rich in factor Xa which act upon endogenous prothrombin. Examination of toxin sequences revealed an extraordinary level of conservation, which indicates that adaptive evolution is not a feature of this toxin type. Consistent with this, the venoms did not display differences on the plasma of different taxa. Examination of the prothrombin target revealed endogenous blood proteins are under extreme negative selection pressure for diversification, this in turn puts a strong negative selection pressure upon the toxins as sequence diversification could result in a drift away from the target. Thus this study reveals that adaptive evolution is not a consistent feature in toxin evolution in cases where the target is under negative selection pressure for diversification. Consistent with this high level of toxin conservation, the antivenom showed extremely high-levels of cross-reactivity. There was however a strong statistical correlation between relative degree of phospholipid-dependence and clotting time, with the least dependent venoms producing faster clotting times than the other venoms even in the presence of phospholipid. The results of this study are not only of interest to evolutionary and ecological disciplines, but also have implications for clinical toxinology. Copyright © 2017 Elsevier Inc. All rights reserved.

A conserved mechanism for replication origin recognition and binding in archaea.

PubMed

Majerník, Alan I; Chong, James P J

2008-01-15

To date, methanogens are the only group within the archaea where firing DNA replication origins have not been demonstrated in vivo. In the present study we show that a previously identified cluster of ORB (origin recognition box) sequences do indeed function as an origin of replication in vivo in the archaeon Methanothermobacter thermautotrophicus. Although the consensus sequence of ORBs in M. thermautotrophicus is somewhat conserved when compared with ORB sequences in other archaea, the Cdc6-1 protein from M. thermautotrophicus (termed MthCdc6-1) displays sequence-specific binding that is selective for the MthORB sequence and does not recognize ORBs from other archaeal species. Stabilization of in vitro MthORB DNA binding by MthCdc6-1 requires additional conserved sequences 3' to those originally described for M. thermautotrophicus. By testing synthetic sequences bearing mutations in the MthORB consensus sequence, we show that Cdc6/ORB binding is critically dependent on the presence of an invariant guanine found in all archaeal ORB sequences. Mutation of a universally conserved arginine residue in the recognition helix of the winged helix domain of archaeal Cdc6-1 shows that specific origin sequence recognition is dependent on the interaction of this arginine residue with the invariant guanine. Recognition of a mutated origin sequence can be achieved by mutation of the conserved arginine residue to a lysine or glutamine residue. Thus despite a number of differences in protein and DNA sequences between species, the mechanism of origin recognition and binding appears to be conserved throughout the archaea.

Genome-Wide Stochastic Adaptive DNA Amplification at Direct and Inverted DNA Repeats in the Parasite Leishmania

PubMed Central

Plourde, Marie; Gingras, Hélène; Roy, Gaétan; Lapointe, Andréanne; Leprohon, Philippe; Papadopoulou, Barbara; Corbeil, Jacques; Ouellette, Marc

2014-01-01

Gene amplification of specific loci has been described in all kingdoms of life. In the protozoan parasite Leishmania, the product of amplification is usually part of extrachromosomal circular or linear amplicons that are formed at the level of direct or inverted repeated sequences. A bioinformatics screen revealed that repeated sequences are widely distributed in the Leishmania genome and the repeats are chromosome-specific, conserved among species, and generally present in low copy number. Using sensitive PCR assays, we provide evidence that the Leishmania genome is continuously being rearranged at the level of these repeated sequences, which serve as a functional platform for constitutive and stochastic amplification (and deletion) of genomic segments in the population. This process is adaptive as the copy number of advantageous extrachromosomal circular or linear elements increases upon selective pressure and is reversible when selection is removed. We also provide mechanistic insights on the formation of circular and linear amplicons through RAD51 recombinase-dependent and -independent mechanisms, respectively. The whole genome of Leishmania is thus stochastically rearranged at the level of repeated sequences, and the selection of parasite subpopulations with changes in the copy number of specific loci is used as a strategy to respond to a changing environment. PMID:24844805

Development of expert system for biobased polymer material selection: food packaging application.

PubMed

Sanyang, M L; Sapuan, S M

2015-10-01

Biobased food packaging materials are gaining more attention owing to their intrinsic biodegradable nature and renewability. Selection of suitable biobased polymers for food packaging applications could be a tedious task with potential mistakes in choosing the best materials. In this paper, an expert system was developed using Exsys Corvid software to select suitable biobased polymer materials for packaging fruits, dry food and dairy products. If - Then rule based system was utilized to accomplish the material selection process whereas a score system was formulated to facilitate the ranking of selected materials. The expert system selected materials that satisfied all constraints and selection results were presented in suitability sequence depending on their scores. The expert system selected polylactic acid (PLA) as the most suitable material.

Technical adequacy of bisulfite sequencing and pyrosequencing for detection of mitochondrial DNA methylation: Sources and avoidance of false-positive detection.

PubMed

Owa, Chie; Poulin, Matthew; Yan, Liying; Shioda, Toshi

2018-01-01

The existence of cytosine methylation in mammalian mitochondrial DNA (mtDNA) is a controversial subject. Because detection of DNA methylation depends on resistance of 5'-modified cytosines to bisulfite-catalyzed conversion to uracil, examined parameters that affect technical adequacy of mtDNA methylation analysis. Negative control amplicons (NCAs) devoid of cytosine methylation were amplified to cover the entire human or mouse mtDNA by long-range PCR. When the pyrosequencing template amplicons were gel-purified after bisulfite conversion, bisulfite pyrosequencing of NCAs did not detect significant levels of bisulfite-resistant cytosines (brCs) at ND1 (7 CpG sites) or CYTB (8 CpG sites) genes (CI95 = 0%-0.94%); without gel-purification, significant false-positive brCs were detected from NCAs (CI95 = 4.2%-6.8%). Bisulfite pyrosequencing of highly purified, linearized mtDNA isolated from human iPS cells or mouse liver detected significant brCs (~30%) in human ND1 gene when the sequencing primer was not selective in bisulfite-converted and unconverted templates. However, repeated experiments using a sequencing primer selective in bisulfite-converted templates almost completely (< 0.8%) suppressed brC detection, supporting the false-positive nature of brCs detected using the non-selective primer. Bisulfite-seq deep sequencing of linearized, gel-purified human mtDNA detected 9.4%-14.8% brCs for 9 CpG sites in ND1 gene. However, because all these brCs were associated with adjacent non-CpG brCs showing the same degrees of bisulfite resistance, DNA methylation in this mtDNA-encoded gene was not confirmed. Without linearization, data generated by bisulfite pyrosequencing or deep sequencing of purified mtDNA templates did not pass the quality control criteria. Shotgun bisulfite sequencing of human mtDNA detected extremely low levels of CpG methylation (<0.65%) over non-CpG methylation (<0.55%). Taken together, our study demonstrates that adequacy of mtDNA methylation analysis using methods dependent on bisulfite conversion needs to be established for each experiment, taking effects of incomplete bisulfite conversion and template impurity or topology into consideration.

Blood from a turnip: tissue origin of low-coverage shotgun sequencing libraries affects recovery of mitogenome sequences

USGS Publications Warehouse

Barker, F. Keith; Oyler-McCance, Sara; Tomback, Diana F.

2015-01-01

Next generation sequencing methods allow rapid, economical accumulation of data that have many applications, even at relatively low levels of genome coverage. However, the utility of shotgun sequencing data sets for specific goals may vary depending on the biological nature of the samples sequenced. We show that the ability to assemble mitogenomes from three avian samples of two different tissue types varies widely. In particular, data with coverage typical of microsatellite development efforts (∼1×) from DNA extracted from avian blood failed to cover even 50% of the mitogenome, relative to at least 500-fold coverage from muscle-derived data. Researchers should consider possible applications of their data and select the tissue source for their work accordingly. Practitioners analyzing low-coverage shotgun sequencing data (including for microsatellite locus development) should consider the potential benefits of mitogenome assembly, including internal barcode verification of species identity, mitochondrial primer development, and phylogenetics.

Movement plans for posture selection do not transfer across hands

PubMed Central

Schütz, Christoph; Schack, Thomas

2015-01-01

In a sequential task, the grasp postures people select depend on their movement history. This motor hysteresis effect results from the reuse of former movement plans and reduces the cognitive cost of movement planning. Movement plans for hand trajectories not only transfer across successive trials, but also across hands. We therefore asked whether such a transfer would also be found in movement plans for hand postures. To this end, we designed a sequential, continuous posture selection task. Participants had to open a column of drawers with cylindrical knobs in ascending and descending sequences. A hand switch was required in each sequence. Hand pro/supination was analyzed directly before and after the hand switch. Results showed that hysteresis effects were present directly before, but absent directly after the hand switch. This indicates that, in the current study, movement plans for hand postures only transfer across trials, but not across hands. PMID:26441734

Selection of homeotic proteins for binding to a human DNA replication origin.

PubMed

de Stanchina, E; Gabellini, D; Norio, P; Giacca, M; Peverali, F A; Riva, S; Falaschi, A; Biamonti, G

2000-06-09

We have previously shown that a cell cycle-dependent nucleoprotein complex assembles in vivo on a 74 bp sequence within the human DNA replication origin associated to the Lamin B2 gene. Here, we report the identification, using a one-hybrid screen in yeast, of three proteins interacting with the 74 bp sequence. All of them, namely HOXA13, HOXC10 and HOXC13, are orthologues of the Abdominal-B gene of Drosophila melanogaster and are members of the homeogene family of developmental regulators. We describe the complete open reading frame sequence of HOXC10 and HOXC13 along with the structure of the HoxC13 gene. The specificity of binding of these two proteins to the Lamin B2 origin is confirmed by both band-shift and in vitro footprinting assays. In addition, the ability of HOXC10 and HOXC13 to increase the activity of a promoter containing the 74 bp sequence, as assayed by CAT-assay experiments, demonstrates a direct interaction of these homeoproteins with the origin sequence in mammalian cells. We also show that HOXC10 expression is cell-type-dependent and positively correlates with cell proliferation. Copyright 2000 Academic Press.

Designing a Bioengine for Detection and Analysis of Base String on an Affected Sequence in High-Concentration Regions

PubMed Central

Mandal, Bijoy Kumar; Kim, Tai-hoon

2013-01-01

We design an Algorithm for bioengine. As a program are enable optimal alignments searching between two sequences, the host sequence (normal plant) as well as query sequence (virus). Searching for homologues has become a routine operation of biological sequences in 4 × 4 combination with different subsequence (word size). This program takes the advantage of the high degree of homology between such sequences to construct an alignment of the matching regions. There is a main aim which is to detect the overlapping reading frames. This program also enables to find out the highly infected colones selection highest matching region with minimum gap or mismatch zones and unique virus colones matches. This is a small, portable, interactive, front-end program intended to be used to find out the regions of matching between host sequence and query subsequences. All the operations are carried out in fraction of seconds, depending on the required task and on the sequence length. PMID:24000321

Orbit Selection for Earth Observation Missions

NASA Technical Reports Server (NTRS)

King, J. C.

1978-01-01

The orbit selection process is simplified for most earth-oriented satellite missions by a restriction to circular orbits, which reduces the primary orbit characteristics to be determined to only two: altitude and inclination. A number of important mission performance characteristics depend on these choices, however, so a major part of the orbit selection task is concerned with developing the correlating relationships in clear and convenient forms to provide a basis for rational orbit selection procedures. The present approach to that task is organized around two major areas of mission performance, orbit plane precession and coverage pattern development, whose dependence on altitude and inclination is delineated graphically in design chart form. These charts provide a visual grasp of the relationships between the quantities cited above, as well as other important mission performance parameters including viewing time of day (solar), sensor swath width (and fields of view), swath sequencing, and pattern repeat condition and repeat periods.

Sequence features of viral and human Internal Ribosome Entry Sites predictive of their activity

PubMed Central

Elias-Kirma, Shani; Nir, Ronit; Segal, Eran

2017-01-01

Translation of mRNAs through Internal Ribosome Entry Sites (IRESs) has emerged as a prominent mechanism of cellular and viral initiation. It supports cap-independent translation of select cellular genes under normal conditions, and in conditions when cap-dependent translation is inhibited. IRES structure and sequence are believed to be involved in this process. However due to the small number of IRESs known, there have been no systematic investigations of the determinants of IRES activity. With the recent discovery of thousands of novel IRESs in human and viruses, the next challenge is to decipher the sequence determinants of IRES activity. We present the first in-depth computational analysis of a large body of IRESs, exploring RNA sequence features predictive of IRES activity. We identified predictive k-mer features resembling IRES trans-acting factor (ITAF) binding motifs across human and viral IRESs, and found that their effect on expression depends on their sequence, number and position. Our results also suggest that the architecture of retroviral IRESs differs from that of other viruses, presumably due to their exposure to the nuclear environment. Finally, we measured IRES activity of synthetically designed sequences to confirm our prediction of increasing activity as a function of the number of short IRES elements. PMID:28922394

Bayesian selection of Markov models for symbol sequences: application to microsaccadic eye movements.

PubMed

Bettenbühl, Mario; Rusconi, Marco; Engbert, Ralf; Holschneider, Matthias

2012-01-01

Complex biological dynamics often generate sequences of discrete events which can be described as a Markov process. The order of the underlying Markovian stochastic process is fundamental for characterizing statistical dependencies within sequences. As an example for this class of biological systems, we investigate the Markov order of sequences of microsaccadic eye movements from human observers. We calculate the integrated likelihood of a given sequence for various orders of the Markov process and use this in a Bayesian framework for statistical inference on the Markov order. Our analysis shows that data from most participants are best explained by a first-order Markov process. This is compatible with recent findings of a statistical coupling of subsequent microsaccade orientations. Our method might prove to be useful for a broad class of biological systems.

Technical Report on Modeling for Quasispecies Abundance Inference with Confidence Intervals from Metagenomic Sequence Data

DOE Office of Scientific and Technical Information (OSTI.GOV)

McLoughlin, K.

2016-01-11

The overall aim of this project is to develop a software package, called MetaQuant, that can determine the constituents of a complex microbial sample and estimate their relative abundances by analysis of metagenomic sequencing data. The goal for Task 1 is to create a generative model describing the stochastic process underlying the creation of sequence read pairs in the data set. The stages in this generative process include the selection of a source genome sequence for each read pair, with probability dependent on its abundance in the sample. The other stages describe the evolution of the source genome from itsmore » nearest common ancestor with a reference genome, breakage of the source DNA into short fragments, and the errors in sequencing the ends of the fragments to produce read pairs.« less

Analysis of Variability in HIV-1 Subtype A Strains in Russia Suggests a Combination of Deep Sequencing and Multitarget RNA Interference for Silencing of the Virus.

PubMed

Kretova, Olga V; Chechetkin, Vladimir R; Fedoseeva, Daria M; Kravatsky, Yuri V; Sosin, Dmitri V; Alembekov, Ildar R; Gorbacheva, Maria A; Gashnikova, Natalya M; Tchurikov, Nickolai A

2017-02-01

Any method for silencing the activity of the HIV-1 retrovirus should tackle the extremely high variability of HIV-1 sequences and mutational escape. We studied sequence variability in the vicinity of selected RNA interference (RNAi) targets from isolates of HIV-1 subtype A in Russia, and we propose that using artificial RNAi is a potential alternative to traditional antiretroviral therapy. We prove that using multiple RNAi targets overcomes the variability in HIV-1 isolates. The optimal number of targets critically depends on the conservation of the target sequences. The total number of targets that are conserved with a probability of 0.7-0.8 should exceed at least 2. Combining deep sequencing and multitarget RNAi may provide an efficient approach to cure HIV/AIDS.

A Nonparametric Approach For Representing Interannual Dependence In Monthly Streamflow Sequences

NASA Astrophysics Data System (ADS)

Sharma, A.; Oneill, R.

The estimation of risks associated with water management plans requires generation of synthetic streamflow sequences. The mathematical algorithms used to generate these sequences at monthly time scales are found lacking in two main respects: inability in preserving dependence attributes particularly at large (seasonal to interannual) time lags; and, a poor representation of observed distributional characteristics, in partic- ular, representation of strong assymetry or multimodality in the probability density function. Proposed here is an alternative that naturally incorporates both observed de- pendence and distributional attributes in the generated sequences. Use of a nonpara- metric framework provides an effective means for representing the observed proba- bility distribution, while the use of a Svariable kernelT ensures accurate modeling of & cedil;streamflow data sets that contain a substantial number of zero flow values. A careful selection of prior flows imparts the appropriate short-term memory, while use of an SaggregateT flow variable allows representation of interannual dependence. The non- & cedil;parametric simulation model is applied to monthly flows from the Beaver River near Beaver, Utah, USA, and the Burrendong dam inflows, New South Wales, Australia. Results indicate that while the use of traditional simulation approaches leads to an inaccurate representation of dependence at long (annual and interannual) time scales, the proposed model can simulate both short and long-term dependence. As a result, the proposed model ensures a significantly improved representation of reservoir storage statistics, particularly for systems influenced by long droughts. It is important to note that the proposed method offers a simpler and better alternative to conventional dis- aggregation models as: (a) a separate annual flow series is not required, (b) stringent assumptions relating annual and monthly flows are not needed, and (c) the method does not require the specification of a "water year", instead ensuring that the sum of any sequence of flows lasting twelve months will result in the type of dependence that is observed in the historical annual flow series.

A DNA enzyme that cleaves RNA

NASA Technical Reports Server (NTRS)

Breaker, R. R.; Joyce, G. F.; Hoyce, G. F. (Principal Investigator)

1994-01-01

BACKGROUND: Several types of RNA enzymes (ribozymes) have been identified in biological systems and generated in the laboratory. Considering the variety of known RNA enzymes and the similarity of DNA and RNA, it is reasonable to imagine that DNA might be able to function as an enzyme as well. No such DNA enzyme has been found in nature, however. We set out to identify a metal-dependent DNA enzyme using in vitro selection methodology. RESULTS: Beginning with a population of 10(14) DNAs containing 50 random nucleotides, we carried out five successive rounds of selective amplification, enriching for individuals that best promote the Pb(2+)-dependent cleavage of a target ribonucleoside 3'-O-P bond embedded within an otherwise all-DNA sequence. By the fifth round, the population as a whole carried out this reaction at a rate of 0.2 min-1. Based on the sequence of 20 individuals isolated from this population, we designed a simplified version of the catalytic domain that operates in an intermolecular context with a turnover rate of 1 min-1. This rate is about 10(5)-fold increased compared to the uncatalyzed reaction. CONCLUSIONS: Using in vitro selection techniques, we obtained a DNA enzyme that catalyzes the Pb(2+)-dependent cleavage of an RNA phosphoester in a reaction that proceeds with rapid turnover. The catalytic rate compares favorably to that of known RNA enzymes. We expect that other examples of DNA enzymes will soon be forthcoming.

Determinants of the rate of protein sequence evolution

PubMed Central

Zhang, Jianzhi; Yang, Jian-Rong

2015-01-01

The rate and mechanism of protein sequence evolution have been central questions in evolutionary biology since the 1960s. Although the rate of protein sequence evolution depends primarily on the level of functional constraint, exactly what constitutes functional constraint has remained unclear. The increasing availability of genomic data has allowed for much needed empirical examinations on the nature of functional constraint. These studies found that the evolutionary rate of a protein is predominantly influenced by its expression level rather than functional importance. A combination of theoretical and empirical analyses have identified multiple mechanisms behind these observations and demonstrated a prominent role that selection against errors in molecular and cellular processes plays in protein evolution. PMID:26055156

Influences of the molecular fuel structure on combustion reactions towards soot precursors in selected alkane and alkene flames.

PubMed

Ruwe, Lena; Moshammer, Kai; Hansen, Nils; Kohse-Höinghaus, Katharina

2018-04-25

In this study, we experimentally investigate the high-temperature oxidation kinetics of n-pentane, 1-pentene and 2-methyl-2-butene (2M2B) in a combustion environment using flame-sampling molecular beam mass spectrometry. The selected C5 fuels are prototypes for linear and branched, saturated and unsaturated fuel components, featuring different C-C and C-H bond structures. It is shown that the formation tendency of species, such as polycyclic aromatic hydrocarbons (PAHs), yielded through mass growth reactions increases drastically in the sequence n-pentane < 1-pentene < 2M2B. This comparative study enables valuable insights into fuel-dependent reaction sequences of the gas-phase combustion mechanism that provide explanations for the observed difference in the PAH formation tendency. First, we investigate the fuel-structure-dependent formation of small hydrocarbon species that are yielded as intermediate species during the fuel decomposition, because these species are at the origin of the subsequent mass growth reaction pathways. Second, we review typical PAH formation reactions inspecting repetitive growth sequences in dependence of the molecular fuel structure. Third, we discuss how differences in the intermediate species pool influence the formation reactions of key aromatic ring species that are important for the PAH growth process underlying soot formation. As a main result it was found that for the fuels featuring a C[double bond, length as m-dash]C double bond, the chemistry of their allylic fuel radicals and their decomposition products strongly influences the combination reactions to the initially formed aromatic ring species and as a consequence, the PAH formation tendency.

Evidence for Context-Dependent Complementarity of Non-Shine-Dalgarno Ribosome Binding Sites to Escherichia coli rRNA

PubMed Central

Barendt, Pamela A.; Shah, Najaf A.; Barendt, Gregory A.; Kothari, Parth A.; Sarkar, Casim A.

2013-01-01

While the ribosome has evolved to function in complex intracellular environments, these contexts do not easily allow for the study of its inherent capabilities. We have used a synthetic, well-defined, Escherichia coli (E. coli)-based translation system in conjunction with ribosome display, a powerful in vitro selection method, to identify ribosome binding sites (RBSs) that can promote the efficient translation of messenger RNAs (mRNAs) with a leader length representative of natural E. coli mRNAs. In previous work, we used a longer leader sequence and unexpectedly recovered highly efficient cytosine-rich sequences with complementarity to the 16S ribosomal RNA (rRNA) and similarity to eukaryotic RBSs. In the current study, Shine-Dalgarno (SD) sequences were prevalent but non-SD sequences were also heavily enriched and were dominated by novel guanine- and uracil-rich motifs which showed statistically significant complementarity to the 16S rRNA. Additionally, only SD motifs exhibited position-dependent decreases in sequence entropy, indicating that non-SD motifs likely operate by increasing the local concentration of ribosomes in the vicinity of the start codon, rather than by a position-dependent mechanism. These results further support the putative generality of mRNA-rRNA complementarity in facilitating mRNA translation, but also suggest that context (e.g., leader length and composition) dictates the specific subset of possible RBSs that are used for efficient translation of a given transcript. PMID:23427812

Selectivity and Efficiency of Late Transgene Expression by Transcriptionally Targeted Oncolytic Adenoviruses Are Dependent on the Transgene Insertion Strategy

PubMed Central

Quirin, Christina; Rohmer, Stanimira; Fernández-Ulibarri, Inés; Behr, Michael; Hesse, Andrea; Engelhardt, Sarah; Erbs, Philippe; Enk, Alexander H.

2011-01-01

Abstract Key challenges facing cancer therapy are the development of tumor-specific drugs and potent multimodal regimens. Oncolytic adenoviruses possess the potential to realize both aims by restricting virus replication to tumors and inserting therapeutic genes into the virus genome, respectively. A major effort in this regard is to express transgenes in a tumor-specific manner without affecting virus replication. Using both luciferase as a sensitive reporter and genetic prodrug activation, we show that promoter control of E1A facilitates highly selective expression of transgenes inserted into the late transcription unit. This, however, required multistep optimization of late transgene expression. Transgene insertion via internal ribosome entry site (IRES), splice acceptor (SA), or viral 2A sequences resulted in replication-dependent expression. Unexpectedly, analyses in appropriate substrates and with matching control viruses revealed that IRES and SA, but not 2A, facilitated indirect transgene targeting via tyrosinase promoter control of E1A. Transgene expression via SA was more selective (up to 1,500-fold) but less effective than via IRES. Notably, we also revealed transgene-dependent interference with splicing. Hence, the prodrug convertase FCU1 (a cytosine deaminase–uracil phosphoribosyltransferase fusion protein) was expressed only after optimizing the sequence surrounding the SA site and mutating a cryptic splice site within the transgene. The resulting tyrosinase promoter-regulated and FCU1-encoding adenovirus combined effective oncolysis with targeted prodrug activation therapy of melanoma. Thus, prodrug activation showed potent bystander killing and increased cytotoxicity of the virus up to 10-fold. We conclude that armed oncolytic viruses can be improved substantially by comparing and optimizing strategies for targeted transgene expression, thereby implementing selective and multimodal cancer therapies. PMID:20939692

Reducing DNA context dependence in bacterial promoters

PubMed Central

Carr, Swati B.; Densmore, Douglas M.

2017-01-01

Variation in the DNA sequence upstream of bacterial promoters is known to affect the expression levels of the products they regulate, sometimes dramatically. While neutral synthetic insulator sequences have been found to buffer promoters from upstream DNA context, there are no established methods for designing effective insulator sequences with predictable effects on expression levels. We address this problem with Degenerate Insulation Screening (DIS), a novel method based on a randomized 36-nucleotide insulator library and a simple, high-throughput, flow-cytometry-based screen that randomly samples from a library of 436 potential insulated promoters. The results of this screen can then be compared against a reference uninsulated device to select a set of insulated promoters providing a precise level of expression. We verify this method by insulating the constitutive, inducible, and repressible promotors of a four transcriptional-unit inverter (NOT-gate) circuit, finding both that order dependence is largely eliminated by insulation and that circuit performance is also significantly improved, with a 5.8-fold mean improvement in on/off ratio. PMID:28422998

Augmented brain function by coordinated reset stimulation with slowly varying sequences.

PubMed

Zeitler, Magteld; Tass, Peter A

2015-01-01

Several brain disorders are characterized by abnormally strong neuronal synchrony. Coordinated Reset (CR) stimulation was developed to selectively counteract abnormal neuronal synchrony by desynchronization. For this, phase resetting stimuli are delivered to different subpopulations in a timely coordinated way. In neural networks with spike timing-dependent plasticity CR stimulation may eventually lead to an anti-kindling, i.e., an unlearning of abnormal synaptic connectivity and abnormal synchrony. The spatiotemporal sequence by which all stimulation sites are stimulated exactly once is called the stimulation site sequence, or briefly sequence. So far, in simulations, pre-clinical and clinical applications CR was applied either with fixed sequences or rapidly varying sequences (RVS). In this computational study we show that appropriate repetition of the sequence with occasional random switching to the next sequence may significantly improve the anti-kindling effect of CR. To this end, a sequence is applied many times before randomly switching to the next sequence. This new method is called SVS CR stimulation, i.e., CR with slowly varying sequences. In a neuronal network with strong short-range excitatory and weak long-range inhibitory dynamic couplings SVS CR stimulation turns out to be superior to CR stimulation with fixed sequences or RVS.

Augmented brain function by coordinated reset stimulation with slowly varying sequences

PubMed Central

Zeitler, Magteld; Tass, Peter A.

2015-01-01

Several brain disorders are characterized by abnormally strong neuronal synchrony. Coordinated Reset (CR) stimulation was developed to selectively counteract abnormal neuronal synchrony by desynchronization. For this, phase resetting stimuli are delivered to different subpopulations in a timely coordinated way. In neural networks with spike timing-dependent plasticity CR stimulation may eventually lead to an anti-kindling, i.e., an unlearning of abnormal synaptic connectivity and abnormal synchrony. The spatiotemporal sequence by which all stimulation sites are stimulated exactly once is called the stimulation site sequence, or briefly sequence. So far, in simulations, pre-clinical and clinical applications CR was applied either with fixed sequences or rapidly varying sequences (RVS). In this computational study we show that appropriate repetition of the sequence with occasional random switching to the next sequence may significantly improve the anti-kindling effect of CR. To this end, a sequence is applied many times before randomly switching to the next sequence. This new method is called SVS CR stimulation, i.e., CR with slowly varying sequences. In a neuronal network with strong short-range excitatory and weak long-range inhibitory dynamic couplings SVS CR stimulation turns out to be superior to CR stimulation with fixed sequences or RVS. PMID:25873867

Design of Cyclic Peptide Based Glucose Receptors and Their Application in Glucose Sensing.

PubMed

Li, Chao; Chen, Xin; Zhang, Fuyuan; He, Xingxing; Fang, Guozhen; Liu, Jifeng; Wang, Shuo

2017-10-03

Glucose assay is of great scientific significance in clinical diagnostics and bioprocess monitoring, and to design a new glucose receptor is necessary for the development of more sensitive, selective, and robust glucose detection techniques. Herein, a series of cyclic peptide (CP) glucose receptors were designed to mimic the binding sites of glucose binding protein (GBP), and CPs' sequence contained amino acid sites Asp, Asn, His, Asp, and Arg, which constituted the first layer interactions of GBP. The properties of these CPs used as a glucose receptor or substitute for the GBP were studied by using a quartz crystal microbalance (QCM) technique. It was found that CPs can form a self-assembled monolayer at the Au quartz electrode surface, and the monolayer's properties were characterized by using cyclic voltammetry, electrochemical impedance spectroscopy, and atomic force microscopy. The CPs' binding affinity to saccharide (i.e., galactose, fructose, lactose, sucrose, and maltose) was investigated, and the CPs' sensitivity and selectivity toward glucose were found to be dependent upon the configuration,i.e., the amino acids sequence of the CPs. The cyclic unit with a cyclo[-CNDNHCRDNDC-] sequence gave the highest selectivity and sensitivity for glucose sensing. This work suggests that a synthetic peptide bearing a particular functional sequence could be applied for developing a new generation of glucose receptors and would find huge application in biological, life science, and clinical diagnostics fields.

Performing SELEX experiments in silico

NASA Astrophysics Data System (ADS)

Wondergem, J. A. J.; Schiessel, H.; Tompitak, M.

2017-11-01

Due to the sequence-dependent nature of the elasticity of DNA, many protein-DNA complexes and other systems in which DNA molecules must be deformed have preferences for the type of DNA sequence they interact with. SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiments and similar sequence selection experiments have been used extensively to examine the (indirect readout) sequence preferences of, e.g., nucleosomes (protein spools around which DNA is wound for compactification) and DNA rings. We show how recently developed computational and theoretical tools can be used to emulate such experiments in silico. Opening up this possibility comes with several benefits. First, it allows us a better understanding of our models and systems, specifically about the roles played by the simulation temperature and the selection pressure on the sequences. Second, it allows us to compare the predictions made by the model of choice with experimental results. We find agreement on important features between predictions of the rigid base-pair model and experimental results for DNA rings and interesting differences that point out open questions in the field. Finally, our simulations allow application of the SELEX methodology to systems that are experimentally difficult to realize because they come with high energetic costs and are therefore unlikely to form spontaneously, such as very short or overwound DNA rings.

A new sequence for single-shot diffusion-weighted NMR spectroscopy by the trace of the diffusion tensor.

PubMed

Valette, Julien; Giraudeau, Céline; Marchadour, Charlotte; Djemai, Boucif; Geffroy, Françoise; Ghaly, Mohamed Ahmed; Le Bihan, Denis; Hantraye, Philippe; Lebon, Vincent; Lethimonnier, Franck

2012-12-01

Diffusion-weighted spectroscopy is a unique tool for exploring the intracellular microenvironment in vivo. In living systems, diffusion may be anisotropic, when biological membranes exhibit particular orientation patterns. In this work, a volume selective diffusion-weighted sequence is proposed, allowing single-shot measurement of the trace of the diffusion tensor, which does not depend on tissue anisotropy. With this sequence, the minimal echo time is only three times the diffusion time. In addition, cross-terms between diffusion gradients and other gradients are cancelled out. An adiabatic version, similar to localization by adiabatic selective refocusing sequence, is then derived, providing partial immunity against cross-terms. Proof of concept is performed ex vivo on chicken skeletal muscle by varying tissue orientation and intra-voxel shim. In vivo performance of the sequence is finally illustrated in a U87 glioblastoma mouse model, allowing the measurement of the trace apparent diffusion coefficient for six metabolites, including J-modulated metabolites. Although measurement performed along three separate orthogonal directions would bring similar accuracy on trace apparent diffusion coefficient under ideal conditions, the method described here should be useful for probing intimate properties of the cells with minimal experimental bias. Copyright © 2012 Wiley Periodicals, Inc.

Engineering of ribozyme-based aminoglycoside switches of gene expression by in vivo genetic selection in Saccharomyces cerevisiae.

PubMed

Klauser, Benedikt; Rehm, Charlotte; Summerer, Daniel; Hartig, Jörg S

2015-01-01

Synthetic RNA-based switches are a growing class of genetic controllers applied in synthetic biology to engineer cellular functions. In this chapter, we detail a protocol for the selection of posttranscriptional controllers of gene expression in yeast using the Schistosoma mansoni hammerhead ribozyme as a central catalytic unit. Incorporation of a small molecule-sensing aptamer domain into the ribozyme renders its activity ligand-dependent. Aptazymes display numerous advantages over conventional protein-based transcriptional controllers, namely, the use of little genomic space for encryption, their modular architecture allowing for easy reprogramming to new inputs, the physical linkage to the message to be controlled, and the ability to function without protein cofactors. Herein, we describe the method to select ribozyme-based switches of gene expression in Saccharomyces cerevisiae that we successfully implemented to engineer neomycin- and theophylline-responsive switches. We also highlight how to adapt the protocol to screen for switches responsive to other ligands. Reprogramming of the sensor unit and incorporation into any RNA of interest enables the fulfillment of a variety of regulatory functions. However, proper functioning of the aptazyme is largely dependent on optimal connection between the aptamer and the catalytic core. We obtained functional switches from a pool of variants carrying randomized connection sequences by an in vivo selection in MaV203 yeast cells that allows screening of a large sequence space of up to 1×10(9) variants. The protocol given explains how to construct aptazyme libraries, carry out the in vivo selection and characterize novel ON- and OFF-switches. © 2015 Elsevier Inc. All rights reserved.

Nonspatial Sequence Coding in CA1 Neurons

PubMed Central

Allen, Timothy A.; Salz, Daniel M.; McKenzie, Sam

2016-01-01

The hippocampus is critical to the memory for sequences of events, a defining feature of episodic memory. However, the fundamental neuronal mechanisms underlying this capacity remain elusive. While considerable research indicates hippocampal neurons can represent sequences of locations, direct evidence of coding for the memory of sequential relationships among nonspatial events remains lacking. To address this important issue, we recorded neural activity in CA1 as rats performed a hippocampus-dependent sequence-memory task. Briefly, the task involves the presentation of repeated sequences of odors at a single port and requires rats to identify each item as “in sequence” or “out of sequence”. We report that, while the animals' location and behavior remained constant, hippocampal activity differed depending on the temporal context of items—in this case, whether they were presented in or out of sequence. Some neurons showed this effect across items or sequence positions (general sequence cells), while others exhibited selectivity for specific conjunctions of item and sequence position information (conjunctive sequence cells) or for specific probe types (probe-specific sequence cells). We also found that the temporal context of individual trials could be accurately decoded from the activity of neuronal ensembles, that sequence coding at the single-cell and ensemble level was linked to sequence memory performance, and that slow-gamma oscillations (20–40 Hz) were more strongly modulated by temporal context and performance than theta oscillations (4–12 Hz). These findings provide compelling evidence that sequence coding extends beyond the domain of spatial trajectories and is thus a fundamental function of the hippocampus. SIGNIFICANCE STATEMENT The ability to remember the order of life events depends on the hippocampus, but the underlying neural mechanisms remain poorly understood. Here we addressed this issue by recording neural activity in hippocampal region CA1 while rats performed a nonspatial sequence memory task. We found that hippocampal neurons code for the temporal context of items (whether odors were presented in the correct or incorrect sequential position) and that this activity is linked with memory performance. The discovery of this novel form of temporal coding in hippocampal neurons advances our fundamental understanding of the neurobiology of episodic memory and will serve as a foundation for our cross-species, multitechnique approach aimed at elucidating the neural mechanisms underlying memory impairments in aging and dementia. PMID:26843637

Network Analysis of Protein Adaptation: Modeling the Functional Impact of Multiple Mutations

PubMed Central

Beleva Guthrie, Violeta; Masica, David L; Fraser, Andrew; Federico, Joseph; Fan, Yunfan; Camps, Manel; Karchin, Rachel

2018-01-01

Abstract The evolution of new biochemical activities frequently involves complex dependencies between mutations and rapid evolutionary radiation. Mutation co-occurrence and covariation have previously been used to identify compensating mutations that are the result of physical contacts and preserve protein function and fold. Here, we model pairwise functional dependencies and higher order interactions that enable evolution of new protein functions. We use a network model to find complex dependencies between mutations resulting from evolutionary trade-offs and pleiotropic effects. We present a method to construct these networks and to identify functionally interacting mutations in both extant and reconstructed ancestral sequences (Network Analysis of Protein Adaptation). The time ordering of mutations can be incorporated into the networks through phylogenetic reconstruction. We apply NAPA to three distantly homologous β-lactamase protein clusters (TEM, CTX-M-3, and OXA-51), each of which has experienced recent evolutionary radiation under substantially different selective pressures. By analyzing the network properties of each protein cluster, we identify key adaptive mutations, positive pairwise interactions, different adaptive solutions to the same selective pressure, and complex evolutionary trajectories likely to increase protein fitness. We also present evidence that incorporating information from phylogenetic reconstruction and ancestral sequence inference can reduce the number of spurious links in the network, whereas preserving overall network community structure. The analysis does not require structural or biochemical data. In contrast to function-preserving mutation dependencies, which are frequently from structural contacts, gain-of-function mutation dependencies are most commonly between residues distal in protein structure. PMID:29522102

Tissue-selective restriction of RNA editing of CaV1.3 by splicing factor SRSF9.

PubMed

Huang, Hua; Kapeli, Katannya; Jin, Wenhao; Wong, Yuk Peng; Arumugam, Thiruma Valavan; Koh, Joanne Huifen; Srimasorn, Sumitra; Mallilankaraman, Karthik; Chua, John Jia En; Yeo, Gene W; Soong, Tuck Wah

2018-05-04

Adenosine DeAminases acting on RNA (ADAR) catalyzes adenosine-to-inosine (A-to-I) conversion within RNA duplex structures. While A-to-I editing is often dynamically regulated in a spatial-temporal manner, the mechanisms underlying its tissue-selective restriction remain elusive. We have previously reported that transcripts of voltage-gated calcium channel CaV1.3 are subject to brain-selective A-to-I RNA editing by ADAR2. Here, we show that editing of CaV1.3 mRNA is dependent on a 40 bp RNA duplex formed between exon 41 and an evolutionarily conserved editing site complementary sequence (ECS) located within the preceding intron. Heterologous expression of a mouse minigene that contained the ECS, intermediate intronic sequence and exon 41 with ADAR2 yielded robust editing. Interestingly, editing of CaV1.3 was potently inhibited by serine/arginine-rich splicing factor 9 (SRSF9). Mechanistically, the inhibitory effect of SRSF9 required direct RNA interaction. Selective down-regulation of SRSF9 in neurons provides a basis for the neuron-specific editing of CaV1.3 transcripts.

Overcoming S-Phase Checkpoint-Mediated Resistance: Sequence-Dependent Synergy of Gemcitabine and SN-38 In Human Carcinoma Cell Lines*

PubMed Central

Gálvez-Peralta, Marina; Dai, Nga T.; Loegering, David A.; Flatten, Karen; Safgren, Stephanie; Wagner, Jill; Ames, Matthew M.; Karnitz, Larry M.; Kaufmann, Scott H.

2008-01-01

Although agents that inhibit DNA synthesis are widely used in the treatment of cancer, the optimal method for combining such agents and the mechanism of their synergy is poorly understood. The present study examined the effects of combining gemcitabine and SN-38 (the active metabolite of irinotecan), two S phase-selective agents that individually have broad antitumor activity, in human cancer cells in vitro. Colony forming assays revealed that simultaneous treatment of Ovcar-5 ovarian cancer cells or BxPC-3 pancreatic cancer cells with gemcitabine and SN-38 resulted in antagonistic effects. In contrast, sequential treatment with the two agents in either order resulted in synergistic antiproliferative effects, although the mechanism of synergy varied with the sequence. In particular, SN-38 arrested cells in S phase, enhanced the accumulation of gemcitabine metabolites and diminished checkpoint kinase 1, thereby sensitizing cells in the SN-38 → gemcitabine sequence. Gemcitabine treatment followed by removal allowed prolonged progression through S phase, contributing to synergy of the gemcitabine → SN-38 sequence. Collectively, these results suggest that S phase selective agents might exhibit more cytotoxicity when administered sequentially rather than simultaneously. PMID:18509065

Deep Sequencing of Random Mutant Libraries Reveals the Active Site of the Narrow Specificity CphA Metallo-β-Lactamase is Fragile to Mutations.

PubMed

Sun, Zhizeng; Mehta, Shrenik C; Adamski, Carolyn J; Gibbs, Richard A; Palzkill, Timothy

2016-09-12

CphA is a Zn(2+)-dependent metallo-β-lactamase that efficiently hydrolyzes only carbapenem antibiotics. To understand the sequence requirements for CphA function, single codon random mutant libraries were constructed for residues in and near the active site and mutants were selected for E. coli growth on increasing concentrations of imipenem, a carbapenem antibiotic. At high concentrations of imipenem that select for phenotypically wild-type mutants, the active-site residues exhibit stringent sequence requirements in that nearly all residues in positions that contact zinc, the substrate, or the catalytic water do not tolerate amino acid substitutions. In addition, at high imipenem concentrations a number of residues that do not directly contact zinc or substrate are also essential and do not tolerate substitutions. Biochemical analysis confirmed that amino acid substitutions at essential positions decreased the stability or catalytic activity of the CphA enzyme. Therefore, the CphA active - site is fragile to substitutions, suggesting active-site residues are optimized for imipenem hydrolysis. These results also suggest that resistance to inhibitors targeted to the CphA active site would be slow to develop because of the strong sequence constraints on function.

U-Groove Aluminum Weld Strength Improvement

NASA Technical Reports Server (NTRS)

Verderaime, V.; Vaughan, R.

1997-01-01

Though butt-welds are among the most preferred joining methods in aerostructures, their strength dependence on inelastic mechanics is generally the least understood. This study investigated experimental strain distributions across a thick aluminum U-grooved weld and identified two weld process considerations for improving the multipass weld strength. One is the source of peaking in which the extreme thermal expansion and contraction gradient of the fusion heat input across the groove tab thickness produces severe angular distortion that induces bending under uniaxial loading. The other is the filler strain hardening decreasing with increasing filler pass sequences, producing the weakest welds on the last weld pass side. Both phenomena are governed by weld pass sequences. Many industrial welding schedules unknowingly compound these effects, which reduce the weld strength. A depeaking index model was developed to select filler pass thickness, pass numbers, and sequences to improve depeaking in the welding process. The result was to select the number and sequence of weld passes to reverse the peaking angle such as to combine the strongest weld pass side with the peaking induced bending tension component side to provide a more uniform stress and stronger weld under axial tensile loading.

Comparison of Methods of Detection of Exceptional Sequences in Prokaryotic Genomes.

PubMed

Rusinov, I S; Ershova, A S; Karyagina, A S; Spirin, S A; Alexeevski, A V

2018-02-01

Many proteins need recognition of specific DNA sequences for functioning. The number of recognition sites and their distribution along the DNA might be of biological importance. For example, the number of restriction sites is often reduced in prokaryotic and phage genomes to decrease the probability of DNA cleavage by restriction endonucleases. We call a sequence an exceptional one if its frequency in a genome significantly differs from one predicted by some mathematical model. An exceptional sequence could be either under- or over-represented, depending on its frequency in comparison with the predicted one. Exceptional sequences could be considered biologically meaningful, for example, as targets of DNA-binding proteins or as parts of abundant repetitive elements. Several methods to predict frequency of a short sequence in a genome, based on actual frequencies of certain its subsequences, are used. The most popular are methods based on Markov chain models. But any rigorous comparison of the methods has not previously been performed. We compared three methods for the prediction of short sequence frequencies: the maximum-order Markov chain model-based method, the method that uses geometric mean of extended Markovian estimates, and the method that utilizes frequencies of all subsequences including discontiguous ones. We applied them to restriction sites in complete genomes of 2500 prokaryotic species and demonstrated that the results depend greatly on the method used: lists of 5% of the most under-represented sites differed by up to 50%. The method designed by Burge and coauthors in 1992, which utilizes all subsequences of the sequence, showed a higher precision than the other two methods both on prokaryotic genomes and randomly generated sequences after computational imitation of selective pressure. We propose this method as the first choice for detection of exceptional sequences in prokaryotic genomes.

Recombination rate and the distribution of transposable elements in the Drosophila melanogaster genome.

PubMed

Rizzon, Carène; Marais, Gabriel; Gouy, Manolo; Biémont, Christian

2002-03-01

We analyzed the distribution of 54 families of transposable elements (TEs; transposons, LTR retrotransposons, and non-LTR retrotransposons) in the chromosomes of Drosophila melanogaster, using data from the sequenced genome. The density of LTR and non-LTR retrotransposons (RNA-based elements) was high in regions with low recombination rates, but there was no clear tendency to parallel the recombination rate. However, the density of transposons (DNA-based elements) was significantly negatively correlated with recombination rate. The accumulation of TEs in regions of reduced recombination rate is compatible with selection acting against TEs, as selection is expected to be weaker in regions with lower recombination. The differences in the relationship between recombination rate and TE density that exist between chromosome arms suggest that TE distribution depends on specific characteristics of the chromosomes (chromatin structure, distribution of other sequences), the TEs themselves (transposition mechanism), and the species (reproductive system, effective population size, etc.), that have differing influences on the effect of natural selection acting against the TE insertions.

Artificial genetic selection for an efficient translation initiation site for expression of human RACK1 gene in Escherichia coli

PubMed Central

Zhelyabovskaya, Olga B.; Berlin, Yuri A.; Birikh, Klara R.

2004-01-01

In bacterial expression systems, translation initiation is usually the rate limiting and the least predictable stage of protein synthesis. Efficiency of a translation initiation site can vary dramatically depending on the sequence context. This is why many standard expression vectors provide very poor expression levels of some genes. This notion persuaded us to develop an artificial genetic selection protocol, which allows one to find for a given target gene an individual efficient ribosome binding site from a random pool. In order to create Darwinian pressure necessary for the genetic selection, we designed a system based on translational coupling, in which microorganism survival in the presence of antibiotic depends on expression of the target gene, while putting no special requirements on this gene. Using this system we obtained superproducing constructs for the human protein RACK1 (receptor for activated C kinase). PMID:15034151

Population-Sequencing as a Biomarker of Burkholderia mallei and Burkholderia pseudomallei Evolution through Microbial Forensic Analysis.

PubMed

Jakupciak, John P; Wells, Jeffrey M; Karalus, Richard J; Pawlowski, David R; Lin, Jeffrey S; Feldman, Andrew B

2013-01-01

Large-scale genomics projects are identifying biomarkers to detect human disease. B. pseudomallei and B. mallei are two closely related select agents that cause melioidosis and glanders. Accurate characterization of metagenomic samples is dependent on accurate measurements of genetic variation between isolates with resolution down to strain level. Often single biomarker sensitivity is augmented by use of multiple or panels of biomarkers. In parallel with single biomarker validation, advances in DNA sequencing enable analysis of entire genomes in a single run: population-sequencing. Potentially, direct sequencing could be used to analyze an entire genome to serve as the biomarker for genome identification. However, genome variation and population diversity complicate use of direct sequencing, as well as differences caused by sample preparation protocols including sequencing artifacts and mistakes. As part of a Department of Homeland Security program in bacterial forensics, we examined how to implement whole genome sequencing (WGS) analysis as a judicially defensible forensic method for attributing microbial sample relatedness; and also to determine the strengths and limitations of whole genome sequence analysis in a forensics context. Herein, we demonstrate use of sequencing to provide genetic characterization of populations: direct sequencing of populations.

Population-Sequencing as a Biomarker of Burkholderia mallei and Burkholderia pseudomallei Evolution through Microbial Forensic Analysis

PubMed Central

Jakupciak, John P.; Wells, Jeffrey M.; Karalus, Richard J.; Pawlowski, David R.; Lin, Jeffrey S.; Feldman, Andrew B.

2013-01-01

Large-scale genomics projects are identifying biomarkers to detect human disease. B. pseudomallei and B. mallei are two closely related select agents that cause melioidosis and glanders. Accurate characterization of metagenomic samples is dependent on accurate measurements of genetic variation between isolates with resolution down to strain level. Often single biomarker sensitivity is augmented by use of multiple or panels of biomarkers. In parallel with single biomarker validation, advances in DNA sequencing enable analysis of entire genomes in a single run: population-sequencing. Potentially, direct sequencing could be used to analyze an entire genome to serve as the biomarker for genome identification. However, genome variation and population diversity complicate use of direct sequencing, as well as differences caused by sample preparation protocols including sequencing artifacts and mistakes. As part of a Department of Homeland Security program in bacterial forensics, we examined how to implement whole genome sequencing (WGS) analysis as a judicially defensible forensic method for attributing microbial sample relatedness; and also to determine the strengths and limitations of whole genome sequence analysis in a forensics context. Herein, we demonstrate use of sequencing to provide genetic characterization of populations: direct sequencing of populations. PMID:24455204

Structural and Functional Basis of the Fidelity of Nucleotide Selection by Flavivirus RNA-Dependent RNA Polymerases

PubMed Central

Canard, Bruno

2018-01-01

Viral RNA-dependent RNA polymerases (RdRps) play a central role not only in viral replication, but also in the genetic evolution of viral RNAs. After binding to an RNA template and selecting 5′-triphosphate ribonucleosides, viral RdRps synthesize an RNA copy according to Watson-Crick base-pairing rules. The copy process sometimes deviates from both the base-pairing rules specified by the template and the natural ribose selectivity and, thus, the process is error-prone due to the intrinsic (in)fidelity of viral RdRps. These enzymes share a number of conserved amino-acid sequence strings, called motifs A–G, which can be defined from a structural and functional point-of-view. A co-relation is gradually emerging between mutations in these motifs and viral genome evolution or observed mutation rates. Here, we review our current knowledge on these motifs and their role on the structural and mechanistic basis of the fidelity of nucleotide selection and RNA synthesis by Flavivirus RdRps. PMID:29385764

Host-Specific and Segment-Specific Evolutionary Dynamics of Avian and Human Influenza A Viruses: A Systematic Review.

PubMed

Kim, Kiyeon; Omori, Ryosuke; Ueno, Keisuke; Iida, Sayaka; Ito, Kimihito

2016-01-01

Understanding the evolutionary dynamics of influenza viruses is essential to control both avian and human influenza. Here, we analyze host-specific and segment-specific Tajima's D trends of influenza A virus through a systematic review using viral sequences registered in the National Center for Biotechnology Information. To avoid bias from viral population subdivision, viral sequences were stratified according to their sampling locations and sampling years. As a result, we obtained a total of 580 datasets each of which consists of nucleotide sequences of influenza A viruses isolated from a single population of hosts at a single sampling site within a single year. By analyzing nucleotide sequences in the datasets, we found that Tajima's D values of viral sequences were different depending on hosts and gene segments. Tajima's D values of viruses isolated from chicken and human samples showed negative, suggesting purifying selection or a rapid population growth of the viruses. The negative Tajima's D values in rapidly growing viral population were also observed in computer simulations. Tajima's D values of PB2, PB1, PA, NP, and M genes of the viruses circulating in wild mallards were close to zero, suggesting that these genes have undergone neutral selection in constant-sized population. On the other hand, Tajima's D values of HA and NA genes of these viruses were positive, indicating HA and NA have undergone balancing selection in wild mallards. Taken together, these results indicated the existence of unknown factors that maintain viral subtypes in wild mallards.

Sequence dependent aggregation of peptides and fibril formation

NASA Astrophysics Data System (ADS)

Hung, Nguyen Ba; Le, Duy-Manh; Hoang, Trinh X.

2017-09-01

Deciphering the links between amino acid sequence and amyloid fibril formation is key for understanding protein misfolding diseases. Here we use Monte Carlo simulations to study the aggregation of short peptides in a coarse-grained model with hydrophobic-polar (HP) amino acid sequences and correlated side chain orientations for hydrophobic contacts. A significant heterogeneity is observed in the aggregate structures and in the thermodynamics of aggregation for systems of different HP sequences and different numbers of peptides. Fibril-like ordered aggregates are found for several sequences that contain the common HPH pattern, while other sequences may form helix bundles or disordered aggregates. A wide variation of the aggregation transition temperatures among sequences, even among those of the same hydrophobic fraction, indicates that not all sequences undergo aggregation at a presumable physiological temperature. The transition is found to be the most cooperative for sequences forming fibril-like structures. For a fibril-prone sequence, it is shown that fibril formation follows the nucleation and growth mechanism. Interestingly, a binary mixture of peptides of an aggregation-prone and a non-aggregation-prone sequence shows the association and conversion of the latter to the fibrillar structure. Our study highlights the role of a sequence in selecting fibril-like aggregates and also the impact of a structural template on fibril formation by peptides of unrelated sequences.

Power Analysis of Artificial Selection Experiments Using Efficient Whole Genome Simulation of Quantitative Traits

PubMed Central

Kessner, Darren; Novembre, John

2015-01-01

Evolve and resequence studies combine artificial selection experiments with massively parallel sequencing technology to study the genetic basis for complex traits. In these experiments, individuals are selected for extreme values of a trait, causing alleles at quantitative trait loci (QTL) to increase or decrease in frequency in the experimental population. We present a new analysis of the power of artificial selection experiments to detect and localize quantitative trait loci. This analysis uses a simulation framework that explicitly models whole genomes of individuals, quantitative traits, and selection based on individual trait values. We find that explicitly modeling QTL provides qualitatively different insights than considering independent loci with constant selection coefficients. Specifically, we observe how interference between QTL under selection affects the trajectories and lengthens the fixation times of selected alleles. We also show that a substantial portion of the genetic variance of the trait (50–100%) can be explained by detected QTL in as little as 20 generations of selection, depending on the trait architecture and experimental design. Furthermore, we show that power depends crucially on the opportunity for recombination during the experiment. Finally, we show that an increase in power is obtained by leveraging founder haplotype information to obtain allele frequency estimates. PMID:25672748

Sequence requirements of oligonucleotide chiral selectors for the capillary electrophoresis resolution of low-affinity DNA binders.

PubMed

Tohala, Luma; Oukacine, Farid; Ravelet, Corinne; Peyrin, Eric

2017-05-01

We recently reported that a great variety of DNA oligonucleotides (ONs) used as chiral selectors in partial-filling capillary electrophoresis (CE) exhibited interesting enantioresolution properties toward low-affinity DNA binders. Herein, the sequence prerequisites of ONs for the CE enantioseparation process were studied. First, the chiral resolution properties of a series of homopolymeric sequences (Poly-dT) of different lengths (from 5 to 60-mer) were investigated. It was shown that the size increase-dependent random coil-like conformation of Poly-dT favorably acted on the apparent selectivity and resolution. The base-unpairing state constituted also an important factor in the chiral resolution ability of ONs as the switch from the single-stranded to double-stranded structure was responsible for a significant decrease in the analyte selectivity range. Finally, the chemical diversity enhanced the enantioresolution ability of single-stranded ONs. The present work could lay the foundation for the design of performant ON chiral selectors for the CE separation of weak DNA binder enantiomers. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

De novo generation of helper virus-satellite chimera RNAs results in disease attenuation and satellite sequence acquisition in a host-dependent manner.

PubMed

Pyle, J D; Scholthof, Karen-Beth G

2018-01-15

Panicum mosaic virus (PMV) is a helper RNA virus for satellite RNAs (satRNAs) and a satellite virus (SPMV). Here, we describe modifications that occur at the 3'-end of a satRNA of PMV, satS. Co-infections of PMV+satS result in attenuation of the disease symptoms induced by PMV alone in Brachypodium distachyon and proso millet. The 375 nt satS acquires ~100-200 nts from the 3'-end of PMV during infection and is associated with decreased abundance of the PMV RNA and capsid protein in millet. PMV-satS chimera RNAs were isolated from native infections of St. Augustinegrass and switchgrass. Phylogenetic analyses revealed that the chimeric RNAs clustered according to the host species from which they were isolated. Additionally, the chimera satRNAs acquired non-viral "linker" sequences in a host-specific manner. These results highlight the dynamic regulation of viral pathogenicity by satellites, and the selective host-dependent, sequence-based pressures for driving satRNA generation and genome compositions. Copyright © 2017 Elsevier Inc. All rights reserved.

DNA Sequence-Mediated, Evolutionarily Rapid Redistribution of Meiotic Recombination Hotspots

PubMed Central

Wahls, Wayne P.; Davidson, Mari K.

2011-01-01

Hotspots regulate the position and frequency of Spo11 (Rec12)-initiated meiotic recombination, but paradoxically they are suicidal and are somehow resurrected elsewhere in the genome. After the DNA sequence-dependent activation of hotspots was discovered in fission yeast, nearly two decades elapsed before the key realizations that (A) DNA site-dependent regulation is broadly conserved and (B) individual eukaryotes have multiple different DNA sequence motifs that activate hotspots. From our perspective, such findings provide a conceptually straightforward solution to the hotspot paradox and can explain other, seemingly complex features of meiotic recombination. We describe how a small number of single-base-pair substitutions can generate hotspots de novo and dramatically alter their distribution in the genome. This model also shows how equilibrium rate kinetics could maintain the presence of hotspots over evolutionary timescales, without strong selective pressures invoked previously, and explains why hotspots localize preferentially to intergenic regions and introns. The model is robust enough to account for all hotspots of humans and chimpanzees repositioned since their divergence from the latest common ancestor. PMID:22084420

RNAi triggered by symmetrically transcribed transgenes in Drosophila melanogaster.

PubMed Central

Giordano, Ennio; Rendina, Rosaria; Peluso, Ivana; Furia, Maria

2002-01-01

Specific silencing of target genes can be induced in a variety of organisms by providing homologous double-stranded RNA molecules. In vivo, these molecules can be generated either by transcription of sequences having an inverted-repeat (IR) configuration or by simultaneous transcription of sense-antisense strands. Since IR constructs are difficult to prepare and can stimulate genomic rearrangements, we investigated the silencing potential of symmetrically transcribed sequences. We report that Drosophila transgenes whose sense-antisense transcription was driven by two convergent arrays of Gal4-dependent UAS sequences can induce specific, dominant, and heritable repression of target genes. This effect is not dependent on a mechanism based on homology-dependent DNA/DNA interactions, but is directly triggered by transcriptional activation and is accompanied by specific depletion of the endogenous target RNA. Tissue-specific induction of these transgenes restricts the target gene silencing to selected body domains, and spreading phenomena described in other cases of post-transcriptional gene silencing (PTGS) were not observed. In addition to providing an additional tool useful for Drosophila functional genomic analysis, these results add further strength to the view that events of sense-antisense transcription may readily account for some, if not all, PTGS-cosuppression phenomena and can potentially play a relevant role in gene regulation. PMID:11861567

Sequence- and Temperature-Dependent Properties of Unfolded and Disordered Proteins from Atomistic Simulations.

PubMed

Zerze, Gül H; Best, Robert B; Mittal, Jeetain

2015-11-19

We use all-atom molecular simulation with explicit solvent to study the properties of selected intrinsically disordered proteins and unfolded states of foldable proteins, which include chain dimensions and shape, secondary structure propensity, solvent accessible surface area, and contact formation. We find that the qualitative scaling behavior of the chains matches expectations from theory under ambient conditions. In particular, unfolded globular proteins tend to be more collapsed under the same conditions than charged disordered sequences of the same length. However, inclusion of explicit solvent in addition naturally captures temperature-dependent solvation effects, which results in an initial collapse of the chains as temperature is increased, in qualitative agreement with experiment. There is a universal origin to the collapse, revealed in the change of hydration of individual residues as a function of temperature: namely, that the initial collapse is driven by unfavorable solvation free energy of individual residues, which in turn has a strong temperature dependence. We also observe that in unfolded globular proteins, increased temperature also initially favors formation of native-like (rather than non-native-like) structure. Our results help to establish how sequence encodes the degree of intrinsic disorder or order as well as its response to changes in environmental conditions.

Unexpected substrate specificity of T4 DNA ligase revealed by in vitro selection

NASA Technical Reports Server (NTRS)

Harada, Kazuo; Orgel, Leslie E.

1993-01-01

We have used in vitro selection techniques to characterize DNA sequences that are ligated efficiently by T4 DNA ligase. We find that the ensemble of selected sequences ligates about 50 times as efficiently as the random mixture of sequences used as the input for selection. Surprisingly many of the selected sequences failed to produce a match at or close to the ligation junction. None of the 20 selected oligomers that we sequenced produced a match two bases upstream from the ligation junction.

Low incidence of DNA sequence variation in human induced pluripotent stem cells generated by non-integrating plasmid expression

PubMed Central

Cheng, Linzhao; Hansen, Nancy F.; Zhao, Ling; Du, Yutao; Zou, Chunlin; Donovan, Frank X.; Chou, Bin-Kuan; Zhou, Guangyu; Li, Shijie; Dowey, Sarah N.; Ye, Zhaohui; Chandrasekharappa, Settara C.; Yang, Huanming; Mullikin, James C.; Liu, P. Paul

2012-01-01

Summary The utility of induced pluripotent stem cells (iPSCs) as models to study diseases and as sources for cell therapy depends on the integrity of their genomes. Despite recent publications of DNA sequence variations in the iPSCs, the true scope of such changes for the entire genome is not clear. Here we report the whole-genome sequencing of three human iPSC lines derived from two cell types of an adult donor by episomal vectors. The vector sequence was undetectable in the deeply sequenced iPSC lines. We identified 1058–1808 heterozygous single nucleotide variants (SNVs), but no copy number variants, in each iPSC line. Six to twelve of these SNVs were within coding regions in each iPSC line, but ~50% of them are synonymous changes and the remaining are not selectively enriched for known genes associated with cancers. Our data thus suggest that episome-mediated reprogramming is not inherently mutagenic during integration-free iPSC induction. PMID:22385660

Is it better to select or to receive? Learning via active and passive hypothesis testing.

PubMed

Markant, Douglas B; Gureckis, Todd M

2014-02-01

People can test hypotheses through either selection or reception. In a selection task, the learner actively chooses observations to test his or her beliefs, whereas in reception tasks data are passively encountered. People routinely use both forms of testing in everyday life, but the critical psychological differences between selection and reception learning remain poorly understood. One hypothesis is that selection learning improves learning performance by enhancing generic cognitive processes related to motivation, attention, and engagement. Alternatively, we suggest that differences between these 2 learning modes derives from a hypothesis-dependent sampling bias that is introduced when a person collects data to test his or her own individual hypothesis. Drawing on influential models of sequential hypothesis-testing behavior, we show that such a bias (a) can lead to the collection of data that facilitates learning compared with reception learning and (b) can be more effective than observing the selections of another person. We then report a novel experiment based on a popular category learning paradigm that compares reception and selection learning. We additionally compare selection learners to a set of "yoked" participants who viewed the exact same sequence of observations under reception conditions. The results revealed systematic differences in performance that depended on the learner's role in collecting information and the abstract structure of the problem.

A graphical language for reliability model generation

NASA Technical Reports Server (NTRS)

Howell, Sandra V.; Bavuso, Salvatore J.; Haley, Pamela J.

1990-01-01

A graphical interface capability of the hybrid automated reliability predictor (HARP) is described. The graphics-oriented (GO) module provides the user with a graphical language for modeling system failure modes through the selection of various fault tree gates, including sequence dependency gates, or by a Markov chain. With this graphical input language, a fault tree becomes a convenient notation for describing a system. In accounting for any sequence dependencies, HARP converts the fault-tree notation to a complex stochastic process that is reduced to a Markov chain which it can then solve for system reliability. The graphics capability is available for use on an IBM-compatible PC, a Sun, and a VAX workstation. The GO module is written in the C programming language and uses the Graphical Kernel System (GKS) standard for graphics implementation. The PC, VAX, and Sun versions of the HARP GO module are currently in beta-testing.

Kakusan4 and Aminosan: two programs for comparing nonpartitioned, proportional and separate models for combined molecular phylogenetic analyses of multilocus sequence data.

PubMed

Tanabe, Akifumi S

2011-09-01

Proportional and separate models able to apply different combination of substitution rate matrix (SRM) and among-site rate variation model (ASRVM) to each locus are frequently used in phylogenetic studies of multilocus data. A proportional model assumes that branch lengths are proportional among partitions and a separate model assumes that each partition has an independent set of branch lengths. However, the selection from among nonpartitioned (i.e., a common combination of models is applied to all-loci concatenated sequences), proportional and separate models is usually based on the researcher's preference rather than on any information criteria. This study describes two programs, 'Kakusan4' (for DNA sequences) and 'Aminosan' (for amino-acid sequences), which allow the selection of evolutionary models based on several types of information criteria. The programs can handle both multilocus and single-locus data, in addition to providing an easy-to-use wizard interface and a noninteractive command line interface. In the case of multilocus data, SRMs and ASRVMs are compared at each locus and at all-loci concatenated sequences, after which nonpartitioned, proportional and separate models are compared based on information criteria. The programs also provide model configuration files for mrbayes, paup*, phyml, raxml and Treefinder to support further phylogenetic analysis using a selected model. When likelihoods are optimized by Treefinder, the best-fit models were found to differ depending on the data set. Furthermore, differences in the information criteria among nonpartitioned, proportional and separate models were much larger than those among the nonpartitioned models. These findings suggest that selecting from nonpartitioned, proportional and separate models results in a better phylogenetic tree. Kakusan4 and Aminosan are available at http://www.fifthdimension.jp/. They are licensed under gnugpl Ver.2, and are able to run on Windows, MacOS X and Linux. © 2011 Blackwell Publishing Ltd.

Systematic Evaluation of the Dependence of Deoxyribozyme Catalysis on Random Region Length

PubMed Central

Velez, Tania E.; Singh, Jaydeep; Xiao, Ying; Allen, Emily C.; Wong, On Yi; Chandra, Madhavaiah; Kwon, Sarah C.; Silverman, Scott K.

2012-01-01

Functional nucleic acids are DNA and RNA aptamers that bind targets, or they are deoxyribozymes and ribozymes that have catalytic activity. These functional DNA and RNA sequences can be identified from random-sequence pools by in vitro selection, which requires choosing the length of the random region. Shorter random regions allow more complete coverage of sequence space but may not permit the structural complexity necessary for binding or catalysis. In contrast, longer random regions are sampled incompletely but may allow adoption of more complicated structures that enable function. In this study, we systematically examined random region length (N20 through N60) for two particular deoxyribozyme catalytic activities, DNA cleavage and tyrosine-RNA nucleopeptide linkage formation. For both activities, we previously identified deoxyribozymes using only N40 regions. In the case of DNA cleavage, here we found that shorter N20 and N30 regions allowed robust catalytic function, either by DNA hydrolysis or by DNA deglycosylation and strand scission via β-elimination, whereas longer N50 and N60 regions did not lead to catalytically active DNA sequences. Follow-up selections with N20, N30, and N40 regions revealed an interesting interplay of metal ion cofactors and random region length. Separately, for Tyr-RNA linkage formation, N30 and N60 regions provided catalytically active sequences, whereas N20 was unsuccessful, and the N40 deoxyribozymes were functionally superior (in terms of rate and yield) to N30 and N60. Collectively, the results indicate that with future in vitro selection experiments for DNA and RNA catalysts, and by extension for aptamers, random region length should be an important experimental variable. PMID:23088677

"FLIPSY"—A New Solvent-Suppression Sequence for Nonexchanging Solutes Offering Improved Integral Accuracy Relative to 1D NOESY

NASA Astrophysics Data System (ADS)

Neuhaus, David; Ismail, Ismail M.; Chung, Chun-Wa

A new method of solvent suppression is described, based on presaturation in combination with volume selection; the name "FLIPSY" is proposed for this sequence. A low-flip-angle pulse is used for excitation, immediately followed by two 180° pulses, each of which is independently phase cycled through Exorcycle. The phase-cycled inversion pulses achieve volume selection in a way similar to the widely used 1D NOESY sequence, thereby largely eliminating any residual "hump" signal from the solvent. The two 180° pulses combine to produce a net 360° rotation for zmagnetization and either a 180° or a 360° rotation for transverse magnetization, depending on the step in the phase cycle. This allows the overall flip angle of the sequence to be controlled by adjusting the length of the initial excitation pulse. It is demonstrated that this property allows one to choose freely a suitable compromise between signal strength and integral accuracy when using FLIPSY, just as when using single-pulse excitation. Such a choice cannot be made when using 1D NOESY, since the effective flip angle in that experiment is always 90°. The application of FLIPSY to recording LC-NMR spectra is demonstrated.

Identification of sequence-structure RNA binding motifs for SELEX-derived aptamers.

PubMed

Hoinka, Jan; Zotenko, Elena; Friedman, Adam; Sauna, Zuben E; Przytycka, Teresa M

2012-06-15

Systematic Evolution of Ligands by EXponential Enrichment (SELEX) represents a state-of-the-art technology to isolate single-stranded (ribo)nucleic acid fragments, named aptamers, which bind to a molecule (or molecules) of interest via specific structural regions induced by their sequence-dependent fold. This powerful method has applications in designing protein inhibitors, molecular detection systems, therapeutic drugs and antibody replacement among others. However, full understanding and consequently optimal utilization of the process has lagged behind its wide application due to the lack of dedicated computational approaches. At the same time, the combination of SELEX with novel sequencing technologies is beginning to provide the data that will allow the examination of a variety of properties of the selection process. To close this gap we developed, Aptamotif, a computational method for the identification of sequence-structure motifs in SELEX-derived aptamers. To increase the chances of identifying functional motifs, Aptamotif uses an ensemble-based approach. We validated the method using two published aptamer datasets containing experimentally determined motifs of increasing complexity. We were able to recreate the author's findings to a high degree, thus proving the capability of our approach to identify binding motifs in SELEX data. Additionally, using our new experimental dataset, we illustrate the application of Aptamotif to elucidate several properties of the selection process.

Efficient engineering of chromosomal ribosome binding site libraries in mismatch repair proficient Escherichia coli.

PubMed

Oesterle, Sabine; Gerngross, Daniel; Schmitt, Steven; Roberts, Tania Michelle; Panke, Sven

2017-09-26

Multiplexed gene expression optimization via modulation of gene translation efficiency through ribosome binding site (RBS) engineering is a valuable approach for optimizing artificial properties in bacteria, ranging from genetic circuits to production pathways. Established algorithms design smart RBS-libraries based on a single partially-degenerate sequence that efficiently samples the entire space of translation initiation rates. However, the sequence space that is accessible when integrating the library by CRISPR/Cas9-based genome editing is severely restricted by DNA mismatch repair (MMR) systems. MMR efficiency depends on the type and length of the mismatch and thus effectively removes potential library members from the pool. Rather than working in MMR-deficient strains, which accumulate off-target mutations, or depending on temporary MMR inactivation, which requires additional steps, we eliminate this limitation by developing a pre-selection rule of genome-library-optimized-sequences (GLOS) that enables introducing large functional diversity into MMR-proficient strains with sequences that are no longer subject to MMR-processing. We implement several GLOS-libraries in Escherichia coli and show that GLOS-libraries indeed retain diversity during genome editing and that such libraries can be used in complex genome editing operations such as concomitant deletions. We argue that this approach allows for stable and efficient fine tuning of chromosomal functions with minimal effort.

Multinuclear NMR Study of the Pressure Dependence for Carbonate Exchange in the UO 2(CO 3) 3 4-(aq) Ion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, Rene L.; Harley, Stephen J.; Ohlin, C. André

2011-09-16

Rates of carbonate exchange by two pH-sensitive pathways between aqueous carbonate ion and UO 2(CO 3) 3 4-(aq) (see picture) are measured by high-pressure NMR. To accomplish this, a custom pulse sequence is employed to achieve selective inversion. Rates of chemical exchange are determined by modeling the return to equilibrium.

Multinuclear NMR study of the pressure dependence for carbonate exchange in the [UO2(CO3)3]4- (aq) ion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Johnson, Rene L.; Harley, S. J.; Ohlin, C. A.

2011-09-16

Rates of carbonate exchange by two pH-sensitive pathways between aqueous carbonate ion and UO₂(CO₃)₃⁴⁻(aq) are measured by high-pressure NMR. To accomplish this, a custom pulse sequence is employed to achieve selective inversion. Rates of chemical exchange are determined by modeling the return to equilibrium.

Multiple bottlenecks in hierarchical control of action sequences: what does "response selection" select in skilled typewriting?

PubMed

Yamaguchi, Motonori; Logan, Gordon D; Li, Vanessa

2013-08-01

Does response selection select words or letters in skilled typewriting? Typing performance involves hierarchically organized control processes: an outer loop that controls word level processing, and an inner loop that controls letter (or keystroke) level processing. The present study addressed whether response selection occurs in the outer loop or the inner loop by using the psychological refractory period (PRP) paradigm in which Task1 required typing single words and Task2 required vocal responses to tones. The number of letters (string length) in the words was manipulated to discriminate selection of words from selection of keystrokes. In Experiment 1, the PRP effect depended on string length of words in Task1, suggesting that response selection occurs in the inner loop. To assess contributions of the outer loop, the influence of string length was examined in a lexical-decision task that also involves word encoding and lexical access (Experiment 2), or to-be-typed words were preexposed so outer-loop processing could finish before typing started (Experiment 3). Response time for Task2 (RT2) did not depend on string length with lexical decision, and RT2 still depended on string length with typing preexposed strings. These results support the inner-loop locus of the PRP effect. In Experiment 4, typing was performed as Task2, and the effect of string length on typing RT interacted with stimulus onset asynchrony superadditively, implying that another bottleneck also exists in the outer loop. We conclude that there are at least two bottleneck processes in skilled typewriting. 2013 APA, all rights reserved

Effect of the 3-halo substitution of the 2'-deoxy aminopyridinyl-pseudocytidine derivatives on the selectivity and stability of antiparallel triplex DNA with a CG inversion site.

PubMed

Wang, Lei; Taniguchi, Yosuke; Okamura, Hidenori; Sasaki, Shigeki

2017-07-15

Triplex formation against a target duplex DNA has the potential to become a tool for the genome research. However, there is an intrinsic restriction on the duplex DNA sequences capable of forming the triplex DNA. Recently, we demonstrated the selective formation of the stable antiparallel triplexes containing the CG inversion sites using the 2'-deoxy-1-methylpseudocytidine derivative (ΨdC), whose amino group was conjugated with the 2-aminopyridine at its 5-position as an additional hydrogen bonding unit (AP-ΨdC). The 1-N of 2-aminopyridine was supposed to be protonated to form the hydrogen bond with the guanine of the CG inversion site. In this study, to test the effect of the 3-substitution of the 2-aminopyridine unit of AP-ΨdC on the triplex stability, we synthesized the 3-halogenated 2-aminopyridine derivatives of AP-ΨdC. The pKa values 1-N of the 2-aminopyridine unit of AP-ΨdC as the monomer nucleoside were determined to be 6.3 for 3-CH 3 ( Me AP-ΨdC), 6.1 for 3-H (AP-ΨdC), 4.3 for 3-Cl ( Cl AP-ΨdC), 4.4 for 3-Br ( Br AP-ΨdC), and 4.7 for 3-I ( I AP-ΨdC), suggesting that all the halogenated AP-ΨdCs are not protonated under neutral conditions. Interestingly, although the recognition selectivity depends on the sequence context, the TFO having the sequence of the 3'-G-( I AP-ΨdC)-A-5' context showed the selective triplex formation with the CG inversion site. These results suggest that the protonation at the 1-N position plays an important role in the stable and selective triplex formation of AP-ΨdC derivatives in any sequences. Copyright © 2017 Elsevier Ltd. All rights reserved.

The dependence of galaxy clustering on stellar mass, star-formation rate and redshift at z = 0.8-2.2, with HiZELS

NASA Astrophysics Data System (ADS)

Cochrane, R. K.; Best, P. N.; Sobral, D.; Smail, I.; Geach, J. E.; Stott, J. P.; Wake, D. A.

2018-04-01

The deep, near-infrared narrow-band survey HiZELS has yielded robust samples of H α-emitting star-forming galaxies within narrow redshift slices at z = 0.8, 1.47 and 2.23. In this paper, we distinguish the stellar mass and star-formation rate (SFR) dependence of the clustering of these galaxies. At high stellar masses (M*/M⊙ ≳ 2 × 1010), where HiZELS selects galaxies close to the so-called star-forming main sequence, the clustering strength is observed to increase strongly with stellar mass (in line with the results of previous studies of mass-selected galaxy samples) and also with SFR. These two dependencies are shown to hold independently. At lower stellar masses, however, where HiZELS probes high specific SFR galaxies, there is little or no dependence of the clustering strength on stellar mass, but the dependence on SFR remains: high-SFR low-mass galaxies are found in more massive dark matter haloes than their lower SFR counterparts. We argue that this is due to environmentally driven star formation in these systems. We apply the same selection criteria to the EAGLE cosmological hydrodynamical simulations. We find that, in EAGLE, the high-SFR low-mass galaxies are central galaxies in more massive dark matter haloes, in which the high SFRs are driven by a (halo-driven) increased gas content.

Evolutionary genetic analyses of MEF2C gene: implications for learning and memory in Homo sapiens.

PubMed

Kalmady, Sunil V; Venkatasubramanian, Ganesan; Arasappa, Rashmi; Rao, Naren P

2013-02-01

MEF2C facilitates context-dependent fear conditioning (CFC) which is a salient aspect of hippocampus-dependent learning and memory. CFC might have played a crucial role in human evolution because of its advantageous influence on survival of species. In this study, we analyzed 23 orthologous mammalian gene sequences of MEF2C gene to examine the evidence for positive selection on this gene in Homo sapiens using Phylogenetic Analysis by Maximum Likelihood (PAML) and HyPhy software. Both PAML Bayes Empirical Bayes (BEB) and HyPhy Fixed Effects Likelihood (FEL) analyses supported significant positive selection on 4 codon sites in H. sapiens. Also, haplotter analysis revealed significant ongoing positive selection on this gene in Central European population. The study findings suggest that adaptive selective pressure on this gene might have influenced human evolution. Further research on this gene might unravel the potential role of this gene in learning and memory as well as its pathogenetic effect in certain hippocampal disorders with evolutionary basis like schizophrenia. Copyright © 2012 Elsevier B.V. All rights reserved.

A confidence interval analysis of sampling effort, sequencing depth, and taxonomic resolution of fungal community ecology in the era of high-throughput sequencing.

PubMed

Oono, Ryoko

2017-01-01

High-throughput sequencing technology has helped microbial community ecologists explore ecological and evolutionary patterns at unprecedented scales. The benefits of a large sample size still typically outweigh that of greater sequencing depths per sample for accurate estimations of ecological inferences. However, excluding or not sequencing rare taxa may mislead the answers to the questions 'how and why are communities different?' This study evaluates the confidence intervals of ecological inferences from high-throughput sequencing data of foliar fungal endophytes as case studies through a range of sampling efforts, sequencing depths, and taxonomic resolutions to understand how technical and analytical practices may affect our interpretations. Increasing sampling size reliably decreased confidence intervals across multiple community comparisons. However, the effects of sequencing depths on confidence intervals depended on how rare taxa influenced the dissimilarity estimates among communities and did not significantly decrease confidence intervals for all community comparisons. A comparison of simulated communities under random drift suggests that sequencing depths are important in estimating dissimilarities between microbial communities under neutral selective processes. Confidence interval analyses reveal important biases as well as biological trends in microbial community studies that otherwise may be ignored when communities are only compared for statistically significant differences.

A confidence interval analysis of sampling effort, sequencing depth, and taxonomic resolution of fungal community ecology in the era of high-throughput sequencing

PubMed Central

2017-01-01

High-throughput sequencing technology has helped microbial community ecologists explore ecological and evolutionary patterns at unprecedented scales. The benefits of a large sample size still typically outweigh that of greater sequencing depths per sample for accurate estimations of ecological inferences. However, excluding or not sequencing rare taxa may mislead the answers to the questions ‘how and why are communities different?’ This study evaluates the confidence intervals of ecological inferences from high-throughput sequencing data of foliar fungal endophytes as case studies through a range of sampling efforts, sequencing depths, and taxonomic resolutions to understand how technical and analytical practices may affect our interpretations. Increasing sampling size reliably decreased confidence intervals across multiple community comparisons. However, the effects of sequencing depths on confidence intervals depended on how rare taxa influenced the dissimilarity estimates among communities and did not significantly decrease confidence intervals for all community comparisons. A comparison of simulated communities under random drift suggests that sequencing depths are important in estimating dissimilarities between microbial communities under neutral selective processes. Confidence interval analyses reveal important biases as well as biological trends in microbial community studies that otherwise may be ignored when communities are only compared for statistically significant differences. PMID:29253889

Time-dependent Data System (TDDS); an interactive program to assemble, manage, and appraise input data and numerical output of flow/transport simulation models

USGS Publications Warehouse

Regan, R.S.; Schaffranek, R.W.; Baltzer, R.A.

1996-01-01

A system of functional utilities and computer routines, collectively identified as the Time-Dependent Data System CI DDS), has been developed and documented by the U.S. Geological Survey. The TDDS is designed for processing time sequences of discrete, fixed-interval, time-varying geophysical data--in particular, hydrologic data. Such data include various, dependent variables and related parameters typically needed as input for execution of one-, two-, and three-dimensional hydrodynamic/transport and associated water-quality simulation models. Such data can also include time sequences of results generated by numerical simulation models. Specifically, TDDS provides the functional capabilities to process, store, retrieve, and compile data in a Time-Dependent Data Base (TDDB) in response to interactive user commands or pre-programmed directives. Thus, the TDDS, in conjunction with a companion TDDB, provides a ready means for processing, preparation, and assembly of time sequences of data for input to models; collection, categorization, and storage of simulation results from models; and intercomparison of field data and simulation results. The TDDS can be used to edit and verify prototype, time-dependent data to affirm that selected sequences of data are accurate, contiguous, and appropriate for numerical simulation modeling. It can be used to prepare time-varying data in a variety of formats, such as tabular lists, sequential files, arrays, graphical displays, as well as line-printer plots of single or multiparameter data sets. The TDDB is organized and maintained as a direct-access data base by the TDDS, thus providing simple, yet efficient, data management and access. A single, easily used, program interface that provides all access to and from a particular TDDB is available for use directly within models, other user-provided programs, and other data systems. This interface, together with each major functional utility of the TDDS, is described and documented in this report.

A computational method for selecting short peptide sequences for inorganic material binding.

PubMed

Nayebi, Niloofar; Cetinel, Sibel; Omar, Sara Ibrahim; Tuszynski, Jack A; Montemagno, Carlo

2017-11-01

Discovering or designing biofunctionalized materials with improved quality highly depends on the ability to manipulate and control the peptide-inorganic interaction. Various peptides can be used as assemblers, synthesizers, and linkers in the material syntheses. In another context, specific and selective material-binding peptides can be used as recognition blocks in mining applications. In this study, we propose a new in silico method to select short 4-mer peptides with high affinity and selectivity for a given target material. This method is illustrated with the calcite (104) surface as an example, which has been experimentally validated. A calcite binding peptide can play an important role in our understanding of biomineralization. A practical aspect of calcite is a need for it to be selectively depressed in mining sites. © 2017 Wiley Periodicals, Inc.

Lactoferricin-related peptides with inhibitory effects on ACE-dependent vasoconstriction.

PubMed

Centeno, José M; Burguete, María C; Castelló-Ruiz, María; Enrique, María; Vallés, Salvador; Salom, Juan B; Torregrosa, Germán; Marcos, José F; Alborch, Enrique; Manzanares, Paloma

2006-07-26

A selection of lactoferricin B (LfcinB)-related peptides with an angiotensin I-converting enzyme (ACE) inhibitory effect have been examined using in vitro and ex vivo functional assays. Peptides that were analyzed included a set of sequence-related antimicrobial hexapeptides previously reported and two representative LfcinB-derived peptides. In vitro assays using hippuryl-L-histidyl-L-leucine (HHL) and angiotensin I as substrates allowed us to select two hexapeptides, PACEI32 (Ac-RKWHFW-NH2) and PACEI34 (Ac-RKWLFW-NH2), and also a LfcinB-derived peptide, LfcinB17-31 (Ac-FKCRRWQWRMKKLGA-NH2). Ex vivo functional assays using rabbit carotid arterial segments showed PACEI32 (both D- and L-enantiomers) and LfcinB17-31 have inhibitory effects on ACE-dependent angiotensin I-induced contraction. None of the peptides exhibited in vitro ACE inhibitory activity using bradykinin as the substrate. In conclusion, three bioactive lactoferricin-related peptides exhibit inhibitory effects on both ACE activity and ACE-dependent vasoconstriction with potential to modulate hypertension that deserves further investigation.

SH2 Domains Recognize Contextual Peptide Sequence Information to Determine Selectivity*

PubMed Central

Liu, Bernard A.; Jablonowski, Karl; Shah, Eshana E.; Engelmann, Brett W.; Jones, Richard B.; Nash, Piers D.

2010-01-01

Selective ligand recognition by modular protein interaction domains is a primary determinant of specificity in signaling pathways. Src homology 2 (SH2) domains fulfill this capacity immediately downstream of tyrosine kinases, acting to recruit their host polypeptides to ligand proteins harboring phosphorylated tyrosine residues. The degree to which SH2 domains are selective and the mechanisms underlying selectivity are fundamental to understanding phosphotyrosine signaling networks. An examination of interactions between 50 SH2 domains and a set of 192 phosphotyrosine peptides corresponding to physiological motifs within FGF, insulin, and IGF-1 receptor pathways indicates that individual SH2 domains have distinct recognition properties and exhibit a remarkable degree of selectivity beyond that predicted by previously described binding motifs. The underlying basis for such selectivity is the ability of SH2 domains to recognize both permissive amino acid residues that enhance binding and non-permissive amino acid residues that oppose binding in the vicinity of the essential phosphotyrosine. Neighboring positions affect one another so local sequence context matters to SH2 domains. This complex linguistics allows SH2 domains to distinguish subtle differences in peptide ligands. This newly appreciated contextual dependence substantially increases the accessible information content embedded in the peptide ligands that can be effectively integrated to determine binding. This concept may serve more broadly as a paradigm for subtle recognition of physiological ligands by protein interaction domains. PMID:20627867

Identification of an HV 1 voltage-gated proton channel in insects.

PubMed

Chaves, Gustavo; Derst, Christian; Franzen, Arne; Mashimo, Yuta; Machida, Ryuichiro; Musset, Boris

2016-04-01

The voltage-gated proton channel 1 (HV 1) is an important component of the cellular proton extrusion machinery and is essential for charge compensation during the respiratory burst of phagocytes. HV 1 has been identified in a wide range of eukaryotes throughout the animal kingdom, with the exception of insects. Therefore, it has been proposed that insects do not possess an HV 1 channel. In the present study, we report the existence of an HV 1-type proton channel in insects. We searched insect transcriptome shotgun assembly (TSA) sequence databases and found putative HV 1 orthologues in various polyneopteran insects. To confirm that these putative HV 1 orthologues were functional channels, we studied the HV 1 channel of Nicoletia phytophila (NpHV 1), an insect of the Zygentoma order, in more detail. NpHV 1 comprises 239 amino acids and is 33% identical to the human voltage-gated proton channel 1. Patch clamp measurements in a heterologous expression system showed proton selectivity, as well as pH- and voltage-dependent gating. Interestingly, NpHV 1 shows slightly enhanced pH-dependent gating compared to the human channel. Mutations in the first transmembrane segment at position 66 (Asp66), the presumed selectivity filter, lead to a loss of proton-selective conduction, confirming the importance of this aspartate residue in voltage-gated proton channels. Nucleotide sequence data have been deposited in the GenBank database under accession number KT780722. © 2016 Federation of European Biochemical Societies.

DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequencesmore » in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.« less

DNA sequence determinants controlling affinity, stability and shape of DNA complexes bound by the nucleoid protein Fis

DOE PAGES

Hancock, Stephen P.; Stella, Stefano; Cascio, Duilio; ...

2016-03-09

The abundant Fis nucleoid protein selectively binds poorly related DNA sequences with high affinities to regulate diverse DNA reactions. Fis binds DNA primarily through DNA backbone contacts and selects target sites by reading conformational properties of DNA sequences, most prominently intrinsic minor groove widths. High-affinity binding requires Fis-stabilized DNA conformational changes that vary depending on DNA sequence. In order to better understand the molecular basis for high affinity site recognition, we analyzed the effects of DNA sequence within and flanking the core Fis binding site on binding affinity and DNA structure. X-ray crystal structures of Fis-DNA complexes containing variable sequencesmore » in the noncontacted center of the binding site or variations within the major groove interfaces show that the DNA can adapt to the Fis dimer surface asymmetrically. We show that the presence and position of pyrimidine-purine base steps within the major groove interfaces affect both local DNA bending and minor groove compression to modulate affinities and lifetimes of Fis-DNA complexes. Sequences flanking the core binding site also modulate complex affinities, lifetimes, and the degree of local and global Fis-induced DNA bending. In particular, a G immediately upstream of the 15 bp core sequence inhibits binding and bending, and A-tracts within the flanking base pairs increase both complex lifetimes and global DNA curvatures. Taken together, our observations support a revised DNA motif specifying high-affinity Fis binding and highlight the range of conformations that Fis-bound DNA can adopt. Lastly, the affinities and DNA conformations of individual Fis-DNA complexes are likely to be tailored to their context-specific biological functions.« less

Process of labeling specific chromosomes using recombinant repetitive DNA

DOEpatents

Moyzis, R.K.; Meyne, J.

1988-02-12

Chromosome preferential nucleotide sequences are first determined from a library of recombinant DNA clones having families of repetitive sequences. Library clones are identified with a low homology with a sequence of repetitive DNA families to which the first clones respectively belong and variant sequences are then identified by selecting clones having a pattern of hybridization with genomic DNA dissimilar to the hybridization pattern shown by the respective families. In another embodiment, variant sequences are selected from a sequence of a known repetitive DNA family. The selected variant sequence is classified as chromosome specific, chromosome preferential, or chromosome nonspecific. Sequences which are classified as chromosome preferential are further sequenced and regions are identified having a low homology with other regions of the chromosome preferential sequence or with known sequences of other family members and consensus sequences of the repetitive DNA families for the chromosome preferential sequences. The selected low homology regions are then hybridized with chromosomes to determine those low homology regions hybridized with a specific chromosome under normal stringency conditions.

A population genetics analysis in clinical isolates of Sporothrix schenckii based on calmodulin and calcium/calmodulin-dependent kinase partial gene sequences.

PubMed

Rangel-Gamboa, Lucia; Martinez-Hernandez, Fernando; Maravilla, Pablo; Flisser, Ana

2018-02-02

Sporotrichosis is a subcutaneous mycosis that is caused by diverse species of Sporothrix. High levels of genetic diversity in Sporothrix isolates have been reported, but few population genetics analyses have been documented. To analyse the genetic variability and population genetics relations of Sporothrix schenckii Mexican clinical isolates and to compare them with other reported isolates. We studied the partial sequences of calmodulin and calcium/calmodulin-dependent kinase genes in 24 isolates; 22 from Mexico, one from Colombia, and one ATCC ® 6331™; the latter was used as a positive control. In total, 24 isolates were analysed. Phylogenetic, haplotype and population genetic analyses were performed with 24 sequences obtained by us and 345 sequences obtained from GenBank. The frequency of S. schenckii sensu stricto was 81% in the 22 Mexican isolates, while the remaining 19% were Sporothrix globosa. Mexican S. schenckii sensu stricto had high genetic diversity and was related to isolates from South America. In contrast, S. globosa showed one haplotype related to isolates from Asia, Brazil, Spain and the USA. In S. schenckii sensu stricto, S. brasiliensis and S. globosa, haplotype polymorphism (θ) values were higher than the nucleotide diversity data (π). In addition, Tajima's D plus Fu and Li's tests analyses displayed negative values, suggesting directional selection and arguing against the model of neutral evolution in these populations. In addition, analyses showed that calcium/calmodulin-dependent kinase was a suitable genetic marker to discriminate between common Sporothrix species. © 2018 Blackwell Verlag GmbH.

A Statistical Guide to the Design of Deep Mutational Scanning Experiments

PubMed Central

Matuszewski, Sebastian; Hildebrandt, Marcel E.; Ghenu, Ana-Hermina; Jensen, Jeffrey D.; Bank, Claudia

2016-01-01

The characterization of the distribution of mutational effects is a key goal in evolutionary biology. Recently developed deep-sequencing approaches allow for accurate and simultaneous estimation of the fitness effects of hundreds of engineered mutations by monitoring their relative abundance across time points in a single bulk competition. Naturally, the achievable resolution of the estimated fitness effects depends on the specific experimental setup, the organism and type of mutations studied, and the sequencing technology utilized, among other factors. By means of analytical approximations and simulations, we provide guidelines for optimizing time-sampled deep-sequencing bulk competition experiments, focusing on the number of mutants, the sequencing depth, and the number of sampled time points. Our analytical results show that sampling more time points together with extending the duration of the experiment improves the achievable precision disproportionately compared with increasing the sequencing depth or reducing the number of competing mutants. Even if the duration of the experiment is fixed, sampling more time points and clustering these at the beginning and the end of the experiment increase experimental power and allow for efficient and precise assessment of the entire range of selection coefficients. Finally, we provide a formula for calculating the 95%-confidence interval for the measurement error estimate, which we implement as an interactive web tool. This allows for quantification of the maximum expected a priori precision of the experimental setup, as well as for a statistical threshold for determining deviations from neutrality for specific selection coefficient estimates. PMID:27412710

Using variable rate models to identify genes under selection in sequence pairs: their validity and limitations for EST sequences.

PubMed

Church, Sheri A; Livingstone, Kevin; Lai, Zhao; Kozik, Alexander; Knapp, Steven J; Michelmore, Richard W; Rieseberg, Loren H

2007-02-01

Using likelihood-based variable selection models, we determined if positive selection was acting on 523 EST sequence pairs from two lineages of sunflower and lettuce. Variable rate models are generally not used for comparisons of sequence pairs due to the limited information and the inaccuracy of estimates of specific substitution rates. However, previous studies have shown that the likelihood ratio test (LRT) is reliable for detecting positive selection, even with low numbers of sequences. These analyses identified 56 genes that show a signature of selection, of which 75% were not identified by simpler models that average selection across codons. Subsequent mapping studies in sunflower show four of five of the positively selected genes identified by these methods mapped to domestication QTLs. We discuss the validity and limitations of using variable rate models for comparisons of sequence pairs, as well as the limitations of using ESTs for identification of positively selected genes.

Functional region prediction with a set of appropriate homologous sequences-an index for sequence selection by integrating structure and sequence information with spatial statistics

PubMed Central

2012-01-01

Background The detection of conserved residue clusters on a protein structure is one of the effective strategies for the prediction of functional protein regions. Various methods, such as Evolutionary Trace, have been developed based on this strategy. In such approaches, the conserved residues are identified through comparisons of homologous amino acid sequences. Therefore, the selection of homologous sequences is a critical step. It is empirically known that a certain degree of sequence divergence in the set of homologous sequences is required for the identification of conserved residues. However, the development of a method to select homologous sequences appropriate for the identification of conserved residues has not been sufficiently addressed. An objective and general method to select appropriate homologous sequences is desired for the efficient prediction of functional regions. Results We have developed a novel index to select the sequences appropriate for the identification of conserved residues, and implemented the index within our method to predict the functional regions of a protein. The implementation of the index improved the performance of the functional region prediction. The index represents the degree of conserved residue clustering on the tertiary structure of the protein. For this purpose, the structure and sequence information were integrated within the index by the application of spatial statistics. Spatial statistics is a field of statistics in which not only the attributes but also the geometrical coordinates of the data are considered simultaneously. Higher degrees of clustering generate larger index scores. We adopted the set of homologous sequences with the highest index score, under the assumption that the best prediction accuracy is obtained when the degree of clustering is the maximum. The set of sequences selected by the index led to higher functional region prediction performance than the sets of sequences selected by other sequence-based methods. Conclusions Appropriate homologous sequences are selected automatically and objectively by the index. Such sequence selection improved the performance of functional region prediction. As far as we know, this is the first approach in which spatial statistics have been applied to protein analyses. Such integration of structure and sequence information would be useful for other bioinformatics problems. PMID:22643026

Comparison of isothermal helicase-dependent amplification and PCR for the detection of Mycobacterium tuberculosis by an electrochemical genomagnetic assay.

PubMed

Barreda-García, Susana; Miranda-Castro, Rebeca; de-Los-Santos-Álvarez, Noemí; Miranda-Ordieres, Arturo J; Lobo-Castañón, M Jesús

2016-12-01

Methods for the early and sensitive detection of pathogenic bacteria suited to low-resource settings could impact diagnosis and management of diseases. Helicase-dependent isothermal amplification (HDA) is an ideal tool for this purpose, especially when combined with a sequence-specific detection method able to improve the selectivity of the assay. The implementation of this approach requires that its analytical performance is shown to be comparable with the gold standard method, polymerase chain reaction (PCR). In this study, we optimize and compare the asymmetric amplification of an 84-base-long DNA sequence specific for Mycobacterium tuberculosis by PCR and HDA, using an electrochemical genomagnetic assay for hybridization-based detection of the obtained single-stranded amplicons. The results indicate the generalizability of the magnetic platform with electrochemical detection for quantifying amplification products without previous purification. Moreover, we demonstrate that under optimal conditions the same gene can be amplified by either PCR or HDA, allowing the detection of as low as 30 copies of the target gene sequence with acceptable reproducibility. Both assays have been applied to the detection of M. tuberculosis in sputum, urine, and pleural fluid samples with comparable results. Simplicity and isothermal nature of HDA offer great potential for the development of point-of-care devices. Graphical Abstract Comparative evaluation of isothermal helicase-dependent amplification and PCR for electrochemical detection of Mycobacterium tuberculosis.

Computational analysis of sequence selection mechanisms.

PubMed

Meyerguz, Leonid; Grasso, Catherine; Kleinberg, Jon; Elber, Ron

2004-04-01

Mechanisms leading to gene variations are responsible for the diversity of species and are important components of the theory of evolution. One constraint on gene evolution is that of protein foldability; the three-dimensional shapes of proteins must be thermodynamically stable. We explore the impact of this constraint and calculate properties of foldable sequences using 3660 structures from the Protein Data Bank. We seek a selection function that receives sequences as input, and outputs survival probability based on sequence fitness to structure. We compute the number of sequences that match a particular protein structure with energy lower than the native sequence, the density of the number of sequences, the entropy, and the "selection" temperature. The mechanism of structure selection for sequences longer than 200 amino acids is approximately universal. For shorter sequences, it is not. We speculate on concrete evolutionary mechanisms that show this behavior.

Stable isotope, site-specific mass tagging for protein identification

DOEpatents

Chen, Xian

2006-10-24

Proteolytic peptide mass mapping as measured by mass spectrometry provides an important method for the identification of proteins, which are usually identified by matching the measured and calculated m/z values of the proteolytic peptides. A unique identification is, however, heavily dependent upon the mass accuracy and sequence coverage of the fragment ions generated by peptide ionization. The present invention describes a method for increasing the specificity, accuracy and efficiency of the assignments of particular proteolytic peptides and consequent protein identification, by the incorporation of selected amino acid residue(s) enriched with stable isotope(s) into the protein sequence without the need for ultrahigh instrumental accuracy. Selected amino acid(s) are labeled with .sup.13C/.sup.15N/.sup.2H and incorporated into proteins in a sequence-specific manner during cell culturing. Each of these labeled amino acids carries a defined mass change encoded in its monoisotopic distribution pattern. Through their characteristic patterns, the peptides with mass tag(s) can then be readily distinguished from other peptides in mass spectra. The present method of identifying unique proteins can also be extended to protein complexes and will significantly increase data search specificity, efficiency and accuracy for protein identifications.

Widespread signatures of local mRNA folding structure selection in four Dengue virus serotypes

PubMed Central

2015-01-01

Background It is known that mRNA folding can affect and regulate various gene expression steps both in living organisms and in viruses. Previous studies have recognized functional RNA structures in the genome of the Dengue virus. However, these studies usually focused either on the viral untranslated regions or on very specific and limited regions at the beginning of the coding sequences, in a limited number of strains, and without considering evolutionary selection. Results Here we performed the first large scale comprehensive genomics analysis of selection for local mRNA folding strength in the Dengue virus coding sequences, based on a total of 1,670 genomes and 4 serotypes. Our analysis identified clusters of positions along the coding regions that may undergo a conserved evolutionary selection for strong or weak local folding maintained across different viral variants. Specifically, 53-66 clusters for strong folding and 49-73 clusters for weak folding (depending on serotype) aggregated of positions with a significant conservation of folding energy signals (related to partially overlapping local genomic regions) were recognized. In addition, up to 7% of these positions were found to be conserved in more than 90% of the viral genomes. Although some of the identified positions undergo frequent synonymous / non-synonymous substitutions, the selection for folding strength therein is preserved, and thus cannot be trivially explained based on sequence conservation alone. Conclusions The fact that many of the positions with significant folding related signals are conserved among different Dengue variants suggests that a better understanding of the mRNA structures in the corresponding regions may promote the development of prospective anti- Dengue vaccination strategies. The comparative genomics approach described here can be employed in the future for detecting functional regions in other pathogens with very high mutations rates. PMID:26449467

Sequence-Dependent Diastereospecific and Diastereodivergent Crosslinking of DNA by Decarbamoylmitomycin C.

PubMed

Aguilar, William; Paz, Manuel M; Vargas, Anayatzinc; Clement, Cristina C; Cheng, Shu-Yuan; Champeil, Elise

2018-04-20

Mitomycin C (MC), a potent antitumor drug, and decarbamoylmitomycin C (DMC), a derivative lacking the carbamoyl group, form highly cytotoxic DNA interstrand crosslinks. The major interstrand crosslink formed by DMC is the C1'' epimer of the major crosslink formed by MC. The molecular basis for the stereochemical configuration exhibited by DMC was investigated using biomimetic synthesis. The formation of DNA-DNA crosslinks by DMC is diastereospecific and diastereodivergent: Only the 1''S-diastereomer of the initially formed monoadduct can form crosslinks at GpC sequences, and only the 1''R-diastereomer of the monoadduct can form crosslinks at CpG sequences. We also show that CpG and GpC sequences react with divergent diastereoselectivity in the first alkylation step: 1"S stereochemistry is favored at GpC sequences and 1''R stereochemistry is favored at CpG sequences. Therefore, the first alkylation step results, at each sequence, in the selective formation of the diastereomer able to generate an interstrand DNA-DNA crosslink after the "second arm" alkylation. Examination of the known DNA adduct pattern obtained after treatment of cancer cell cultures with DMC indicates that the GpC sequence is the major target for the formation of DNA-DNA crosslinks in vivo by this drug. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

On site DNA barcoding by nanopore sequencing

PubMed Central

Menegon, Michele; Cantaloni, Chiara; Rodriguez-Prieto, Ana; Centomo, Cesare; Abdelfattah, Ahmed; Rossato, Marzia; Bernardi, Massimo; Xumerle, Luciano; Loader, Simon; Delledonne, Massimo

2017-01-01

Biodiversity research is becoming increasingly dependent on genomics, which allows the unprecedented digitization and understanding of the planet’s biological heritage. The use of genetic markers i.e. DNA barcoding, has proved to be a powerful tool in species identification. However, full exploitation of this approach is hampered by the high sequencing costs and the absence of equipped facilities in biodiversity-rich countries. In the present work, we developed a portable sequencing laboratory based on the portable DNA sequencer from Oxford Nanopore Technologies, the MinION. Complementary laboratory equipment and reagents were selected to be used in remote and tough environmental conditions. The performance of the MinION sequencer and the portable laboratory was tested for DNA barcoding in a mimicking tropical environment, as well as in a remote rainforest of Tanzania lacking electricity. Despite the relatively high sequencing error-rate of the MinION, the development of a suitable pipeline for data analysis allowed the accurate identification of different species of vertebrates including amphibians, reptiles and mammals. In situ sequencing of a wild frog allowed us to rapidly identify the species captured, thus confirming that effective DNA barcoding in the field is possible. These results open new perspectives for real-time-on-site DNA sequencing thus potentially increasing opportunities for the understanding of biodiversity in areas lacking conventional laboratory facilities. PMID:28977016

Longitudinal Antigenic Sequences and Sites from Intra-Host Evolution (LASSIE) identifies immune-selected HIV variants

DOE PAGES

Hraber, Peter; Korber, Bette; Wagh, Kshitij; ...

2015-10-21

Within-host genetic sequencing from samples collected over time provides a dynamic view of how viruses evade host immunity. Immune-driven mutations might stimulate neutralization breadth by selecting antibodies adapted to cycles of immune escape that generate within-subject epitope diversity. Comprehensive identification of immune-escape mutations is experimentally and computationally challenging. With current technology, many more viral sequences can readily be obtained than can be tested for binding and neutralization, making down-selection necessary. Typically, this is done manually, by picking variants that represent different time-points and branches on a phylogenetic tree. Such strategies are likely to miss many relevant mutations and combinations ofmore » mutations, and to be redundant for other mutations. Longitudinal Antigenic Sequences and Sites from Intrahost Evolution (LASSIE) uses transmitted founder loss to identify virus “hot-spots” under putative immune selection and chooses sequences that represent recurrent mutations in selected sites. LASSIE favors earliest sequences in which mutations arise. Here, with well-characterized longitudinal Env sequences, we confirmed selected sites were concentrated in antibody contacts and selected sequences represented diverse antigenic phenotypes. Finally, practical applications include rapidly identifying immune targets under selective pressure within a subject, selecting minimal sets of reagents for immunological assays that characterize evolving antibody responses, and for immunogens in polyvalent “cocktail” vaccines.« less

Absence of positive selection on CenH3 in Luzula suggests that holokinetic chromosomes may suppress centromere drive.

PubMed

Zedek, František; Bureš, Petr

2016-12-01

The centromere drive theory explains diversity of eukaryotic centromeres as a consequence of the recurrent conflict between centromeric repeats and centromeric histone H3 (CenH3), in which selfish centromeres exploit meiotic asymmetry and CenH3 evolves adaptively to counterbalance deleterious consequences of driving centromeres. Accordingly, adaptively evolving CenH3 has so far been observed only in eukaryotes with asymmetric meiosis. However, if such evolution is a consequence of centromere drive, it should depend not only on meiotic asymmetry but also on monocentric or holokinetic chromosomal structure. Selective pressures acting on CenH3 have never been investigated in organisms with holokinetic meiosis despite the fact that holokinetic chromosomes have been hypothesized to suppress centromere drive. Therefore, the present study evaluates selective pressures acting on the CenH3 gene in holokinetic organisms for the first time, specifically in the representatives of the plant genus Luzula (Juncaceae), in which the kinetochore formation is not co-localized with any type of centromeric repeat. PCR, cloning and sequencing, and database searches were used to obtain coding CenH3 sequences from Luzula species. Codon substitution models were employed to infer selective regimes acting on CenH3 in Luzula KEY RESULTS: In addition to the two previously published CenH3 sequences from L. nivea, 16 new CenH3 sequences have been isolated from 12 Luzula species. Two CenH3 isoforms in Luzula that originated by a duplication event prior to the divergence of analysed species were found. No signs of positive selection acting on CenH3 in Luzula were detected. Instead, evidence was found that selection on CenH3 of Luzula might have been relaxed. The results indicate that holokinetism itself may suppress centromere drive and, therefore, holokinetic chromosomes might have evolved as a defence against centromere drive. © The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

The red-sequence of 72 WINGS local galaxy clusters

NASA Astrophysics Data System (ADS)

Valentinuzzi, T.; Poggianti, B. M.; Fasano, G.; D'Onofrio, M.; Moretti, A.; Ramella, M.; Biviano, A.; Fritz, J.; Varela, J.; Bettoni, D.; Vulcani, B.; Moles, M.; Couch, W. J.; Dressler, A.; Kjærgaard, P.; Omizzolo, A.; Cava, A.

2011-12-01

We study the color - magnitude red sequence and blue fraction of 72 X-ray selected galaxy clusters at z = 0.04-0.07 from the WINGS survey, searching for correlations between the characteristics of the red sequence (RS) and the environment. We consider the slope and scatter of the red sequence, the number ratio of red luminous-to-faint galaxies, the blue fraction, and the fractions of ellipticals, S0s, and spirals that compose the RS. None of these quantities correlate with the cluster velocity dispersion, X-ray luminosity, number of cluster substructures, BCG prevalence over next brightest galaxies, and the spatial concentration of ellipticals. The properties of the RS, instead, depend strongly on local galaxy density. Higher density regions have a smaller RS scatter, a higher luminous-to-faint ratio, a lower blue fraction, and a lower spiral fraction on the RS. Our results clearly illustrate the prominent effect of the local density in setting the epoch when galaxies become passive and join the red sequence, as opposed to the mass of the galaxy host structure.

Niche construction, sources of selection and trait coevolution.

PubMed

Laland, Kevin; Odling-Smee, John; Endler, John

2017-10-06

Organisms modify and choose components of their local environments. This 'niche construction' can alter ecological processes, modify natural selection and contribute to inheritance through ecological legacies. Here, we propose that niche construction initiates and modifies the selection directly affecting the constructor, and on other species, in an orderly, directed and sustained manner. By dependably generating specific environmental states, niche construction co-directs adaptive evolution by imposing a consistent statistical bias on selection. We illustrate how niche construction can generate this evolutionary bias by comparing it with artificial selection. We suggest that it occupies the middle ground between artificial and natural selection. We show how the perspective leads to testable predictions related to: (i) reduced variance in measures of responses to natural selection in the wild; (ii) multiple trait coevolution, including the evolution of sequences of traits and patterns of parallel evolution; and (iii) a positive association between niche construction and biodiversity. More generally, we submit that evolutionary biology would benefit from greater attention to the diverse properties of all sources of selection.

Therapeutic magnetic microcarriers characterization by measuring magnetophoretic attributes

NASA Astrophysics Data System (ADS)

Vidal Ibacache, Guillermo

Micro/nano robots are considered a promising approach to conduct minimally invasive interventions. We have proposed to embed magnetic nanoparticles in therapeutic or diagnostic agents in order to magnetically control them. A modified clinical Magnetic Resonance Imaging (MRI) scanner is used to provide the driving force that allows these magnetically embedded microcarriers to navigate the vascular human network. By using specific Magnetic Resonance (MR) gradient sequences this method has been validated in previous research works. Magnetophoresis is the term used to describe the fact that a magnetic particle changes its trajectory under the influence of a magnetic force while being carried by a fluid flow. This movement depends on the particle's magnetic characteristics, the particle's geometric shape, the fluid flow's attributes and other factors. In our proposed method, magnetic microcarriers can be produced in several different ways, and so their response will differ to the same magnetic force and fluid flow conditions. The outcome of the therapeutic treatment using our method depends on the adequate selection of the therapeutic and/or diagnosis agents to be used. The selected therapeutic and/or diagnosis magnetic microcarrier also influences the selection of the MR gradient sequence that best fit for a given treatment. This master's thesis presents the design of a device intended to assess the magnetophoretic properties of magnetic therapeutic microcarriers and/or diagnostic agents. Such characterization is essential for determining the optimal sequences of magnetic gradients to deflect their trajectory through relatively complex vascular networks in order to reach a pre-defined target. A microfluidic device was fabricated to validate the design. Magnetophoretic velocities are measured and a simple tracking method is proposed. The preliminary experimental results indicate that, despite some limitations, the proposed technique has the potential to be appropriate to characterize any drug and/or diagnosis magnetic microcarrier containing different magnetic nanoparticle content.

Kit for detecting nucleic acid sequences using competitive hybridization probes

DOEpatents

Lucas, Joe N.; Straume, Tore; Bogen, Kenneth T.

2001-01-01

A kit is provided for detecting a target nucleic acid sequence in a sample, the kit comprising: a first hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a first portion of the target sequence, the first hybridization probe including a first complexing agent for forming a binding pair with a second complexing agent; and a second hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a second portion of the target sequence to which the first hybridization probe does not selectively hybridize, the second hybridization probe including a detectable marker; a third hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a first portion of the target sequence, the third hybridization probe including the same detectable marker as the second hybridization probe; and a fourth hybridization probe which includes a nucleic acid sequence that is sufficiently complementary to selectively hybridize to a second portion of the target sequence to which the third hybridization probe does not selectively hybridize, the fourth hybridization probe including the first complexing agent for forming a binding pair with the second complexing agent; wherein the first and second hybridization probes are capable of simultaneously hybridizing to the target sequence and the third and fourth hybridization probes are capable of simultaneously hybridizing to the target sequence, the detectable marker is not present on the first or fourth hybridization probes and the first, second, third, and fourth hybridization probes each include a competitive nucleic acid sequence which is sufficiently complementary to a third portion of the target sequence that the competitive sequences of the first, second, third, and fourth hybridization probes compete with each other to hybridize to the third portion of the target sequence.

A privacy-preserving solution for compressed storage and selective retrieval of genomic data.

PubMed

Huang, Zhicong; Ayday, Erman; Lin, Huang; Aiyar, Raeka S; Molyneaux, Adam; Xu, Zhenyu; Fellay, Jacques; Steinmetz, Lars M; Hubaux, Jean-Pierre

2016-12-01

In clinical genomics, the continuous evolution of bioinformatic algorithms and sequencing platforms makes it beneficial to store patients' complete aligned genomic data in addition to variant calls relative to a reference sequence. Due to the large size of human genome sequence data files (varying from 30 GB to 200 GB depending on coverage), two major challenges facing genomics laboratories are the costs of storage and the efficiency of the initial data processing. In addition, privacy of genomic data is becoming an increasingly serious concern, yet no standard data storage solutions exist that enable compression, encryption, and selective retrieval. Here we present a privacy-preserving solution named SECRAM (Selective retrieval on Encrypted and Compressed Reference-oriented Alignment Map) for the secure storage of compressed aligned genomic data. Our solution enables selective retrieval of encrypted data and improves the efficiency of downstream analysis (e.g., variant calling). Compared with BAM, the de facto standard for storing aligned genomic data, SECRAM uses 18% less storage. Compared with CRAM, one of the most compressed nonencrypted formats (using 34% less storage than BAM), SECRAM maintains efficient compression and downstream data processing, while allowing for unprecedented levels of security in genomic data storage. Compared with previous work, the distinguishing features of SECRAM are that (1) it is position-based instead of read-based, and (2) it allows random querying of a subregion from a BAM-like file in an encrypted form. Our method thus offers a space-saving, privacy-preserving, and effective solution for the storage of clinical genomic data. © 2016 Huang et al.; Published by Cold Spring Harbor Laboratory Press.

The optimal sequence and selection of screening test items to predict fall risk in older disabled women: the Women's Health and Aging Study.

PubMed

Lamb, Sarah E; McCabe, Chris; Becker, Clemens; Fried, Linda P; Guralnik, Jack M

2008-10-01

Falls are a major cause of disability, dependence, and death in older people. Brief screening algorithms may be helpful in identifying risk and leading to more detailed assessment. Our aim was to determine the most effective sequence of falls screening test items from a wide selection of recommended items including self-report and performance tests, and to compare performance with other published guidelines. Data were from a prospective, age-stratified, cohort study. Participants were 1002 community-dwelling women aged 65 years old or older, experiencing at least some mild disability. Assessments of fall risk factors were conducted in participants' homes. Fall outcomes were collected at 6 monthly intervals. Algorithms were built for prediction of any fall over a 12-month period using tree classification with cross-set validation. Algorithms using performance tests provided the best prediction of fall events, and achieved moderate to strong performance when compared to commonly accepted benchmarks. The items selected by the best performing algorithm were the number of falls in the last year and, in selected subpopulations, frequency of difficulty balancing while walking, a 4 m walking speed test, body mass index, and a test of knee extensor strength. The algorithm performed better than that from the American Geriatric Society/British Geriatric Society/American Academy of Orthopaedic Surgeons and other guidance, although these findings should be treated with caution. Suggestions are made on the type, number, and sequence of tests that could be used to maximize estimation of the probability of falling in older disabled women.

A privacy-preserving solution for compressed storage and selective retrieval of genomic data

PubMed Central

Huang, Zhicong; Ayday, Erman; Lin, Huang; Aiyar, Raeka S.; Molyneaux, Adam; Xu, Zhenyu; Hubaux, Jean-Pierre

2016-01-01

In clinical genomics, the continuous evolution of bioinformatic algorithms and sequencing platforms makes it beneficial to store patients’ complete aligned genomic data in addition to variant calls relative to a reference sequence. Due to the large size of human genome sequence data files (varying from 30 GB to 200 GB depending on coverage), two major challenges facing genomics laboratories are the costs of storage and the efficiency of the initial data processing. In addition, privacy of genomic data is becoming an increasingly serious concern, yet no standard data storage solutions exist that enable compression, encryption, and selective retrieval. Here we present a privacy-preserving solution named SECRAM (Selective retrieval on Encrypted and Compressed Reference-oriented Alignment Map) for the secure storage of compressed aligned genomic data. Our solution enables selective retrieval of encrypted data and improves the efficiency of downstream analysis (e.g., variant calling). Compared with BAM, the de facto standard for storing aligned genomic data, SECRAM uses 18% less storage. Compared with CRAM, one of the most compressed nonencrypted formats (using 34% less storage than BAM), SECRAM maintains efficient compression and downstream data processing, while allowing for unprecedented levels of security in genomic data storage. Compared with previous work, the distinguishing features of SECRAM are that (1) it is position-based instead of read-based, and (2) it allows random querying of a subregion from a BAM-like file in an encrypted form. Our method thus offers a space-saving, privacy-preserving, and effective solution for the storage of clinical genomic data. PMID:27789525

Non-invasive MRI detection of individual pellets in the human stomach.

PubMed

Knörgen, Manfred; Spielmann, Rolf Peter; Abdalla, Ahmed; Metz, Hendrik; Mäder, Karsten

2010-01-01

MRI is a powerful and non-invasive method to follow the fate of oral drug delivery systems in humans. Until now, most MRI studies focused on monolithic dosage forms (tablets and capsules). Small-sized multi-particulate drug delivery systems are very difficult to detect due to the poor differentiation between the delivery system and the food. A new approach was developed to overcome the described difficulties and permit the selective imaging of small multi-particulate dosage forms within the stomach. We took advantage of the different sensitivities to susceptibility artefacts of T(2)-weighted spin-echo sequences and T(2)-weighted gradient echo pulse sequences. Using a combination of both methods within a breath hold followed by a specific mathematical image analysis involving co-registration, motion correction, voxel-by-voxel comparison of the maps from different pulse sequences and graphic 2D-/3D-presentation, we were able to obtain pictures with a high sensitivity due to susceptibility effects caused by a 1% magnetite load. By means of the new imaging sequence, single pellets as small as 1mm can be detected with high selectivity within surrounding heterogeneous food in the human stomach. The developed method greatly expands the use of MRI to study the fate of oral multi-particulate drug delivery systems and their food dependency in men. Copyright 2009 Elsevier B.V. All rights reserved.

Development of Scoring Functions for Antibody Sequence Assessment and Optimization

PubMed Central

Seeliger, Daniel

2013-01-01

Antibody development is still associated with substantial risks and difficulties as single mutations can radically change molecule properties like thermodynamic stability, solubility or viscosity. Since antibody generation methodologies cannot select and optimize for molecule properties which are important for biotechnological applications, careful sequence analysis and optimization is necessary to develop antibodies that fulfil the ambitious requirements of future drugs. While efforts to grab the physical principles of undesired molecule properties from the very bottom are becoming increasingly powerful, the wealth of publically available antibody sequences provides an alternative way to develop early assessment strategies for antibodies using a statistical approach which is the objective of this paper. Here, publically available sequences were used to develop heuristic potentials for the framework regions of heavy and light chains of antibodies of human and murine origin. The potentials take into account position dependent probabilities of individual amino acids but also conditional probabilities which are inevitable for sequence assessment and optimization. It is shown that the potentials derived from human sequences clearly distinguish between human sequences and sequences from mice and, hence, can be used as a measure of humaness which compares a given sequence with the phenotypic pool of human sequences instead of comparing sequence identities to germline genes. Following this line, it is demonstrated that, using the developed potentials, humanization of an antibody can be described as a simple mathematical optimization problem and that the in-silico generated framework variants closely resemble native sequences in terms of predicted immunogenicity. PMID:24204701

COACH: profile-profile alignment of protein families using hidden Markov models.

PubMed

Edgar, Robert C; Sjölander, Kimmen

2004-05-22

Alignments of two multiple-sequence alignments, or statistical models of such alignments (profiles), have important applications in computational biology. The increased amount of information in a profile versus a single sequence can lead to more accurate alignments and more sensitive homolog detection in database searches. Several profile-profile alignment methods have been proposed and have been shown to improve sensitivity and alignment quality compared with sequence-sequence methods (such as BLAST) and profile-sequence methods (e.g. PSI-BLAST). Here we present a new approach to profile-profile alignment we call Comparison of Alignments by Constructing Hidden Markov Models (HMMs) (COACH). COACH aligns two multiple sequence alignments by constructing a profile HMM from one alignment and aligning the other to that HMM. We compare the alignment accuracy of COACH with two recently published methods: Yona and Levitt's prof_sim and Sadreyev and Grishin's COMPASS. On two sets of reference alignments selected from the FSSP database, we find that COACH is able, on average, to produce alignments giving the best coverage or the fewest errors, depending on the chosen parameter settings. COACH is freely available from www.drive5.com/lobster

Key Aspects of Nucleic Acid Library Design for in Vitro Selection

PubMed Central

Vorobyeva, Maria A.; Davydova, Anna S.; Vorobjev, Pavel E.; Pyshnyi, Dmitrii V.; Venyaminova, Alya G.

2018-01-01

Nucleic acid aptamers capable of selectively recognizing their target molecules have nowadays been established as powerful and tunable tools for biospecific applications, be it therapeutics, drug delivery systems or biosensors. It is now generally acknowledged that in vitro selection enables one to generate aptamers to almost any target of interest. However, the success of selection and the affinity of the resulting aptamers depend to a large extent on the nature and design of an initial random nucleic acid library. In this review, we summarize and discuss the most important features of the design of nucleic acid libraries for in vitro selection such as the nature of the library (DNA, RNA or modified nucleotides), the length of a randomized region and the presence of fixed sequences. We also compare and contrast different randomization strategies and consider computer methods of library design and some other aspects. PMID:29401748

Functional metagenomic selection of RubisCOs from uncultivated bacteria

USGS Publications Warehouse

Varaljay, Vanessa A; Satagopan, Sriram; North, Justin A.; Witteveen, Briana; Dourado, Manuella N.; Anantharaman, Karthik; Arbing, Mark A.; McCann, Shelley; Oremland, Ronald S.; Banfield, Jillian F.; Wrighton, Kelly C.; Tabita, F. Robert

2016-01-01

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a critical yet severely inefficient enzyme that catalyses the fixation of virtually all of the carbon found on Earth. Here, we report a functional metagenomic selection that recovers physiologically active RubisCO molecules directly from uncultivated and largely unknown members of natural microbial communities. Selection is based on CO2-dependent growth in a host strain capable of expressing environmental deoxyribonucleic acid (DNA), precluding the need for pure cultures or screening of recombinant clones for enzymatic activity. Seventeen functional RubisCO-encoded sequences were selected using DNA extracted from soil and river autotrophic enrichments, a photosynthetic biofilm and a subsurface groundwater aquifer. Notably, three related form II RubisCOs were recovered which share high sequence similarity with metagenomic scaffolds from uncultivated members of theGallionellaceae family. One of the Gallionellaceae RubisCOs was purified and shown to possessCO2/O2 specificity typical of form II enzymes. X-ray crystallography determined that this enzyme is a hexamer, only the second form II multimer ever solved and the first RubisCO structure obtained from an uncultivated bacterium. Functional metagenomic selection leverages natural biological diversity and billions of years of evolution inherent in environmental communities, providing a new window into the discovery of CO2-fixing enzymes not previously characterized.

Expert systems for fault diagnosis in nuclear reactor control

NASA Astrophysics Data System (ADS)

Jalel, N. A.; Nicholson, H.

1990-11-01

An expert system for accident analysis and fault diagnosis for the Loss Of Fluid Test (LOFT) reactor, a small scale pressurized water reactor, was developed for a personal computer. The knowledge of the system is presented using a production rule approach with a backward chaining inference engine. The data base of the system includes simulated dependent state variables of the LOFT reactor model. Another system is designed to assist the operator in choosing the appropriate cooling mode and to diagnose the fault in the selected cooling system. The response tree, which is used to provide the link between a list of very specific accident sequences and a set of generic emergency procedures which help the operator in monitoring system status, and to differentiate between different accident sequences and select the correct procedures, is used to build the system knowledge base. Both systems are written in TURBO PROLOG language and can be run on an IBM PC compatible with 640k RAM, 40 Mbyte hard disk and color graphics.

DNA targeting specificity of RNA-guided Cas9 nucleases.

PubMed

Hsu, Patrick D; Scott, David A; Weinstein, Joshua A; Ran, F Ann; Konermann, Silvana; Agarwala, Vineeta; Li, Yinqing; Fine, Eli J; Wu, Xuebing; Shalem, Ophir; Cradick, Thomas J; Marraffini, Luciano A; Bao, Gang; Zhang, Feng

2013-09-01

The Streptococcus pyogenes Cas9 (SpCas9) nuclease can be efficiently targeted to genomic loci by means of single-guide RNAs (sgRNAs) to enable genome editing. Here, we characterize SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. Our study evaluates >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that SpCas9-mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. To facilitate mammalian genome engineering applications, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.

Antisense phosphorothioate oligonucleotides: selective killing of the intracellular parasite Leishmania amazonensis.

PubMed

Ramazeilles, C; Mishra, R K; Moreau, S; Pascolo, E; Toulmé, J J

1994-08-16

We targeted the mini-exon sequence, present at the 5' end of every mRNA of the protozoan parasite Leishmania amazonensis, by phosphorothioate oligonucleotides. A complementary 16-mer (16PS) was able to kill amastigotes--the intracellular stage of the parasite--in murine macrophages in culture. After 24 hr of incubation with 10 microM 16PS, about 30% infected macrophages were cured. The oligomer 16PS acted through antisense hybridization in a sequence-dependent way; no effect on parasites was observed with noncomplementary phosphorothioate oligonucleotides. The antisense oligonucleotide 16PS was a selective killer of the protozoans without any detrimental effect to the host macrophage. Using 16PS linked to a palmitate chain, which enabled it to complex with low density lipoproteins, improved the leishmanicidal efficiency on intracellular amastigotes, probably due to increased endocytosis. Phosphorothioate oligonucleotides complementary to the intron part of the mini-exon pre-RNA were also effective, suggesting that antisense oligomers could prevent trans-splicing in these parasites.

The evolution of transcriptional regulation in eukaryotes

NASA Technical Reports Server (NTRS)

Wray, Gregory A.; Hahn, Matthew W.; Abouheif, Ehab; Balhoff, James P.; Pizer, Margaret; Rockman, Matthew V.; Romano, Laura A.

2003-01-01

Gene expression is central to the genotype-phenotype relationship in all organisms, and it is an important component of the genetic basis for evolutionary change in diverse aspects of phenotype. However, the evolution of transcriptional regulation remains understudied and poorly understood. Here we review the evolutionary dynamics of promoter, or cis-regulatory, sequences and the evolutionary mechanisms that shape them. Existing evidence indicates that populations harbor extensive genetic variation in promoter sequences, that a substantial fraction of this variation has consequences for both biochemical and organismal phenotype, and that some of this functional variation is sorted by selection. As with protein-coding sequences, rates and patterns of promoter sequence evolution differ considerably among loci and among clades for reasons that are not well understood. Studying the evolution of transcriptional regulation poses empirical and conceptual challenges beyond those typically encountered in analyses of coding sequence evolution: promoter organization is much less regular than that of coding sequences, and sequences required for the transcription of each locus reside at multiple other loci in the genome. Because of the strong context-dependence of transcriptional regulation, sequence inspection alone provides limited information about promoter function. Understanding the functional consequences of sequence differences among promoters generally requires biochemical and in vivo functional assays. Despite these challenges, important insights have already been gained into the evolution of transcriptional regulation, and the pace of discovery is accelerating.

Uncommonly isolated clinical Pseudomonas: identification and phylogenetic assignation.

PubMed

Mulet, M; Gomila, M; Ramírez, A; Cardew, S; Moore, E R B; Lalucat, J; García-Valdés, E

2017-02-01

Fifty-two Pseudomonas strains that were difficult to identify at the species level in the phenotypic routine characterizations employed by clinical microbiology laboratories were selected for genotypic-based analysis. Species level identifications were done initially by partial sequencing of the DNA dependent RNA polymerase sub-unit D gene (rpoD). Two other gene sequences, for the small sub-unit ribosonal RNA (16S rRNA) and for DNA gyrase sub-unit B (gyrB) were added in a multilocus sequence analysis (MLSA) study to confirm the species identifications. These sequences were analyzed with a collection of reference sequences from the type strains of 161 Pseudomonas species within an in-house multi-locus sequence analysis database. Whole-cell matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses of these strains complemented the DNA sequenced-based phylogenetic analyses and were observed to be in accordance with the results of the sequence data. Twenty-three out of 52 strains were assigned to 12 recognized species not commonly detected in clinical specimens and 29 (56 %) were considered representatives of at least ten putative new species. Most strains were distributed within the P. fluorescens and P. aeruginosa lineages. The value of rpoD sequences in species-level identifications for Pseudomonas is emphasized. The correct species identifications of clinical strains is essential for establishing the intrinsic antibiotic resistance patterns and improved treatment plans.

On causal roles and selected effects: our genome is mostly junk.

PubMed

Doolittle, W Ford; Brunet, Tyler D P

2017-12-05

The idea that much of our genome is irrelevant to fitness-is not the product of positive natural selection at the organismal level-remains viable. Claims to the contrary, and specifically that the notion of "junk DNA" should be abandoned, are based on conflating meanings of the word "function". Recent estimates suggest that perhaps 90% of our DNA, though biochemically active, does not contribute to fitness in any sequence-dependent way, and possibly in no way at all. Comparisons to vertebrates with much larger and smaller genomes (the lungfish and the pufferfish) strongly align with such a conclusion, as they have done for the last half-century.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kielmas, Martyna; Szewczuk, Zbigniew; Stefanowicz, Piotr, E-mail: Piotr.stefanowicz@chem.uni.wroc.pl

Highlights: •The glycation of fibrinogen was investigated by isotopic labeling method. •The potential glycation sites in fibrinogen were identified. •Human serum albumin (HSA) inhibits the glycation of fibrinogen. •The effect of HSA on fibrinogen glycation is sequence-dependent. -- Abstract: Although in vivo glycation proceeds in complex mixture of proteins, previous studies did not take in consideration the influence of protein–protein interaction on Maillard reaction. The aim of our study was to test the influence of human serum albumin (HSA) on glycation of fibrinogen. The isotopic labeling using [{sup 13}C{sub 6}] glucose combined with LC-MS were applied as tool for identificationmore » possible glycation sites in fibrinogen and for evaluation the effect of HSA on the glycation level of selected amino acids in fibrinogen. The obtained data indicate that the addition of HSA protects the fibrinogen from glycation. The level of glycation in presence of HSA is reduced by 30–60% and depends on the location of glycated residue in sequence of protein.« less

Mechanism for Coordinated RNA Packaging and Genome Replication by Rotavirus Polymerase VP1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, Xiaohui; McDonald, Sarah M.; Tortorici, M. Alejandra

2009-04-08

Rotavirus RNA-dependent RNA polymerase VP1 catalyzes RNA synthesis within a subviral particle. This activity depends on core shell protein VP2. A conserved sequence at the 3' end of plus-strand RNA templates is important for polymerase association and genome replication. We have determined the structure of VP1 at 2.9 {angstrom} resolution, as apoenzyme and in complex with RNA. The cage-like enzyme is similar to reovirus {lambda}3, with four tunnels leading to or from a central, catalytic cavity. A distinguishing characteristic of VP1 is specific recognition, by conserved features of the template-entry channel, of four bases, UGUG, in the conserved 3' sequence.more » Well-defined interactions with these bases position the RNA so that its 3' end overshoots the initiating register, producing a stable but catalytically inactive complex. We propose that specific 3' end recognition selects rotavirus RNA for packaging and that VP2 activates the autoinhibited VP1/RNA complex to coordinate packaging and genome replication.« less

Deconstruction of the Ras switching cycle through saturation mutagenesis

PubMed Central

Bandaru, Pradeep; Shah, Neel H; Bhattacharyya, Moitrayee; Barton, John P; Kondo, Yasushi; Cofsky, Joshua C; Gee, Christine L; Chakraborty, Arup K; Kortemme, Tanja; Ranganathan, Rama; Kuriyan, John

2017-01-01

Ras proteins are highly conserved signaling molecules that exhibit regulated, nucleotide-dependent switching between active and inactive states. The high conservation of Ras requires mechanistic explanation, especially given the general mutational tolerance of proteins. Here, we use deep mutational scanning, biochemical analysis and molecular simulations to understand constraints on Ras sequence. Ras exhibits global sensitivity to mutation when regulated by a GTPase activating protein and a nucleotide exchange factor. Removing the regulators shifts the distribution of mutational effects to be largely neutral, and reveals hotspots of activating mutations in residues that restrain Ras dynamics and promote the inactive state. Evolutionary analysis, combined with structural and mutational data, argue that Ras has co-evolved with its regulators in the vertebrate lineage. Overall, our results show that sequence conservation in Ras depends strongly on the biochemical network in which it operates, providing a framework for understanding the origin of global selection pressures on proteins. DOI: http://dx.doi.org/10.7554/eLife.27810.001 PMID:28686159

Structural hot spots for the solubility of globular proteins

PubMed Central

Ganesan, Ashok; Siekierska, Aleksandra; Beerten, Jacinte; Brams, Marijke; Van Durme, Joost; De Baets, Greet; Van der Kant, Rob; Gallardo, Rodrigo; Ramakers, Meine; Langenberg, Tobias; Wilkinson, Hannah; De Smet, Frederik; Ulens, Chris; Rousseau, Frederic; Schymkowitz, Joost

2016-01-01

Natural selection shapes protein solubility to physiological requirements and recombinant applications that require higher protein concentrations are often problematic. This raises the question whether the solubility of natural protein sequences can be improved. We here show an anti-correlation between the number of aggregation prone regions (APRs) in a protein sequence and its solubility, suggesting that mutational suppression of APRs provides a simple strategy to increase protein solubility. We show that mutations at specific positions within a protein structure can act as APR suppressors without affecting protein stability. These hot spots for protein solubility are both structure and sequence dependent but can be computationally predicted. We demonstrate this by reducing the aggregation of human α-galactosidase and protective antigen of Bacillus anthracis through mutation. Our results indicate that many proteins possess hot spots allowing to adapt protein solubility independently of structure and function. PMID:26905391

Using the self-select paradigm to delineate the nature of speech motor programming.

PubMed

Wright, David L; Robin, Don A; Rhee, Jooyhun; Vaculin, Amber; Jacks, Adam; Guenther, Frank H; Fox, Peter T

2009-06-01

The authors examined the involvement of 2 speech motor programming processes identified by S. T. Klapp (1995, 2003) during the articulation of utterances differing in syllable and sequence complexity. According to S. T. Klapp, 1 process, INT, resolves the demands of the programmed unit, whereas a second process, SEQ, oversees the serial order demands of longer sequences. A modified reaction time paradigm was used to assess INT and SEQ demands. Specifically, syllable complexity was dependent on syllable structure, whereas sequence complexity involved either repeated or unique syllabi within an utterance. INT execution was slowed when articulating single syllables in the form CCCV compared to simpler CV syllables. Planning unique syllables within a multisyllabic utterance rather than repetitions of the same syllable slowed INT but not SEQ. The INT speech motor programming process, important for mental syllabary access, is sensitive to changes in both syllable structure and the number of unique syllables in an utterance.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Xiang; Zhang, Shuai; Jiao, Fang

Two-step nucleation pathways in which disordered, amorphous, or dense liquid states precede appearance of crystalline phases have been reported for a wide range of materials, but the dynamics of such pathways are poorly understood. Moreover, whether these pathways are general features of crystallizing systems or a consequence of system-specific structural details that select for direct vs two-step processes is unknown. Using atomic force microscopy to directly observe crystallization of sequence-defined polymers, we show that crystallization pathways are indeed sequence dependent. When a short hydrophobic region is added to a sequence that directly forms crystalline particles, crystallization instead follows a two-stepmore » pathway that begins with creation of disordered clusters of 10-20 molecules and is characterized by highly non-linear crystallization kinetics in which clusters transform into ordered structures that then enter the growth phase. The results shed new light on non-classical crystallization mechanisms and have implications for design of self-assembling polymer systems.« less

Hierarchic models for laminated plates. Ph.D. Thesis

NASA Technical Reports Server (NTRS)

Actis, Ricardo Luis

1991-01-01

Structural plates and shells are three-dimensional bodies, one dimension of which happens to be much smaller than the other two. Thus, the quality of a plate or shell model must be judged on the basis of how well its exact solution approximates the corresponding three-dimensional problem. Of course, the exact solution depends not only on the choice of the model but also on the topology, material properties, loading and constraints. The desired degree of approximation depends on the analyst's goals in performing the analysis. For these reasons models have to be chosen adaptively. Hierarchic sequences of models make adaptive selection of the model which is best suited for the purposes of a particular analysis possible. The principles governing the formulation of hierarchic models for laminated plates are presented. The essential features of the hierarchic models described models are: (1) the exact solutions corresponding to the hierarchic sequence of models converge to the exact solution of the corresponding problem of elasticity for a fixed laminate thickness; and (2) the exact solution of each model converges to the same limit as the exact solution of the corresponding problem of elasticity with respect to the laminate thickness approaching zero. The formulation is based on one parameter (beta) which characterizes the hierarchic sequence of models, and a set of constants whose influence was assessed by a numerical sensitivity study. The recommended selection of these constants results in the number of fields increasing by three for each increment in the power of beta. Numerical examples analyzed with the proposed sequence of models are included and good correlation with the reference solutions was found. Results were obtained for laminated strips (plates in cylindrical bending) and for square and rectangular plates with uniform loading and with homogeneous boundary conditions. Cross-ply and angle-ply laminates were evaluated and the results compared with those of MSC/PROBE. Hierarchic models make the computation of any engineering data possible to an arbitrary level of precision within the framework of the theory of elasticity.

Expression and permeation properties of the K(+) channel Kir7.1 in the retinal pigment epithelium.

PubMed

Shimura, M; Yuan, Y; Chang, J T; Zhang, S; Campochiaro, P A; Zack, D J; Hughes, B A

2001-03-01

Bovine Kir7.1 clones were obtained from a retinal pigment epithelium (RPE)-subtracted cDNA library. Human RPE cDNA library screening resulted in clones encoding full-length human Kir7.1. Northern blot analysis indicated that bovine Kir7.1 is highly expressed in the RPE. Human Kir7.1 channels were expressed in Xenopus oocytes and studied using the two-electrode voltage-clamp technique. The macroscopic Kir7.1 conductance exhibited mild inward rectification and an inverse dependence on extracellular K+ concentration ([K+]o). The selectivity sequence based on permeability ratios was K+ (1.0) approximately Rb+ (0.89) > Cs+ (0.013) > Na+ (0.003) approximately Li+ (0.001) and the sequence based on conductance ratios was Rb+ (9.5) > K+ (1.0) > Na+ (0.458) > Cs+ (0.331) > Li+ (0.139). Non-stationary noise analysis of Rb+ currents in cell-attached patches yielded a unitary conductance for Kir7.1 of approximately 2 pS. In whole-cell recordings from freshly isolated bovine RPE cells, the predominant current was a mild inwardly rectifying K+ current that exhibited an inverse dependence of conductance on [K+]o. The selectivity sequence based on permeability ratios was K+ (1.0) approximately Rb+ (0.89) > Cs+ (0.021) > Na+ (0.003) approximately Li+ (0.002) and the sequence based on conductance ratios was Rb+ (8.9) > K+ (1.0) > Na+ (0.59) > Cs+ (0.23) > Li+ (0.08). In cell-attached recordings with Rb+ in the pipette, inwardly rectifying currents were observed in nine of 12 patches of RPE apical membrane but in only one of 13 basolateral membrane patches. Non-stationary noise analysis of Rb+ currents in cell-attached apical membrane patches yielded a unitary conductance for RPE Kir of approximately 2 pS. On the basis of this molecular and electrophysiological evidence, we conclude that Kir7.1 channel subunits comprise the K+ conductance of the RPE apical membrane.

Bayesian network analyses of resistance pathways against efavirenz and nevirapine

PubMed Central

Deforche, Koen; Camacho, Ricardo J.; Grossman, Zehave; Soares, Marcelo A.; Laethem, Kristel Van; Katzenstein, David A.; Harrigan, P. Richard; Kantor, Rami; Shafer, Robert; Vandamme, Anne-Mieke

2016-01-01

Objective To clarify the role of novel mutations selected by treatment with efavirenz or nevirapine, and investigate the influence of HIV-1 subtype on nonnucleoside reverse transcriptase inhibitor (nNRTI) resistance pathways. Design By finding direct dependencies between treatment-selected mutations, the involvement of these mutations as minor or major resistance mutations against efavirenz, nevirapine, or coadministrated nucleoside analogue reverse transcriptase inhibitors (NRTIs) is hypothesized. In addition, direct dependencies were investigated between treatment-selected mutations and polymorphisms, some of which are linked with subtype, and between NRTI and nNRTI resistance pathways. Methods Sequences from a large collaborative database of various subtypes were jointly analyzed to detect mutations selected by treatment. Using Bayesian network learning, direct dependencies were investigated between treatment-selected mutations, NRTI and nNRTI treatment history, and known NRTI resistance mutations. Results Several novel minor resistance mutations were found: 28K and 196R (for resistance against efavirenz), 101H and 138Q (nevirapine), and 31L (lamivudine). Robust interactions between NRTI mutations (65R, 74V, 75I/M, and 184V) and nNRTI resistance mutations (100I, 181C, 190E and 230L) may affect resistance development to particular treatment combinations. For example, an interaction between 65R and 181C predicts that the nevirapine and tenofovir and lamivudine/emtricitabine combination should be more prone to failure than efavirenz and tenofovir and lamivudine/emtricitabine. Conclusion Bayesian networks were helpful in untangling the selection of mutations by NRTI versus nNRTI treatment, and in discovering interactions between resistance mutations within and between these two classes of inhibitors. PMID:18832874

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hraber, Peter; Korber, Bette; Wagh, Kshitij

Within-host genetic sequencing from samples collected over time provides a dynamic view of how viruses evade host immunity. Immune-driven mutations might stimulate neutralization breadth by selecting antibodies adapted to cycles of immune escape that generate within-subject epitope diversity. Comprehensive identification of immune-escape mutations is experimentally and computationally challenging. With current technology, many more viral sequences can readily be obtained than can be tested for binding and neutralization, making down-selection necessary. Typically, this is done manually, by picking variants that represent different time-points and branches on a phylogenetic tree. Such strategies are likely to miss many relevant mutations and combinations ofmore » mutations, and to be redundant for other mutations. Longitudinal Antigenic Sequences and Sites from Intrahost Evolution (LASSIE) uses transmitted founder loss to identify virus “hot-spots” under putative immune selection and chooses sequences that represent recurrent mutations in selected sites. LASSIE favors earliest sequences in which mutations arise. Here, with well-characterized longitudinal Env sequences, we confirmed selected sites were concentrated in antibody contacts and selected sequences represented diverse antigenic phenotypes. Finally, practical applications include rapidly identifying immune targets under selective pressure within a subject, selecting minimal sets of reagents for immunological assays that characterize evolving antibody responses, and for immunogens in polyvalent “cocktail” vaccines.« less

Sequence Alignment to Predict Across Species Susceptibility ...

EPA Pesticide Factsheets

Conservation of a molecular target across species can be used as a line-of-evidence to predict the likelihood of chemical susceptibility. The web-based Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool was developed to simplify, streamline, and quantitatively assess protein sequence/structural similarity across taxonomic groups as a means to predict relative intrinsic susceptibility. The intent of the tool is to allow for evaluation of any potential protein target, so it is amenable to variable degrees of protein characterization, depending on available information about the chemical/protein interaction and the molecular target itself. To allow for flexibility in the analysis, a layered strategy was adopted for the tool. The first level of the SeqAPASS analysis compares primary amino acid sequences to a query sequence, calculating a metric for sequence similarity (including detection of candidate orthologs), the second level evaluates sequence similarity within selected domains (e.g., ligand-binding domain, DNA binding domain), and the third level of analysis compares individual amino acid residue positions identified as being of importance for protein conformation and/or ligand binding upon chemical perturbation. Each level of the SeqAPASS analysis provides increasing evidence to apply toward rapid, screening-level assessments of probable cross species susceptibility. Such analyses can support prioritization of chemicals for further ev

A multi-model approach to nucleic acid-based drug development.

PubMed

Gautherot, Isabelle; Sodoyer, Regís

2004-01-01

With the advent of functional genomics and the shift of interest towards sequence-based therapeutics, the past decades have witnessed intense research efforts on nucleic acid-mediated gene regulation technologies. Today, RNA interference is emerging as a groundbreaking discovery, holding promise for development of genetic modulators of unprecedented potency. Twenty-five years after the discovery of antisense RNA and ribozymes, gene control therapeutics are still facing developmental difficulties, with only one US FDA-approved antisense drug currently available in the clinic. Limited predictability of target site selection models is recognized as one major stumbling block that is shared by all of the so-called complementary technologies, slowing the progress towards a commercial product. Currently employed in vitro systems for target site selection include RNAse H-based mapping, antisense oligonucleotide microarrays, and functional screening approaches using libraries of catalysts with randomized target-binding arms to identify optimal ribozyme/DNAzyme cleavage sites. Individually, each strategy has its drawbacks from a drug development perspective. Utilization of message-modulating sequences as therapeutic agents requires that their action on a given target transcript meets criteria of potency and selectivity in the natural physiological environment. In addition to sequence-dependent characteristics, other factors will influence annealing reactions and duplex stability, as well as nucleic acid-mediated catalysis. Parallel consideration of physiological selection systems thus appears essential for screening for nucleic acid compounds proposed for therapeutic applications. Cellular message-targeting studies face issues relating to efficient nucleic acid delivery and appropriate analysis of response. For reliability and simplicity, prokaryotic systems can provide a rapid and cost-effective means of studying message targeting under pseudo-cellular conditions, but such approaches also have limitations. To streamline nucleic acid drug discovery, we propose a multi-model strategy integrating high-throughput-adapted bacterial screening, followed by reporter-based and/or natural cellular models and potentially also in vitro assays for characterization of the most promising candidate sequences, before final in vivo testing.

Ribozyme-based aminoglycoside switches of gene expression engineered by genetic selection in S. cerevisiae.

PubMed

Klauser, Benedikt; Atanasov, Janina; Siewert, Lena K; Hartig, Jörg S

2015-05-15

Systems for conditional gene expression are powerful tools in basic research as well as in biotechnology. For future applications, it is of great importance to engineer orthogonal genetic switches that function reliably in diverse contexts. RNA-based switches have the advantage that effector molecules interact immediately with regulatory modules inserted into the target RNAs, getting rid of the need of transcription factors usually mediating genetic control. Artificial riboswitches are characterized by their simplicity and small size accompanied by a high degree of modularity. We have recently reported a series of hammerhead ribozyme-based artificial riboswitches that allow for post-transcriptional regulation of gene expression via switching mRNA, tRNA, or rRNA functions. A more widespread application was so far hampered by moderate switching performances and a limited set of effector molecules available. Here, we report the re-engineering of hammerhead ribozymes in order to respond efficiently to aminoglycoside antibiotics. We first established an in vivo selection protocol in Saccharomyces cerevisiae that enabled us to search large sequence spaces for optimized switches. We then envisioned and characterized a novel strategy of attaching the aptamer to the ribozyme catalytic core, increasing the design options for rendering the ribozyme ligand-dependent. These innovations enabled the development of neomycin-dependent RNA modules that switch gene expression up to 25-fold. The presented aminoglycoside-responsive riboswitches belong to the best-performing RNA-based genetic regulators reported so far. The developed in vivo selection protocol should allow for sampling of large sequence spaces for engineering of further optimized riboswitches.

Sequence Dependent Interactions Between DNA and Single-Walled Carbon Nanotubes

NASA Astrophysics Data System (ADS)

Roxbury, Daniel

It is known that single-stranded DNA adopts a helical wrap around a single-walled carbon nanotube (SWCNT), forming a water-dispersible hybrid molecule. The ability to sort mixtures of SWCNTs based on chirality (electronic species) has recently been demonstrated using special short DNA sequences that recognize certain matching SWCNTs of specific chirality. This thesis investigates the intricacies of DNA-SWCNT sequence-specific interactions through both experimental and molecular simulation studies. The DNA-SWCNT binding strengths were experimentally quantified by studying the kinetics of DNA replacement by a surfactant on the surface of particular SWCNTs. Recognition ability was found to correlate strongly with measured binding strength, e.g. DNA sequence (TAT)4 was found to bind 20 times stronger to the (6,5)-SWCNT than sequence (TAT)4T. Next, using replica exchange molecular dynamics (REMD) simulations, equilibrium structures formed by (a) single-strands and (b) multiple-strands of 12-mer oligonucleotides adsorbed on various SWCNTs were explored. A number of structural motifs were discovered in which the DNA strand wraps around the SWCNT and 'stitches' to itself via hydrogen bonding. Great variability among equilibrium structures was observed and shown to be directly influenced by DNA sequence and SWCNT type. For example, the (6,5)-SWCNT DNA recognition sequence, (TAT)4, was found to wrap in a tight single-stranded right-handed helical conformation. In contrast, DNA sequence T12 forms a beta-barrel left-handed structure on the same SWCNT. These are the first theoretical indications that DNA-based SWCNT selectivity can arise on a molecular level. In a biomedical collaboration with the Mayo Clinic, pathways for DNA-SWCNT internalization into healthy human endothelial cells were explored. Through absorbance spectroscopy, TEM imaging, and confocal fluorescence microscopy, we showed that intracellular concentrations of SWCNTs far exceeded those of the incubation solution, which suggested an energy-dependent pathway. Additionally, by means of pharmacological inhibition and vector-induced gene knockout studies, the DNA-SWCNTs were shown to enter the cells via Rac1-mediated macropinocytosis.

The impact of single nucleotide polymorphism in monomeric alpha-amylase inhibitor genes from wild emmer wheat, primarily from Israel and Golan

PubMed Central

2010-01-01

Background Various enzyme inhibitors act on key insect gut digestive hydrolases, including alpha-amylases and proteinases. Alpha-amylase inhibitors have been widely investigated for their possible use in strengthening a plant's defense against insects that are highly dependent on starch as an energy source. We attempted to unravel the diversity of monomeric alpha-amylase inhibitor genes of Israeli and Golan Heights' wild emmer wheat with different ecological factors (e.g., geography, water, and temperature). Population methods that analyze the nature and frequency of allele diversity within a species and the codon analysis method (comparing patterns of synonymous and non-synonymous changes in protein coding sequences) were used to detect natural selection. Results Three hundred and forty-eight sequences encoding monomeric alpha-amylase inhibitors (WMAI) were obtained from 14 populations of wild emmer wheat. The frequency of SNPs in WMAI genes was 1 out of 16.3 bases, where 28 SNPs were detected in the coding sequence. The results of purifying and the positive selection hypothesis (p < 0.05) showed that the sequences of WMAI were contributed by both natural selection and co-evolution, which ensured conservation of protein function and inhibition against diverse insect amylases. The majority of amino acid substitutions occurred at the C-terminal (positive selection domain), which ensured the stability of WMAI. SNPs in this gene could be classified into several categories associated with water, temperature, and geographic factors, respectively. Conclusions Great diversity at the WMAI locus, both between and within populations, was detected in the populations of wild emmer wheat. It was revealed that WMAI were naturally selected for across populations by a ratio of dN/dS as expected. Ecological factors, singly or in combination, explained a significant proportion of the variations in the SNPs. A sharp genetic divergence over very short geographic distances compared to a small genetic divergence between large geographic distances also suggested that the SNPs were subjected to natural selection, and ecological factors had an important evolutionary role in polymorphisms at this locus. According to population and codon analysis, these results suggested that monomeric alpha-amylase inhibitors are adaptively selected under different environmental conditions. PMID:20534122

A Statistical Guide to the Design of Deep Mutational Scanning Experiments.

PubMed

Matuszewski, Sebastian; Hildebrandt, Marcel E; Ghenu, Ana-Hermina; Jensen, Jeffrey D; Bank, Claudia

2016-09-01

The characterization of the distribution of mutational effects is a key goal in evolutionary biology. Recently developed deep-sequencing approaches allow for accurate and simultaneous estimation of the fitness effects of hundreds of engineered mutations by monitoring their relative abundance across time points in a single bulk competition. Naturally, the achievable resolution of the estimated fitness effects depends on the specific experimental setup, the organism and type of mutations studied, and the sequencing technology utilized, among other factors. By means of analytical approximations and simulations, we provide guidelines for optimizing time-sampled deep-sequencing bulk competition experiments, focusing on the number of mutants, the sequencing depth, and the number of sampled time points. Our analytical results show that sampling more time points together with extending the duration of the experiment improves the achievable precision disproportionately compared with increasing the sequencing depth or reducing the number of competing mutants. Even if the duration of the experiment is fixed, sampling more time points and clustering these at the beginning and the end of the experiment increase experimental power and allow for efficient and precise assessment of the entire range of selection coefficients. Finally, we provide a formula for calculating the 95%-confidence interval for the measurement error estimate, which we implement as an interactive web tool. This allows for quantification of the maximum expected a priori precision of the experimental setup, as well as for a statistical threshold for determining deviations from neutrality for specific selection coefficient estimates. Copyright © 2016 by the Genetics Society of America.

Evolution of ribozymes in the presence of a mineral surface

PubMed Central

Stephenson, James D.; Popović, Milena; Bristow, Thomas F.

2016-01-01

Mineral surfaces are often proposed as the sites of critical processes in the emergence of life. Clay minerals in particular are thought to play significant roles in the origin of life including polymerizing, concentrating, organizing, and protecting biopolymers. In these scenarios, the impact of minerals on biopolymer folding is expected to influence evolutionary processes. These processes include both the initial emergence of functional structures in the presence of the mineral and the subsequent transition away from the mineral-associated niche. The initial evolution of function depends upon the number and distribution of sequences capable of functioning in the presence of the mineral, and the transition to new environments depends upon the overlap between sequences that evolve on the mineral surface and sequences that can perform the same functions in the mineral's absence. To examine these processes, we evolved self-cleaving ribozymes in vitro in the presence or absence of Na-saturated montmorillonite clay mineral particles. Starting from a shared population of random sequences, RNA populations were evolved in parallel, along separate evolutionary trajectories. Comparative sequence analysis and activity assays show that the impact of this clay mineral on functional structure selection was minimal; it neither prevented common structures from emerging, nor did it promote the emergence of new structures. This suggests that montmorillonite does not improve RNA's ability to evolve functional structures; however, it also suggests that RNAs that do evolve in contact with montmorillonite retain the same structures in mineral-free environments, potentially facilitating an evolutionary transition away from a mineral-associated niche. PMID:27793980

Whole-Gene Positive Selection, Elevated Synonymous Substitution Rates, Duplication, and Indel Evolution of the Chloroplast clpP1 Gene

PubMed Central

Erixon, Per; Oxelman, Bengt

2008-01-01

Background Synonymous DNA substitution rates in the plant chloroplast genome are generally relatively slow and lineage dependent. Non-synonymous rates are usually even slower due to purifying selection acting on the genes. Positive selection is expected to speed up non-synonymous substitution rates, whereas synonymous rates are expected to be unaffected. Until recently, positive selection has seldom been observed in chloroplast genes, and large-scale structural rearrangements leading to gene duplications are hitherto supposed to be rare. Methodology/Principle Findings We found high substitution rates in the exons of the plastid clpP1 gene in Oenothera (the Evening Primrose family) and three separate lineages in the tribe Sileneae (Caryophyllaceae, the Carnation family). Introns have been lost in some of the lineages, but where present, the intron sequences have substitution rates similar to those found in other introns of their genomes. The elevated substitution rates of clpP1 are associated with statistically significant whole-gene positive selection in three branches of the phylogeny. In two of the lineages we found multiple copies of the gene. Neighboring genes present in the duplicated fragments do not show signs of elevated substitution rates or positive selection. Although non-synonymous substitutions account for most of the increase in substitution rates, synonymous rates are also markedly elevated in some lineages. Whereas plant clpP1 genes experiencing negative (purifying) selection are characterized by having very conserved lengths, genes under positive selection often have large insertions of more or less repetitive amino acid sequence motifs. Conclusions/Significance We found positive selection of the clpP1 gene in various plant lineages to correlated with repeated duplication of the clpP1 gene and surrounding regions, repetitive amino acid sequences, and increase in synonymous substitution rates. The present study sheds light on the controversial issue of whether negative or positive selection is to be expected after gene duplications by providing evidence for the latter alternative. The observed increase in synonymous substitution rates in some of the lineages indicates that the detection of positive selection may be obscured under such circumstances. Future studies are required to explore the functional significance of the large inserted repeated amino acid motifs, as well as the possibility that synonymous substitution rates may be affected by positive selection. PMID:18167545

OptSSeq: High-throughput sequencing readout of growth enrichment defines optimal gene expression elements for homoethanologenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghosh, Indro Neil; Landick, Robert

The optimization of synthetic pathways is a central challenge in metabolic engineering. OptSSeq (Optimization by Selection and Sequencing) is one approach to this challenge. OptSSeq couples selection of optimal enzyme expression levels linked to cell growth rate with high-throughput sequencing to track enrichment of gene expression elements (promoters and ribosomebinding sites) from a combinatorial library. OptSSeq yields information on both optimal and suboptimal enzyme levels, and helps identify constraints that limit maximal product formation. Here we report a proof-of-concept implementation of OptSSeq using homoethanologenesis, a two-step pathway consisting of pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh) that converts pyruvate tomore » ethanol and is naturally optimized in the bacterium Zymomonas mobilis. We used OptSSeq to determine optimal gene expression elements and enzyme levels for Z. mobilis Pdc, AdhA, and AdhB expressed in Escherichia coli. By varying both expression signals and gene order, we identified an optimal solution using only Pdc and AdhB. We resolved current uncertainty about the functions of the Fe 2+-dependent AdhB and Zn 2+- dependent AdhA by showing that AdhB is preferred over AdhA for rapid growth in both E. coli and Z. mobilis. Finally, by comparing predictions of growth-linked metabolic flux to enzyme synthesis costs, we established that optimal E. coli homoethanologenesis was achieved by our best pdc-adhB expression cassette and that the remaining constraints lie in the E. coli metabolic network or inefficient Pdc or AdhB function in E. coli. Furthermore, OptSSeq is a general tool for synthetic biology to tune enzyme levels in any pathway whose optimal function can be linked to cell growth or survival.« less

Selective amplification of an mRNA and related pseudogene for a human ADP-ribosylation factor, a guanine nucleotide-dependent protein activator of cholera toxin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Monaco, L.; Murtagh, J.J.; Newman, K.B.

1990-03-01

ADP-ribosylation factors (ARFs) are {approx}20-kDa proteins that act as GTP-dependent allosteric activators of cholera toxin. With deoxyinosine-containing degenerate oligonucleotide primers corresponding to conserved GTP-binding domains in ARFs, the polymerase chain reaction (PCR) was used to amplify simultaneously from human DNA portions of three ARF genes that include codons for 102 amino acids, with intervening sequences. Amplification products that differed in size because of differences in intron sizes were separated by agarose gel electrophoresis. One amplified DNA contained no introns and had a sequence different from those of known AFRs. Based on this sequence, selective oligonucleotide probes were prepared and usedmore » to isolate clone {Psi}ARF 4, a putative ARF pseudogene, from a human genomic library in {lambda} phage EMBL3. Reverse transcription-PCR was then used to clone from human poly(A){sup +} RNA the cDNA corresponding to the expressed homolog of {Psi}ARF 4, referred to as human ARF 4. It appears that {Psi}ARF 4 arose during human evolution by integration of processed ARF 4 mRNA into the genome. Human ARF 4 differs from previously identified mammalian ARFs 1, 2, and 3. Hybridization of ARF 4-specific oligonucleotide probes with human, bovine, and rat RNA revealed a single 1.8-kilobase mRNA, which was clearly distinguished from the 1.9-kilobase mRNA for ARF 1 in these tissues. The PCR provides a powerful tool for investigating diversity in this and other multigene families, especially with primers targeted at domains believed to have functional significance.« less

OptSSeq: High-throughput sequencing readout of growth enrichment defines optimal gene expression elements for homoethanologenesis

DOE PAGES

Ghosh, Indro Neil; Landick, Robert

2016-07-16

The optimization of synthetic pathways is a central challenge in metabolic engineering. OptSSeq (Optimization by Selection and Sequencing) is one approach to this challenge. OptSSeq couples selection of optimal enzyme expression levels linked to cell growth rate with high-throughput sequencing to track enrichment of gene expression elements (promoters and ribosomebinding sites) from a combinatorial library. OptSSeq yields information on both optimal and suboptimal enzyme levels, and helps identify constraints that limit maximal product formation. Here we report a proof-of-concept implementation of OptSSeq using homoethanologenesis, a two-step pathway consisting of pyruvate decarboxylase (Pdc) and alcohol dehydrogenase (Adh) that converts pyruvate tomore » ethanol and is naturally optimized in the bacterium Zymomonas mobilis. We used OptSSeq to determine optimal gene expression elements and enzyme levels for Z. mobilis Pdc, AdhA, and AdhB expressed in Escherichia coli. By varying both expression signals and gene order, we identified an optimal solution using only Pdc and AdhB. We resolved current uncertainty about the functions of the Fe 2+-dependent AdhB and Zn 2+- dependent AdhA by showing that AdhB is preferred over AdhA for rapid growth in both E. coli and Z. mobilis. Finally, by comparing predictions of growth-linked metabolic flux to enzyme synthesis costs, we established that optimal E. coli homoethanologenesis was achieved by our best pdc-adhB expression cassette and that the remaining constraints lie in the E. coli metabolic network or inefficient Pdc or AdhB function in E. coli. Furthermore, OptSSeq is a general tool for synthetic biology to tune enzyme levels in any pathway whose optimal function can be linked to cell growth or survival.« less

Construction of random sheared fosmid library from Chinese cabbage and its use for Brassica rapa genome sequencing project.

PubMed

Park, Tae-Ho; Park, Beom-Seok; Kim, Jin-A; Hong, Joon Ki; Jin, Mina; Seol, Young-Joo; Mun, Jeong-Hwan

2011-01-01

As a part of the Multinational Genome Sequencing Project of Brassica rapa, linkage group R9 and R3 were sequenced using a bacterial artificial chromosome (BAC) by BAC strategy. The current physical contigs are expected to cover approximately 90% euchromatins of both chromosomes. As the project progresses, BAC selection for sequence extension becomes more limited because BAC libraries are restriction enzyme-specific. To support the project, a random sheared fosmid library was constructed. The library consists of 97536 clones with average insert size of approximately 40 kb corresponding to seven genome equivalents, assuming a Chinese cabbage genome size of 550 Mb. The library was screened with primers designed at the end of sequences of nine points of scaffold gaps where BAC clones cannot be selected to extend the physical contigs. The selected positive clones were end-sequenced to check the overlap between the fosmid clones and the adjacent BAC clones. Nine fosmid clones were selected and fully sequenced. The sequences revealed two completed gap filling and seven sequence extensions, which can be used for further selection of BAC clones confirming that the fosmid library will facilitate the sequence completion of B. rapa. Copyright © 2011. Published by Elsevier Ltd.

OrthoSelect: a protocol for selecting orthologous groups in phylogenomics.

PubMed

Schreiber, Fabian; Pick, Kerstin; Erpenbeck, Dirk; Wörheide, Gert; Morgenstern, Burkhard

2009-07-16

Phylogenetic studies using expressed sequence tags (EST) are becoming a standard approach to answer evolutionary questions. Such studies are usually based on large sets of newly generated, unannotated, and error-prone EST sequences from different species. A first crucial step in EST-based phylogeny reconstruction is to identify groups of orthologous sequences. From these data sets, appropriate target genes are selected, and redundant sequences are eliminated to obtain suitable sequence sets as input data for tree-reconstruction software. Generating such data sets manually can be very time consuming. Thus, software tools are needed that carry out these steps automatically. We developed a flexible and user-friendly software pipeline, running on desktop machines or computer clusters, that constructs data sets for phylogenomic analyses. It automatically searches assembled EST sequences against databases of orthologous groups (OG), assigns ESTs to these predefined OGs, translates the sequences into proteins, eliminates redundant sequences assigned to the same OG, creates multiple sequence alignments of identified orthologous sequences and offers the possibility to further process this alignment in a last step by excluding potentially homoplastic sites and selecting sufficiently conserved parts. Our software pipeline can be used as it is, but it can also be adapted by integrating additional external programs. This makes the pipeline useful for non-bioinformaticians as well as to bioinformatic experts. The software pipeline is especially designed for ESTs, but it can also handle protein sequences. OrthoSelect is a tool that produces orthologous gene alignments from assembled ESTs. Our tests show that OrthoSelect detects orthologs in EST libraries with high accuracy. In the absence of a gold standard for orthology prediction, we compared predictions by OrthoSelect to a manually created and published phylogenomic data set. Our tool was not only able to rebuild the data set with a specificity of 98%, but it detected four percent more orthologous sequences. Furthermore, the results OrthoSelect produces are in absolut agreement with the results of other programs, but our tool offers a significant speedup and additional functionality, e.g. handling of ESTs, computing sequence alignments, and refining them. To our knowledge, there is currently no fully automated and freely available tool for this purpose. Thus, OrthoSelect is a valuable tool for researchers in the field of phylogenomics who deal with large quantities of EST sequences. OrthoSelect is written in Perl and runs on Linux/Mac OS X. The tool can be downloaded at (http://gobics.de/fabian/orthoselect.php).

SamSelect: a sample sequence selection algorithm for quorum planted motif search on large DNA datasets.

PubMed

Yu, Qiang; Wei, Dingbang; Huo, Hongwei

2018-06-18

Given a set of t n-length DNA sequences, q satisfying 0 < q ≤ 1, and l and d satisfying 0 ≤ d < l < n, the quorum planted motif search (qPMS) finds l-length strings that occur in at least qt input sequences with up to d mismatches and is mainly used to locate transcription factor binding sites in DNA sequences. Existing qPMS algorithms have been able to efficiently process small standard datasets (e.g., t = 20 and n = 600), but they are too time consuming to process large DNA datasets, such as ChIP-seq datasets that contain thousands of sequences or more. We analyze the effects of t and q on the time performance of qPMS algorithms and find that a large t or a small q causes a longer computation time. Based on this information, we improve the time performance of existing qPMS algorithms by selecting a sample sequence set D' with a small t and a large q from the large input dataset D and then executing qPMS algorithms on D'. A sample sequence selection algorithm named SamSelect is proposed. The experimental results on both simulated and real data show (1) that SamSelect can select D' efficiently and (2) that the qPMS algorithms executed on D' can find implanted or real motifs in a significantly shorter time than when executed on D. We improve the ability of existing qPMS algorithms to process large DNA datasets from the perspective of selecting high-quality sample sequence sets so that the qPMS algorithms can find motifs in a short time in the selected sample sequence set D', rather than take an unfeasibly long time to search the original sequence set D. Our motif discovery method is an approximate algorithm.

Intelligent fault diagnosis of rolling bearings using an improved deep recurrent neural network

NASA Astrophysics Data System (ADS)

Jiang, Hongkai; Li, Xingqiu; Shao, Haidong; Zhao, Ke

2018-06-01

Traditional intelligent fault diagnosis methods for rolling bearings heavily depend on manual feature extraction and feature selection. For this purpose, an intelligent deep learning method, named the improved deep recurrent neural network (DRNN), is proposed in this paper. Firstly, frequency spectrum sequences are used as inputs to reduce the input size and ensure good robustness. Secondly, DRNN is constructed by the stacks of the recurrent hidden layer to automatically extract the features from the input spectrum sequences. Thirdly, an adaptive learning rate is adopted to improve the training performance of the constructed DRNN. The proposed method is verified with experimental rolling bearing data, and the results confirm that the proposed method is more effective than traditional intelligent fault diagnosis methods.

Spatial structure of disordered proteins dictates conductance and selectivity in nuclear pore complex mimics

PubMed Central

Frey, Steffen; Dwarkasing, Arvind; Versloot, Roderick; van der Giessen, Erik

2018-01-01

Nuclear pore complexes (NPCs) lined with intrinsically disordered FG-domains act as selective gatekeepers for molecular transport between the nucleus and the cytoplasm in eukaryotic cells. The underlying physical mechanism of the intriguing selectivity is still under debate. Here, we probe the transport of ions and transport receptors through biomimetic NPCs consisting of Nsp1 domains attached to the inner surface of solid-state nanopores. We examine both wildtype FG-domains and hydrophilic SG-mutants. FG-nanopores showed a clear selectivity as transport receptors can translocate across the pore whereas other proteins cannot. SG mutant pores lack such selectivity. To unravel this striking difference, we present coarse-grained molecular dynamics simulations that reveal that FG-pores exhibit a high-density, nonuniform protein distribution, in contrast to a uniform and significantly less-dense protein distribution in the SG-mutant. We conclude that the sequence-dependent density distribution of disordered proteins inside the NPC plays a key role for its conductivity and selective permeability. PMID:29442997

Sequence Learning and Selection Difficulty

ERIC Educational Resources Information Center

Rowland, Lee A.; Shanks, David R.

2006-01-01

The authors studied the role of attention as a selection mechanism in implicit learning by examining the effect on primary sequence learning of performing a demanding target-selection task. Participants were trained on probabilistic sequences in a novel version of the serial reaction time (SRT) task, with dual- and triple-stimulus participants…

Artificial selection increased body weight but induced increase of runs of homozygosity in Hanwoo cattle

PubMed Central

Kim, Kwondo; Jung, Jaehoon; Caetano-Anollés, Kelsey; Sung, Samsun; Yoo, DongAhn; Choi, Bong-Hwan; Kim, Hyung-Chul; Jeong, Jin-Young; Cho, Yong-Min; Park, Eung-Woo; Choi, Tae-Jeong; Park, Byoungho; Lim, Dajeong

2018-01-01

Artificial selection has been demonstrated to have a rapid and significant effect on the phenotype and genome of an organism. However, most previous studies on artificial selection have focused solely on genomic sequences modified by artificial selection or genomic sequences associated with a specific trait. In this study, we generated whole genome sequencing data of 126 cattle under artificial selection, and 24,973,862 single nucleotide variants to investigate the relationship among artificial selection, genomic sequences and trait. Using runs of homozygosity detected by the variants, we showed increase of inbreeding for decades, and at the same time demonstrated a little influence of recent inbreeding on body weight. Also, we could identify ~0.2 Mb runs of homozygosity segment which may be created by recent artificial selection. This approach may aid in development of genetic markers directly influenced by artificial selection, and provide insight into the process of artificial selection. PMID:29561881

DNA sequence-dependent mechanics and protein-assisted bending in repressor-mediated loop formation

PubMed Central

Boedicker, James Q.; Garcia, Hernan G.; Johnson, Stephanie; Phillips, Rob

2014-01-01

As the chief informational molecule of life, DNA is subject to extensive physical manipulations. The energy required to deform double-helical DNA depends on sequence, and this mechanical code of DNA influences gene regulation, such as through nucleosome positioning. Here we examine the sequence-dependent flexibility of DNA in bacterial transcription factor-mediated looping, a context for which the role of sequence remains poorly understood. Using a suite of synthetic constructs repressed by the Lac repressor and two well-known sequences that show large flexibility differences in vitro, we make precise statistical mechanical predictions as to how DNA sequence influences loop formation and test these predictions using in vivo transcription and in vitro single-molecule assays. Surprisingly, sequence-dependent flexibility does not affect in vivo gene regulation. By theoretically and experimentally quantifying the relative contributions of sequence and the DNA-bending protein HU to DNA mechanical properties, we reveal that bending by HU dominates DNA mechanics and masks intrinsic sequence-dependent flexibility. Such a quantitative understanding of how mechanical regulatory information is encoded in the genome will be a key step towards a predictive understanding of gene regulation at single-base pair resolution. PMID:24231252

Improvement and efficient display of Bacillus thuringiensis toxins on M13 phages and ribosomes.

PubMed

Pacheco, Sabino; Cantón, Emiliano; Zuñiga-Navarrete, Fernando; Pecorari, Frédéric; Bravo, Alejandra; Soberón, Mario

2015-12-01

Bacillus thuringiensis (Bt) produces insecticidal proteins that have been used worldwide in the control of insect-pests in crops and vectors of human diseases. However, different insect species are poorly controlled by the available Bt toxins or have evolved resistance to these toxins. Evolution of Bt toxicity could provide novel toxins to control insect pests. To this aim, efficient display systems to select toxins with increased binding to insect membranes or midgut proteins involved in toxicity are likely to be helpful. Here we describe two display systems, phage display and ribosome display, that allow the efficient display of two non-structurally related Bt toxins, Cry1Ac and Cyt1Aa. Improved display of Cry1Ac and Cyt1Aa on M13 phages was achieved by changing the commonly used peptide leader sequence of the coat pIII-fusion protein, that relies on the Sec translocation pathway, for a peptide leader sequence that relies on the signal recognition particle pathway (SRP) and by using a modified M13 helper phage (Phaberge) that has an amber mutation in its pIII genomic sequence and preferentially assembles using the pIII-fusion protein. Also, both Cry1Ac and Cyt1Aa were efficiently displayed on ribosomes, which could allow the construction of large libraries of variants. Furthermore, Cry1Ac or Cyt1Aa displayed on M13 phages or ribosomes were specifically selected from a mixture of both toxins depending on which antigen was immobilized for binding selection. These improved systems may allow the selection of Cry toxin variants with improved insecticidal activities that could counter insect resistances.

Morphological transformations of diblock copolymers in binary solvents: A simulation study

NASA Astrophysics Data System (ADS)

Wang, Zheng; Yin, Yuhua; Jiang, Run; Li, Baohui

2017-12-01

Morphological transformations of amphiphilic AB diblock copolymers in mixtures of a common solvent (S1) and a selective solvent (S2) for the B block are studied using the simulated annealing method. We focus on the morphological transformation depending on the fraction of the selective solvent C S2, the concentration of the polymer C p , and the polymer-solvent interactions ɛ ij ( i = A, B; j = S1, S2). Morphology diagrams are constructed as functions of C p , C S2, and/or ɛ AS2. The copolymer morphological sequence from dissolved → sphere → rod → ring/cage → vesicle is obtained upon increasing C S2 at a fixed C p . This morphology sequence is consistent with previous experimental observations. It is found that the selectivity of the selective solvent affects the self-assembled microstructure significantly. In particular, when the interaction ɛ BS2 is negative, aggregates of stacked lamellae dominate the diagram. The mechanisms of aggregate transformation and the formation of stacked lamellar aggregates are discussed by analyzing variations of the average contact numbers of the A or B monomers with monomers and with molecules of the two types of solvent, as well as the mean square end-to-end distances of chains. It is found that the basic morphological sequence of spheres to rods to vesicles and the stacked lamellar aggregates result from competition between the interfacial energy and the chain conformational entropy. Analysis of the vesicle structure reveals that the vesicle size increases with increasing C p or with decreasing C S2, but remains almost unchanged with variations in ɛ AS2.

Alcohol and aldehyde dehydrogenase polymorphisms in Chinese and Indian populations.

PubMed

Tan, Ene-Choo; Lim, Leslie; Leong, Jern-Yi; Lim, Jing-Yan; Lee, Arthur; Yang, Jun; Tan, Chay-Hoon; Winslow, Munidasa

2010-01-01

The association between two functional polymorphisms in alcohol dehydrogenase (ADH2/ADH1B) and aldehyde dehydrogenase (ALDH2) genes and alcohol dependence was examined in 182 Chinese and Indian patients undergoing treatment for alcohol dependence and 184 screened control subjects from Singapore. All subjects were screened by the Alcohol Use Disorders Identification Test (AUDIT). Patients were also administered the Severity of Alcohol Dependence Questionnaire (SADQ). Polymorphisms were genotyped by allele-specific polymerase chain reaction and selected genotypes confirmed by DNA sequencing or restriction fragment length polymorphism. Our results showed that frequencies of ADH1B*2 and ALDH2*2 were higher in controls compared to alcohol-dependent subjects for both Chinese and Indians. Frequencies of these two alleles were also higher in the 104 Chinese controls compared to the 80 Indian controls. None of the eight Chinese who were homozygous for both protective alleles was alcohol dependent. The higher frequencies of the protective alleles could explain the lower rate of alcohol dependence in Chinese.

Design of a fast echo matching algorithm to reduce crosstalk with Doppler shifts in ultrasonic ranging

NASA Astrophysics Data System (ADS)

Liu, Lei; Guo, Rui; Wu, Jun-an

2017-02-01

Crosstalk is a main factor for wrong distance measurement by ultrasonic sensors, and this problem becomes more difficult to deal with under Doppler effects. In this paper, crosstalk reduction with Doppler shifts on small platforms is focused on, and a fast echo matching algorithm (FEMA) is proposed on the basis of chaotic sequences and pulse coding technology, then verified through applying it to match practical echoes. Finally, we introduce how to select both better mapping methods for chaotic sequences, and algorithm parameters for higher achievable maximum of cross-correlation peaks. The results indicate the following: logistic mapping is preferred to generate good chaotic sequences, with high autocorrelation even when the length is very limited; FEMA can not only match echoes and calculate distance accurately with an error degree mostly below 5%, but also generates nearly the same calculation cost level for static or kinematic ranging, much lower than that by direct Doppler compensation (DDC) with the same frequency compensation step; The sensitivity to threshold value selection and performance of FEMA depend significantly on the achievable maximum of cross-correlation peaks, and a higher peak is preferred, which can be considered as a criterion for algorithm parameter optimization under practical conditions.

Wi-Fi location fingerprinting using an intelligent checkpoint sequence

NASA Astrophysics Data System (ADS)

Retscher, Günther; Hofer, Hannes

2017-09-01

For Wi-Fi positioning location fingerprinting is very common but has the disadvantage that it is very labour consuming for the establishment of a database (DB) with received signal strength (RSS) scans measured on a large number of known reference points (RPs). To overcome this drawback a novel approach is developed which uses a logical sequence of intelligent checkpoints (iCPs) instead of RPs distributed in a regular grid. The iCPs are the selected RPs which have to be passed along the way for navigation from a start point A to the destination B. They are twofold intelligent because of the fact that they depend on their meaningful selection and because of their logical sequence in their correct order. Thus, always the following iCP is known due to a vector graph allocation in the DB and only a small limited number of iCPs needs to be tested when matching the current RSS scans. This reduces the required processing time significantly. It is proven that the iCP approach achieves a higher success rate than conventional approaches. In average correct matching results of 90.0% were achieved using a joint DB including RSS scans of all employed smartphones. An even higher success rate is achieved if the same mobile device is used in both the training and positioning phase.

Selecting sequence variants to improve genomic predictions for dairy cattle

USDA-ARS?s Scientific Manuscript database

Millions of genetic variants have been identified by population-scale sequencing projects, but subsets are needed for routine genomic predictions or to include on genotyping arrays. Methods of selecting sequence variants were compared using both simulated sequence genotypes and actual data from run ...

Locating Sequence on FPC Maps and Selecting a Minimal Tiling Path

PubMed Central

Engler, Friedrich W.; Hatfield, James; Nelson, William; Soderlund, Carol A.

2003-01-01

This study discusses three software tools, the first two aid in integrating sequence with an FPC physical map and the third automatically selects a minimal tiling path given genomic draft sequence and BAC end sequences. The first tool, FSD (FPC Simulated Digest), takes a sequenced clone and adds it back to the map based on a fingerprint generated by an in silico digest of the clone. This allows verification of sequenced clone positions and the integration of sequenced clones that were not originally part of the FPC map. The second tool, BSS (Blast Some Sequence), takes a query sequence and positions it on the map based on sequence associated with the clones in the map. BSS has multiple uses as follows: (1) When the query is a file of marker sequences, they can be added as electronic markers. (2) When the query is draft sequence, the results of BSS can be used to close gaps in a sequenced clone or the physical map. (3) When the query is a sequenced clone and the target is BAC end sequences, one may select the next clone for sequencing using both sequence comparison results and map location. (4) When the query is whole-genome draft sequence and the target is BAC end sequences, the results can be used to select many clones for a minimal tiling path at once. The third tool, pickMTP, automates the majority of this last usage of BSS. Results are presented using the rice FPC map, BAC end sequences, and whole-genome shotgun from Syngenta. PMID:12915486

Prospective identification of parasitic sequences in phage display screens

PubMed Central

Matochko, Wadim L.; Cory Li, S.; Tang, Sindy K.Y.; Derda, Ratmir

2014-01-01

Phage display empowered the development of proteins with new function and ligands for clinically relevant targets. In this report, we use next-generation sequencing to analyze phage-displayed libraries and uncover a strong bias induced by amplification preferences of phage in bacteria. This bias favors fast-growing sequences that collectively constitute <0.01% of the available diversity. Specifically, a library of 109 random 7-mer peptides (Ph.D.-7) includes a few thousand sequences that grow quickly (the ‘parasites’), which are the sequences that are typically identified in phage display screens published to date. A similar collapse was observed in other libraries. Using Illumina and Ion Torrent sequencing and multiple biological replicates of amplification of Ph.D.-7 library, we identified a focused population of 770 ‘parasites’. In all, 197 sequences from this population have been identified in literature reports that used Ph.D.-7 library. Many of these enriched sequences have confirmed function (e.g. target binding capacity). The bias in the literature, thus, can be viewed as a selection with two different selection pressures: (i) target-binding selection, and (ii) amplification-induced selection. Enrichment of parasitic sequences could be minimized if amplification bias is removed. Here, we demonstrate that emulsion amplification in libraries of ∼106 diverse clones prevents the biased selection of parasitic clones. PMID:24217917

Reconstruction of DNA sequences using genetic algorithms and cellular automata: towards mutation prediction?

PubMed

Mizas, Ch; Sirakoulis, G Ch; Mardiris, V; Karafyllidis, I; Glykos, N; Sandaltzopoulos, R

2008-04-01

Change of DNA sequence that fuels evolution is, to a certain extent, a deterministic process because mutagenesis does not occur in an absolutely random manner. So far, it has not been possible to decipher the rules that govern DNA sequence evolution due to the extreme complexity of the entire process. In our attempt to approach this issue we focus solely on the mechanisms of mutagenesis and deliberately disregard the role of natural selection. Hence, in this analysis, evolution refers to the accumulation of genetic alterations that originate from mutations and are transmitted through generations without being subjected to natural selection. We have developed a software tool that allows modelling of a DNA sequence as a one-dimensional cellular automaton (CA) with four states per cell which correspond to the four DNA bases, i.e. A, C, T and G. The four states are represented by numbers of the quaternary number system. Moreover, we have developed genetic algorithms (GAs) in order to determine the rules of CA evolution that simulate the DNA evolution process. Linear evolution rules were considered and square matrices were used to represent them. If DNA sequences of different evolution steps are available, our approach allows the determination of the underlying evolution rule(s). Conversely, once the evolution rules are deciphered, our tool may reconstruct the DNA sequence in any previous evolution step for which the exact sequence information was unknown. The developed tool may be used to test various parameters that could influence evolution. We describe a paradigm relying on the assumption that mutagenesis is governed by a near-neighbour-dependent mechanism. Based on the satisfactory performance of our system in the deliberately simplified example, we propose that our approach could offer a starting point for future attempts to understand the mechanisms that govern evolution. The developed software is open-source and has a user-friendly graphical input interface.

A statistical evaluation of effective time constants of random telegraph noise with various operation timings of in-pixel source follower transistors

NASA Astrophysics Data System (ADS)

Yonezawa, A.; Kuroda, R.; Teramoto, A.; Obara, T.; Sugawa, S.

2014-03-01

We evaluated effective time constants of random telegraph noise (RTN) with various operation timings of in-pixel source follower transistors statistically, and discuss the dependency of RTN time constants on the duty ratio (on/off ratio) of MOSFET which is controlled by the gate to source voltage (VGS). Under a general readout operation of CMOS image sensor (CIS), the row selected pixel-source followers (SFs) turn on and not selected pixel-SFs operate at different bias conditions depending on the select switch position; when select switch locate in between the SF driver and column output line, SF drivers nearly turn off. The duty ratio and cyclic period of selected time of SF driver depends on the operation timing determined by the column read out sequence. By changing the duty ratio from 1 to 7.6 x 10-3, time constant ratio of RTN (time to capture <τc<)/(time to emission <τe<) of a part of MOSFETs increased while RTN amplitudes were almost the same regardless of the duty ratio. In these MOSFETs, <τc< increased and the majority of <τe< decreased and the minority of <τe< increased by decreasing the duty ratio. The same tendencies of behaviors of <τc< and <τe< were obtained when VGS was decreased. This indicates that the effective <τc< and <τe< converge to those under off state as duty ratio decreases. These results are important for the noise reduction, detection and analysis of in pixel-SF with RTN.

Accuracy of the high-throughput amplicon sequencing to identify species within the genus Aspergillus.

PubMed

Lee, Seungeun; Yamamoto, Naomichi

2015-12-01

This study characterized the accuracy of high-throughput amplicon sequencing to identify species within the genus Aspergillus. To this end, we sequenced the internal transcribed spacer 1 (ITS1), β-tubulin (BenA), and calmodulin (CaM) gene encoding sequences as DNA markers from eight reference Aspergillus strains with known identities using 300-bp sequencing on the Illumina MiSeq platform, and compared them with the BLASTn outputs. The identifications with the sequences longer than 250 bp were accurate at the section rank, with some ambiguities observed at the species rank due to mostly cross detection of sibling species. Additionally, in silico analysis was performed to predict the identification accuracy for all species in the genus Aspergillus, where 107, 210, and 187 species were predicted to be identifiable down to the species rank based on ITS1, BenA, and CaM, respectively. Finally, air filter samples were analysed to quantify the relative abundances of Aspergillus species in outdoor air. The results were reproducible across biological duplicates both at the species and section ranks, but not strongly correlated between ITS1 and BenA, suggesting the Aspergillus detection can be taxonomically biased depending on the selection of the DNA markers and/or primers. Copyright © 2015 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Complete genome sequence of a Chinese isolate of pepper vein yellows virus and evolutionary analysis based on the CP, MP and RdRp coding regions.

PubMed

Liu, Maoyan; Liu, Xiangning; Li, Xun; Zhang, Deyong; Dai, Liangyin; Tang, Qianjun

2016-03-01

The genome sequence of pepper vein yellows virus (PeVYV) (PeVYV-HN, accession number KP326573), isolated from pepper plants (Capsicum annuum L.) grown at the Hunan Vegetables Institute (Changsha, Hunan, China), was determined by deep sequencing of small RNAs. The PeVYV-HN genome consists of 6244 nucleotides, contains six open reading frames (ORFs), and is similar to that of an isolate (AB594828) from Japan. Its genomic organization is similar to that of members of the genus Polerovirus. Sequence analysis revealed that PeVYV-HN shared 92% sequence identity with the Japanese PeVYV genome at both the nucleotide and amino acid levels. Evolutionary analysis based on the coat protein (CP), movement protein (MP), and RNA-dependent RNA polymerase (RdRP) showed that PeVYV could be divided into two major lineages corresponding to their geographical origins. The Asian isolates have a higher population expansion frequency than the African isolates. Negative selection and genetic drift (founder effect) were found to be the potential drivers of the molecular evolution of PeVYV. Moreover, recombination was not the distinct cause of PeVYV evolution. This is the first report of a complete genomic sequence of PeVYV in China.

Experimental and Theoretical Studies on the Nazarov Cyclization/Wagner-Meerwein Rearrangement Sequence

PubMed Central

Lebœuf, David; Ciesielski, Jennifer

2012-01-01

Highly functionalized cyclopentenones can be generated stereospecifically by a chemoselective copper(II)-mediated Nazarov/Wagner-Meerwein rearrangement sequence of divinyl ketones. A detailed investigation of this sequence is described including a study of substrate scope and limitations. After the initial 4π electrocyclization, this reaction proceeds via two different sequential [1,2]-shifts, with selectivity that depends upon either migratory ability or the steric bulkiness of the substituents at C1 and C5. This methodology allows the creation of vicinal stereogenic centers, including adjacent quaternary centers. This sequence can also be achieved by using a catalytic amount of copper(II) in combination with NaBAr4f, a weak Lewis acid. During the study of the scope of the reaction, a partial or complete E / Z isomerization of the enone moiety was observed in some cases prior to the cyclization, which resulted in a mixture of diastereomeric products. Use of a Cu(II)-bisoxazoline complex prevented the isomerization, allowing high diastereoselectivity to be obtained in all substrate types. In addition, the reaction sequence was studied by DFT computations at the UB3LYP/6-31G(d,p) level, which are consistent with the proposed sequences observed, including E / Z isomerizations and chemoselective Wagner-Meerwein shifts. PMID:22471833

G-Quadruplex Induction by the Hairpin Pyrrole-Imidazole Polyamide Dimer.

PubMed

Obata, Shunsuke; Asamitsu, Sefan; Hashiya, Kaori; Bando, Toshikazu; Sugiyama, Hiroshi

2018-02-06

The G-quadruplex (G4) is one type of higher-order structure of nucleic acids and is thought to play important roles in various biological events such as regulation of transcription and inhibition of DNA replication. Pyrrole-imidazole polyamides (PIPs) are programmable small molecules that can sequence-specifically bind with high affinity to the minor groove of double-stranded DNA (dsDNA). Herein, we designed head-to-head hairpin PIP dimers and their target dsDNA in a model G4-forming sequence. Using an electrophoresis mobility shift assay and transcription arrest assay, we found that PIP dimers could induce the structural change to G4 DNA from dsDNA through the recognition by one PIP dimer molecule of two duplex-binding sites flanking both ends of the G4-forming sequence. This induction ability was dependent on linker length. This is the first study to induce G4 formation using PIPs, which are known to be dsDNA binders. The results reported here suggest that selective G4 induction in native sequences may be achieved with PIP dimers by applying the same design strategy.

Association mining of dependency between time series

NASA Astrophysics Data System (ADS)

Hafez, Alaaeldin

2001-03-01

Time series analysis is considered as a crucial component of strategic control over a broad variety of disciplines in business, science and engineering. Time series data is a sequence of observations collected over intervals of time. Each time series describes a phenomenon as a function of time. Analysis on time series data includes discovering trends (or patterns) in a time series sequence. In the last few years, data mining has emerged and been recognized as a new technology for data analysis. Data Mining is the process of discovering potentially valuable patterns, associations, trends, sequences and dependencies in data. Data mining techniques can discover information that many traditional business analysis and statistical techniques fail to deliver. In this paper, we adapt and innovate data mining techniques to analyze time series data. By using data mining techniques, maximal frequent patterns are discovered and used in predicting future sequences or trends, where trends describe the behavior of a sequence. In order to include different types of time series (e.g. irregular and non- systematic), we consider past frequent patterns of the same time sequences (local patterns) and of other dependent time sequences (global patterns). We use the word 'dependent' instead of the word 'similar' for emphasis on real life time series where two time series sequences could be completely different (in values, shapes, etc.), but they still react to the same conditions in a dependent way. In this paper, we propose the Dependence Mining Technique that could be used in predicting time series sequences. The proposed technique consists of three phases: (a) for all time series sequences, generate their trend sequences, (b) discover maximal frequent trend patterns, generate pattern vectors (to keep information of frequent trend patterns), use trend pattern vectors to predict future time series sequences.

A general framework for optimization of probes for gene expression microarray and its application to the fungus Podospora anserina.

PubMed

Bidard, Frédérique; Imbeaud, Sandrine; Reymond, Nancie; Lespinet, Olivier; Silar, Philippe; Clavé, Corinne; Delacroix, Hervé; Berteaux-Lecellier, Véronique; Debuchy, Robert

2010-06-18

The development of new microarray technologies makes custom long oligonucleotide arrays affordable for many experimental applications, notably gene expression analyses. Reliable results depend on probe design quality and selection. Probe design strategy should cope with the limited accuracy of de novo gene prediction programs, and annotation up-dating. We present a novel in silico procedure which addresses these issues and includes experimental screening, as an empirical approach is the best strategy to identify optimal probes in the in silico outcome. We used four criteria for in silico probe selection: cross-hybridization, hairpin stability, probe location relative to coding sequence end and intron position. This latter criterion is critical when exon-intron gene structure predictions for intron-rich genes are inaccurate. For each coding sequence (CDS), we selected a sub-set of four probes. These probes were included in a test microarray, which was used to evaluate the hybridization behavior of each probe. The best probe for each CDS was selected according to three experimental criteria: signal-to-noise ratio, signal reproducibility, and representative signal intensities. This procedure was applied for the development of a gene expression Agilent platform for the filamentous fungus Podospora anserina and the selection of a single 60-mer probe for each of the 10,556 P. anserina CDS. A reliable gene expression microarray version based on the Agilent 44K platform was developed with four spot replicates of each probe to increase statistical significance of analysis.

Affinity selection of Nipah and Hendra virus-related vaccine candidates from a complex random peptide library displayed on bacteriophage virus-like particles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Peabody, David S.; Chackerian, Bryce; Ashley, Carlee

The invention relates to virus-like particles of bacteriophage MS2 (MS2 VLPs) displaying peptide epitopes or peptide mimics of epitopes of Nipah Virus envelope glycoprotein that elicit an immune response against Nipah Virus upon vaccination of humans or animals. Affinity selection on Nipah Virus-neutralizing monoclonal antibodies using random sequence peptide libraries on MS2 VLPs selected peptides with sequence similarity to peptide sequences found within the envelope glycoprotein of Nipah itself, thus identifying the epitopes the antibodies recognize. The selected peptide sequences themselves are not necessarily identical in all respects to a sequence within Nipah Virus glycoprotein, and therefore may be referredmore » to as epitope mimics VLPs displaying these epitope mimics can serve as vaccine. On the other hand, display of the corresponding wild-type sequence derived from Nipah Virus and corresponding to the epitope mapped by affinity selection, may also be used as a vaccine.« less

A cost effective 5΄ selective single cell transcriptome profiling approach with improved UMI design

PubMed Central

Arguel, Marie-Jeanne; LeBrigand, Kevin; Paquet, Agnès; Ruiz García, Sandra; Zaragosi, Laure-Emmanuelle; Waldmann, Rainer

2017-01-01

Abstract Single cell RNA sequencing approaches are instrumental in studies of cell-to-cell variability. 5΄ selective transcriptome profiling approaches allow simultaneous definition of the transcription start size and have advantages over 3΄ selective approaches which just provide internal sequences close to the 3΄ end. The only currently existing 5΄ selective approach requires costly and labor intensive fragmentation and cell barcoding after cDNA amplification. We developed an optimized 5΄ selective workflow where all the cell indexing is done prior to fragmentation. With our protocol, cell indexing can be performed in the Fluidigm C1 microfluidic device, resulting in a significant reduction of cost and labor. We also designed optimized unique molecular identifiers that show less sequence bias and vulnerability towards sequencing errors resulting in an improved accuracy of molecule counting. We provide comprehensive experimental workflows for Illumina and Ion Proton sequencers that allow single cell sequencing in a cost range comparable to qPCR assays. PMID:27940562

SU-G-IeP1-08: MR Geometric Distortion Dependency On Imaging Sequence, Acquisition Orientation and Receiver Bandwidth of a Dedicated 1.5T MR-Simulator

DOE Office of Scientific and Technical Information (OSTI.GOV)

Law, M; Yuan, J; Wong, O

Purpose: To investigate the 3D geometric distortion of four potential MR sequences for radiotheraptic applications, and its dependency on sequence-type, acquisition-orientation and receiver-bandwidth from a dedicated 1.5T 700mm-wide bore MR-simulator (Magnetom-Aera, Sienmens Healthcare, Erlangen, Germany), using a large customized geometric accuracy phantom. Methods: This work studied 3D gradient-echo (VIBE) and spin-echo (SPACE) sequences for anatomical imaging; a specific ultra-short-TE sequence (PETRA) potentially for bone imaging and MR-based dosimetry; and a motion-insensitive sequence (BLADE) for dynamic applications like 4D-MRI. Integrated geometric-correction was employed, three orthogonal acquisition-orientations and up to three receiver-bandwidths were used, yielding 27 acquisitions for testing (Table 1a).A customizedmore » geometric accuracy phantom (polyurethane, MR/CT invisible, W×L×H:55×55×32.5cm3) was constructed and filled with 3892 spherical markers (6mm diameter, MR/CT visible) arranged on a 25mm-interval 3D isotropic-grid (Fig.1). The marker positions in MR images were quantitatively calculated and compared against those in the CT-reference using customized MatLab scripts. Results: The average distortion within various diameter-of-spherical-volumes (DSVs) and the usable DSVs under various distortion limits were measured (Tables 1b-c). It was observed that distortions fluctuated when sequence-type, acquisition-orientation or receiver-bandwidth changed (e.g. within 300mm-DSV, the lowest/highest average distortions of VIBE were 0.40mm/0.59mm, a 47.5% difference). According to AAPM-TG66 (<1mm distortion, left-most column of Table 1c), PETRA (Largest-DSV:253.9mm) has the potential on brain treatment, while BLADE (Largest-DSV:207.2mm) may need improvement for thoracic/abdominal applications. The results of VIBE (Largest-DSVs:294.3mm, the best among tested acquisitions) and SPACE (Largest-DSVs:267.7mm) suggests their potentials on head and neck applications. These Largest-DSVs were attained on different acquisition-orientations and receiver-bandwidths. Conclusion: Geometric distortion was shown to be dependent on sequence-type, acquisition-orientation and receiver-bandwidth. In the experiment, no configuration in any one of these factors could consistently reduce distortion while the others were varying. The distortion analysis result is a valuable guideline for sequence selection and optimization for MR-aided radiotherapy applications.« less

Fungal antagonists of the plant pathogen Rhizoctonia solani: selection, control efficacy and influence on the indigenous microbial community.

PubMed

Grosch, Rita; Scherwinski, Katja; Lottmann, Jana; Berg, Gabriele

2006-12-01

A broad spectrum of fungal antagonists was evaluated as potential biocontrol agents (BCAs) against the soil-borne pathogen Rhizoctonia solani using a new combination of in vitro and in vivo assays. The in vitro characterisation of diverse parameters including the ability to parasitise mycelium and to inhibit the germination of Rhizoctonia sclerotia at different temperatures resulted in the selection of six potential fungal antagonists. These were genotypically characterised by their BOX-PCR fingerprints, and identified as Trichoderma reesei and T. viride by partial 18S rDNA sequencing. When potato sprouts were treated with Trichoderma, all isolates significantly reduced the incidence of Rhizoctonia symptoms. Evaluated under growth chamber conditions, the selected Trichoderma isolates either partly or completely controlled the dry mass loss of lettuce caused by R. solani. Furthermore, the antagonistic Trichoderma strains were active under field conditions. To analyse the effect of Trichoderma treatment on indigenous root-associated microbial communities, we performed a DNA-dependent SSCP (Single-Strand Conformation Polymorphism) analysis of 16S rDNA/ITS sequences. In this first assessment study for Trichoderma it was shown that the pathogen and the vegetation time had much more influence on the composition of the microbiota than the BCA treatment. After evaluation of all results, three Trichoderma strains originally isolated from Rhizoctonia sclerotia were selected as promising BCAs.

Dynamic peptide libraries for the discovery of supramolecular nanomaterials

NASA Astrophysics Data System (ADS)

Pappas, Charalampos G.; Shafi, Ramim; Sasselli, Ivan R.; Siccardi, Henry; Wang, Tong; Narang, Vishal; Abzalimov, Rinat; Wijerathne, Nadeesha; Ulijn, Rein V.

2016-11-01

Sequence-specific polymers, such as oligonucleotides and peptides, can be used as building blocks for functional supramolecular nanomaterials. The design and selection of suitable self-assembling sequences is, however, challenging because of the vast combinatorial space available. Here we report a methodology that allows the peptide sequence space to be searched for self-assembling structures. In this approach, unprotected homo- and heterodipeptides (including aromatic, aliphatic, polar and charged amino acids) are subjected to continuous enzymatic condensation, hydrolysis and sequence exchange to create a dynamic combinatorial peptide library. The free-energy change associated with the assembly process itself gives rise to selective amplification of self-assembling candidates. By changing the environmental conditions during the selection process, different sequences and consequent nanoscale morphologies are selected.

Dynamic peptide libraries for the discovery of supramolecular nanomaterials.

PubMed

Pappas, Charalampos G; Shafi, Ramim; Sasselli, Ivan R; Siccardi, Henry; Wang, Tong; Narang, Vishal; Abzalimov, Rinat; Wijerathne, Nadeesha; Ulijn, Rein V

2016-11-01

Sequence-specific polymers, such as oligonucleotides and peptides, can be used as building blocks for functional supramolecular nanomaterials. The design and selection of suitable self-assembling sequences is, however, challenging because of the vast combinatorial space available. Here we report a methodology that allows the peptide sequence space to be searched for self-assembling structures. In this approach, unprotected homo- and heterodipeptides (including aromatic, aliphatic, polar and charged amino acids) are subjected to continuous enzymatic condensation, hydrolysis and sequence exchange to create a dynamic combinatorial peptide library. The free-energy change associated with the assembly process itself gives rise to selective amplification of self-assembling candidates. By changing the environmental conditions during the selection process, different sequences and consequent nanoscale morphologies are selected.

DHS Student Report

DOE Office of Scientific and Technical Information (OSTI.GOV)

. Wynne, E K

Throughout this project I have been involved in every step of the protocol. After proper training, I was introduced to the necessary lab techniques for the project. From then on it has been my responsibility to perform the necessary tasks to identify and isolate the mutants. This includes carrying out a detailed protocol of mixing reagents, streaking and incubating plates, inoculating cultures and evaluating any results in order to guide my actions for the next antibiotic concentration level. Simultaneously, I have been running PCR and sequencing reactions on all mutants in order to obtain the genetic sequence of the genesmore » of interest for comparison. Once I have the gene sequences of interest I am able, with the aid of a sequencing program (Sequencher 4.2.2), to analyze the sequences of the mutants against that of a wild type strain. This entails aligning the DNA sequences of a given gene for each of the mutants and locating any base changes from the wild types bacteria's genes. These polymorphisms allow me to identify the QRDR for that particular gene. Depending on whether the polymorphism occurred at a low antibiotic concentration level or high concentration level, we can evaluate whether that change is necessary for low or high-level quinolone resistance. Finally, I will compare the polymorphisms of each mutant at a given antibiotic selection level and evaluate whether B. anthracis consistently acquires resistance through the same polymorphisms or whether the resistance mechanism varies with each new mutant strain. Currently, I am analyzing the sequence data for stage one mutants, while simultaneously continuing the lab work necessary to select for stage two mutants. After I have left, the personnel at the lab that I've been working with at LLNL will continue this project. By the end of this experiment, we hope to corroborate the suggested mechanisms of resistance typically employed by B. anthracis Sterne at different resistance levels. Furthermore, if the mechanism is determined by one of the following genes: gyrA, gyrB, parC, parE we will be able to pinpoint which base pair changes are necessary for acquiring a given resistance level. Hopefully from these data researchers will be better able to determine an appropriate action should quinolone resistant strains of B. anthracis arise in either by natural evolution or selection in a laboratory.« less

Comparison of MR imaging sequences for liver and head and neck interventions: is there a single optimal sequence for all purposes?

PubMed

Boll, Daniel T; Lewin, Jonathan S; Duerk, Jeffrey L; Aschoff, Andrik J; Merkle, Elmar M

2004-05-01

To compare the appropriate pulse sequences for interventional device guidance during magnetic resonance (MR) imaging at 0.2 T and to evaluate the dependence of sequence selection on the anatomic region of the procedure. Using a C-arm 0.2 T system, four interventional MR sequences were applied in 23 liver cases and during MR-guided neck interventions in 13 patients. The imaging protocol consisted of: multislice turbo spin echo (TSE) T2w, sequential-slice fast imaging with steady precession (FISP), a time-reversed version of FISP (PSIF), and FISP with balanced gradients in all spatial directions (True-FISP) sequences. Vessel conspicuity was rated and contrast-to-noise ratio (CNR) was calculated for each sequence and a differential receiver operating characteristic was performed. Liver findings were detected in 96% using the TSE sequence. PSIF, FISP, and True-FISP imaging showed lesions in 91%, 61%, and 65%, respectively. The TSE sequence offered the best CNR, followed by PSIF imaging. Differential receiver operating characteristic analysis also rated TSE and PSIF to be the superior sequences. Lesions in the head and neck were detected in all cases by TSE and FISP, in 92% using True-FISP, and in 84% using PSIF. True-FISP offered the best CNR, followed by TSE imaging. Vessels appeared bright on FISP and True-FISP imaging and dark on the other sequences. In interventional MR imaging, no single sequence fits all purposes. Image guidance for interventional MR during liver procedures is best achieved by PSIF or TSE, whereas biopsies in the head and neck are best performed using FISP or True-FISP sequences.

Comparison of single cell sequencing data between two whole genome amplification methods on two sequencing platforms.

PubMed

Chen, DaYang; Zhen, HeFu; Qiu, Yong; Liu, Ping; Zeng, Peng; Xia, Jun; Shi, QianYu; Xie, Lin; Zhu, Zhu; Gao, Ya; Huang, GuoDong; Wang, Jian; Yang, HuanMing; Chen, Fang

2018-03-21

Research based on a strategy of single-cell low-coverage whole genome sequencing (SLWGS) has enabled better reproducibility and accuracy for detection of copy number variations (CNVs). The whole genome amplification (WGA) method and sequencing platform are critical factors for successful SLWGS (<0.1 × coverage). In this study, we compared single cell and multiple cells sequencing data produced by the HiSeq2000 and Ion Proton platforms using two WGA kits and then comprehensively evaluated the GC-bias, reproducibility, uniformity and CNV detection among different experimental combinations. Our analysis demonstrated that the PicoPLEX WGA Kit resulted in higher reproducibility, lower sequencing error frequency but more GC-bias than the GenomePlex Single Cell WGA Kit (WGA4 kit) independent of the cell number on the HiSeq2000 platform. While on the Ion Proton platform, the WGA4 kit (both single cell and multiple cells) had higher uniformity and less GC-bias but lower reproducibility than those of the PicoPLEX WGA Kit. Moreover, on these two sequencing platforms, depending on cell number, the performance of the two WGA kits was different for both sensitivity and specificity on CNV detection. The results can help researchers who plan to use SLWGS on single or multiple cells to select appropriate experimental conditions for their applications.

Chromosome specific repetitive DNA sequences

DOEpatents

Moyzis, Robert K.; Meyne, Julianne

1991-01-01

A method is provided for determining specific nucleotide sequences useful in forming a probe which can identify specific chromosomes, preferably through in situ hybridization within the cell itself. In one embodiment, chromosome preferential nucleotide sequences are first determined from a library of recombinant DNA clones having families of repetitive sequences. Library clones are identified with a low homology with a sequence of repetitive DNA families to which the first clones respectively belong and variant sequences are then identified by selecting clones having a pattern of hybridization with genomic DNA dissimilar to the hybridization pattern shown by the respective families. In another embodiment, variant sequences are selected from a sequence of a known repetitive DNA family. The selected variant sequence is classified as chromosome specific, chromosome preferential, or chromosome nonspecific. Sequences which are classified as chromosome preferential are further sequenced and regions are identified having a low homology with other regions of the chromosome preferential sequence or with known sequences of other family me This invention is the result of a contract with the Department of Energy (Contract No. W-7405-ENG-36).

Algorithm for Video Summarization of Bronchoscopy Procedures

PubMed Central

2011-01-01

Background The duration of bronchoscopy examinations varies considerably depending on the diagnostic and therapeutic procedures used. It can last more than 20 minutes if a complex diagnostic work-up is included. With wide access to videobronchoscopy, the whole procedure can be recorded as a video sequence. Common practice relies on an active attitude of the bronchoscopist who initiates the recording process and usually chooses to archive only selected views and sequences. However, it may be important to record the full bronchoscopy procedure as documentation when liability issues are at stake. Furthermore, an automatic recording of the whole procedure enables the bronchoscopist to focus solely on the performed procedures. Video recordings registered during bronchoscopies include a considerable number of frames of poor quality due to blurry or unfocused images. It seems that such frames are unavoidable due to the relatively tight endobronchial space, rapid movements of the respiratory tract due to breathing or coughing, and secretions which occur commonly in the bronchi, especially in patients suffering from pulmonary disorders. Methods The use of recorded bronchoscopy video sequences for diagnostic, reference and educational purposes could be considerably extended with efficient, flexible summarization algorithms. Thus, the authors developed a prototype system to create shortcuts (called summaries or abstracts) of bronchoscopy video recordings. Such a system, based on models described in previously published papers, employs image analysis methods to exclude frames or sequences of limited diagnostic or education value. Results The algorithm for the selection or exclusion of specific frames or shots from video sequences recorded during bronchoscopy procedures is based on several criteria, including automatic detection of "non-informative", frames showing the branching of the airways and frames including pathological lesions. Conclusions The paper focuses on the challenge of generating summaries of bronchoscopy video recordings. PMID:22185344

Exome sequencing of a multigenerational human pedigree.

PubMed

Hedges, Dale J; Hedges, Dale; Burges, Dan; Powell, Eric; Almonte, Cherylyn; Huang, Jia; Young, Stuart; Boese, Benjamin; Schmidt, Mike; Pericak-Vance, Margaret A; Martin, Eden; Zhang, Xinmin; Harkins, Timothy T; Züchner, Stephan

2009-12-14

Over the next few years, the efficient use of next-generation sequencing (NGS) in human genetics research will depend heavily upon the effective mechanisms for the selective enrichment of genomic regions of interest. Recently, comprehensive exome capture arrays have become available for targeting approximately 33 Mb or approximately 180,000 coding exons across the human genome. Selective genomic enrichment of the human exome offers an attractive option for new experimental designs aiming to quickly identify potential disease-associated genetic variants, especially in family-based studies. We have evaluated a 2.1 M feature human exome capture array on eight individuals from a three-generation family pedigree. We were able to cover up to 98% of the targeted bases at a long-read sequence read depth of > or = 3, 86% at a read depth of > or = 10, and over 50% of all targets were covered with > or = 20 reads. We identified up to 14,284 SNPs and small indels per individual exome, with up to 1,679 of these representing putative novel polymorphisms. Applying the conservative genotype calling approach HCDiff, the average rate of detection of a variant allele based on Illumina 1 M BeadChips genotypes was 95.2% at > or = 10x sequence. Further, we propose an advantageous genotype calling strategy for low covered targets that empirically determines cut-off thresholds at a given coverage depth based on existing genotype data. Application of this method was able to detect >99% of SNPs covered > or = 8x. Our results offer guidance for "real-world" applications in human genetics and provide further evidence that microarray-based exome capture is an efficient and reliable method to enrich for chromosomal regions of interest in next-generation sequencing experiments.

Community-Level Analysis of psbA Gene Sequences and Irgarol Tolerance in Marine Periphyton▿

PubMed Central

Eriksson, K. M.; Clarke, A. K.; Franzen, L.-G.; Kuylenstierna, M.; Martinez, K.; Blanck, H.

2009-01-01

This study analyzes psbA gene sequences, predicted D1 protein sequences, species relative abundance, and pollution-induced community tolerance in marine periphyton communities exposed to the antifouling compound Irgarol 1051. The mechanism of action of Irgarol is the inhibition of photosynthetic electron transport at photosystem II by binding to the D1 protein. The metagenome of the communities was used to produce clone libraries containing fragments of the psbA gene encoding the D1 protein. Community tolerance was quantified with a short-term test for the inhibition of photosynthesis. The communities were established in a continuous flow of natural seawater through microcosms with or without added Irgarol. The selection pressure from Irgarol resulted in an altered species composition and an inducted community tolerance to Irgarol. Moreover, there was a very high diversity in the psbA gene sequences in the periphyton, and the composition of psbA and D1 fragments within the communities was dramatically altered by increased Irgarol exposure. Even though tolerance to this type of compound in land plants often depends on a single amino acid substitution (Ser264→Gly) in the D1 protein, this was not the case for marine periphyton species. Instead, the tolerance mechanism likely involves increased degradation of D1. When we compared sequences from low and high Irgarol exposure, differences in nonconserved amino acids were found only in the so-called PEST region of D1, which is involved in regulating its degradation. Our results suggest that environmental contamination with Irgarol has led to selection for high-turnover D1 proteins in marine periphyton communities at the west coast of Sweden. PMID:19088321

The Influence of Primary and Secondary DNA Structure in Deletion and Duplication between Direct Repeats in Escherichia Coli

PubMed Central

Trinh, T. Q.; Sinden, R. R.

1993-01-01

We describe a system to measure the frequency of both deletions and duplications between direct repeats. Short 17- and 18-bp palindromic and nonpalindromic DNA sequences were cloned into the EcoRI site within the chloramphenicol acetyltransferase gene of plasmids pBR325 and pJT7. This creates an insert between direct repeated EcoRI sites and results in a chloramphenicol-sensitive phenotype. Selection for chloramphenicol resistance was utilized to select chloramphenicol resistant revertants that included those with precise deletion of the insert from plasmid pBR325 and duplication of the insert in plasmid pJT7. The frequency of deletion or duplication varied more than 500-fold depending on the sequence of the short sequence inserted into the EcoRI site. For the nonpalindromic inserts, multiple internal direct repeats and the length of the direct repeats appear to influence the frequency of deletion. Certain palindromic DNA sequences with the potential to form DNA hairpin structures that might stabilize the misalignment of direct repeats had a high frequency of deletion. Other DNA sequences with the potential to form structures that might destabilize misalignment of direct repeats had a very low frequency of deletion. Duplication mutations occurred at the highest frequency when the DNA between the direct repeats contained no direct or inverted repeats. The presence of inverted repeats dramatically reduced the frequency of duplications. The results support the slippage-misalignment model, suggesting that misalignment occurring during DNA replication leads to deletion and duplication mutations. The results also support the idea that the formation of DNA secondary structures during DNA replication can facilitate and direct specific mutagenic events. PMID:8325478

Deep sequencing in library selection projects: what insight does it bring?

PubMed

Glanville, J; D'Angelo, S; Khan, T A; Reddy, S T; Naranjo, L; Ferrara, F; Bradbury, A R M

2015-08-01

High throughput sequencing is poised to change all aspects of the way antibodies and other binders are discovered and engineered. Millions of available sequence reads provide an unprecedented sampling depth able to guide the design and construction of effective, high quality naïve libraries containing tens of billions of unique molecules. Furthermore, during selections, high throughput sequencing enables quantitative tracing of enriched clones and position-specific guidance to amino acid variation under positive selection during antibody engineering. Successful application of the technologies relies on specific PCR reagent design, correct sequencing platform selection, and effective use of computational tools and statistical measures to remove error, identify antibodies, estimate diversity, and extract signatures of selection from the clone down to individual structural positions. Here we review these considerations and discuss some of the remaining challenges to the widespread adoption of the technology. Copyright © 2015 Elsevier Ltd. All rights reserved.

Deep sequencing in library selection projects: what insight does it bring?

PubMed Central

Glanville, J; D’Angelo, S; Khan, T.A.; Reddy, S. T.; Naranjo, L.; Ferrara, F.; Bradbury, A.R.M.

2015-01-01

High throughput sequencing is poised to change all aspects of the way antibodies and other binders are discovered and engineered. Millions of available sequence reads provide an unprecedented sampling depth able to guide the design and construction of effective, high quality naïve libraries containing tens of billions of unique molecules. Furthermore, during selections, high throughput sequencing enables quantitative tracing of enriched clones and position-specific guidance to amino acid variation under positive selection during antibody engineering. Successful application of the technologies relies on specific PCR reagent design, correct sequencing platform selection, and effective use of computational tools and statistical measures to remove error, identify antibodies, estimate diversity, and extract signatures of selection from the clone down to individual structural positions. Here we review these considerations and discuss some of the remaining challenges to the widespread adoption of the technology. PMID:26451649

Microbial diversity of a Camembert-type cheese using freeze-dried Tibetan kefir coculture as starter culture by culture-dependent and culture-independent methods.

PubMed

Mei, Jun; Guo, Qizhen; Wu, Yan; Li, Yunfei

2014-01-01

The biochemical changes occurring during cheese ripening are directly and indirectly dependent on the microbial associations of starter cultures. Freeze-dried Tibetan kefir coculture was used as a starter culture in the Camembert-type cheese production for the first time. Therefore, it's necessary to elucidate the stability, organization and identification of the dominant microbiota presented in the cheese. Bacteria and yeasts were subjected to culture-dependent on selective media and culture-independent polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis and sequencing of dominant bands to assess the microbial structure and dynamics through ripening. In further studies, kefir grains were observed using scanning electron microscopy (SEM) methods. A total of 147 bacteria and 129 yeasts were obtained from the cheese during ripening. Lactobacillus paracasei represents the most commonly identified lactic acid bacteria isolates, with 59 of a total of 147 isolates, followed by Lactococcus lactis (29 isolates). Meanwhile, Kazachstania servazzii (51 isolates) represented the mainly identified yeast isolate, followed by Saccharomyces cerevisiae (40 isolates). However, some lactic acid bacteria detected by sequence analysis of DGGE bands were not recovered by plating. The yeast S. cerevisiae and K. servazzii are described for the first time with kefir starter culture. SEM showed that the microbiota were dominated by a variety of lactobacilli (long and curved) cells growing in close association with a few yeasts in the inner portion of the grain and the short lactobacilli were observed along with yeast cells on the exterior portion. Results indicated that conventional culture method and PCR-DGGE should be combined to describe in maximal detail the microbiological composition in the cheese during ripening. The data could help in the selection of appropriate commercial starters for Camembert-type cheese.

Microbial Diversity of a Camembert-Type Cheese Using Freeze-Dried Tibetan Kefir Coculture as Starter Culture by Culture-Dependent and Culture-Independent Methods

PubMed Central

Mei, Jun; Guo, Qizhen; Wu, Yan; Li, Yunfei

2014-01-01

The biochemical changes occurring during cheese ripening are directly and indirectly dependent on the microbial associations of starter cultures. Freeze-dried Tibetan kefir coculture was used as a starter culture in the Camembert-type cheese production for the first time. Therefore, it's necessary to elucidate the stability, organization and identification of the dominant microbiota presented in the cheese. Bacteria and yeasts were subjected to culture-dependent on selective media and culture-independent polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis and sequencing of dominant bands to assess the microbial structure and dynamics through ripening. In further studies, kefir grains were observed using scanning electron microscopy (SEM) methods. A total of 147 bacteria and 129 yeasts were obtained from the cheese during ripening. Lactobacillus paracasei represents the most commonly identified lactic acid bacteria isolates, with 59 of a total of 147 isolates, followed by Lactococcus lactis (29 isolates). Meanwhile, Kazachstania servazzii (51 isolates) represented the mainly identified yeast isolate, followed by Saccharomyces cerevisiae (40 isolates). However, some lactic acid bacteria detected by sequence analysis of DGGE bands were not recovered by plating. The yeast S. cerevisiae and K. servazzii are described for the first time with kefir starter culture. SEM showed that the microbiota were dominated by a variety of lactobacilli (long and curved) cells growing in close association with a few yeasts in the inner portion of the grain and the short lactobacilli were observed along with yeast cells on the exterior portion. Results indicated that conventional culture method and PCR-DGGE should be combined to describe in maximal detail the microbiological composition in the cheese during ripening. The data could help in the selection of appropriate commercial starters for Camembert-type cheese. PMID:25360757

A sequence-dependent rigid-base model of DNA

NASA Astrophysics Data System (ADS)

Gonzalez, O.; Petkevičiutė, D.; Maddocks, J. H.

2013-02-01

A novel hierarchy of coarse-grain, sequence-dependent, rigid-base models of B-form DNA in solution is introduced. The hierarchy depends on both the assumed range of energetic couplings, and the extent of sequence dependence of the model parameters. A significant feature of the models is that they exhibit the phenomenon of frustration: each base cannot simultaneously minimize the energy of all of its interactions. As a consequence, an arbitrary DNA oligomer has an intrinsic or pre-existing stress, with the level of this frustration dependent on the particular sequence of the oligomer. Attention is focussed on the particular model in the hierarchy that has nearest-neighbor interactions and dimer sequence dependence of the model parameters. For a Gaussian version of this model, a complete coarse-grain parameter set is estimated. The parameterized model allows, for an oligomer of arbitrary length and sequence, a simple and explicit construction of an approximation to the configuration-space equilibrium probability density function for the oligomer in solution. The training set leading to the coarse-grain parameter set is itself extracted from a recent and extensive database of a large number of independent, atomic-resolution molecular dynamics (MD) simulations of short DNA oligomers immersed in explicit solvent. The Kullback-Leibler divergence between probability density functions is used to make several quantitative assessments of our nearest-neighbor, dimer-dependent model, which is compared against others in the hierarchy to assess various assumptions pertaining both to the locality of the energetic couplings and to the level of sequence dependence of its parameters. It is also compared directly against all-atom MD simulation to assess its predictive capabilities. The results show that the nearest-neighbor, dimer-dependent model can successfully resolve sequence effects both within and between oligomers. For example, due to the presence of frustration, the model can successfully predict the nonlocal changes in the minimum energy configuration of an oligomer that are consequent upon a local change of sequence at the level of a single point mutation.

A sequence-dependent rigid-base model of DNA.

PubMed

Gonzalez, O; Petkevičiūtė, D; Maddocks, J H

2013-02-07

A novel hierarchy of coarse-grain, sequence-dependent, rigid-base models of B-form DNA in solution is introduced. The hierarchy depends on both the assumed range of energetic couplings, and the extent of sequence dependence of the model parameters. A significant feature of the models is that they exhibit the phenomenon of frustration: each base cannot simultaneously minimize the energy of all of its interactions. As a consequence, an arbitrary DNA oligomer has an intrinsic or pre-existing stress, with the level of this frustration dependent on the particular sequence of the oligomer. Attention is focussed on the particular model in the hierarchy that has nearest-neighbor interactions and dimer sequence dependence of the model parameters. For a Gaussian version of this model, a complete coarse-grain parameter set is estimated. The parameterized model allows, for an oligomer of arbitrary length and sequence, a simple and explicit construction of an approximation to the configuration-space equilibrium probability density function for the oligomer in solution. The training set leading to the coarse-grain parameter set is itself extracted from a recent and extensive database of a large number of independent, atomic-resolution molecular dynamics (MD) simulations of short DNA oligomers immersed in explicit solvent. The Kullback-Leibler divergence between probability density functions is used to make several quantitative assessments of our nearest-neighbor, dimer-dependent model, which is compared against others in the hierarchy to assess various assumptions pertaining both to the locality of the energetic couplings and to the level of sequence dependence of its parameters. It is also compared directly against all-atom MD simulation to assess its predictive capabilities. The results show that the nearest-neighbor, dimer-dependent model can successfully resolve sequence effects both within and between oligomers. For example, due to the presence of frustration, the model can successfully predict the nonlocal changes in the minimum energy configuration of an oligomer that are consequent upon a local change of sequence at the level of a single point mutation.

Animal selection for whole genome sequencing by quantifying the unique contribution of homozygous haplotypes sequenced

USDA-ARS?s Scientific Manuscript database

Major whole genome sequencing projects promise to identify rare and causal variants within livestock species; however, the efficient selection of animals for sequencing remains a major problem within these surveys. The goal of this project was to develop a library of high accuracy genetic variants f...

Longitudinal stability of MRI for mapping brain change using tensor-based morphometry.

PubMed

Leow, Alex D; Klunder, Andrea D; Jack, Clifford R; Toga, Arthur W; Dale, Anders M; Bernstein, Matt A; Britson, Paula J; Gunter, Jeffrey L; Ward, Chadwick P; Whitwell, Jennifer L; Borowski, Bret J; Fleisher, Adam S; Fox, Nick C; Harvey, Danielle; Kornak, John; Schuff, Norbert; Studholme, Colin; Alexander, Gene E; Weiner, Michael W; Thompson, Paul M

2006-06-01

Measures of brain change can be computed from sequential MRI scans, providing valuable information on disease progression, e.g., for patient monitoring and drug trials. Tensor-based morphometry (TBM) creates maps of these brain changes, visualizing the 3D profile and rates of tissue growth or atrophy, but its sensitivity depends on the contrast and geometric stability of the images. As part of the Alzheimer's Disease Neuroimaging Initiative (ADNI), 17 normal elderly subjects were scanned twice (at a 2-week interval) with several 3D 1.5 T MRI pulse sequences: high and low flip angle SPGR/FLASH (from which Synthetic T1 images were generated), MP-RAGE, IR-SPGR (N = 10) and MEDIC (N = 7) scans. For each subject and scan type, a 3D deformation map aligned baseline and follow-up scans, computed with a nonlinear, inverse-consistent elastic registration algorithm. Voxelwise statistics, in ICBM stereotaxic space, visualized the profile of mean absolute change and its cross-subject variance; these maps were then compared using permutation testing. Image stability depended on: (1) the pulse sequence; (2) the transmit/receive coil type (birdcage versus phased array); (3) spatial distortion corrections (using MEDIC sequence information); (4) B1-field intensity inhomogeneity correction (using N3). SPGR/FLASH images acquired using a birdcage coil had least overall deviation. N3 correction reduced coil type and pulse sequence differences and improved scan reproducibility, except for Synthetic T1 images (which were intrinsically corrected for B1-inhomogeneity). No strong evidence favored B0 correction. Although SPGR/FLASH images showed least deviation here, pulse sequence selection for the ADNI project was based on multiple additional image analyses, to be reported elsewhere.

Longitudinal stability of MRI for mapping brain change using tensor-based morphometry

PubMed Central

Leow, Alex D.; Klunder, Andrea D.; Jack, Clifford R.; Toga, Arthur W.; Dale, Anders M.; Bernstein, Matt A.; Britson, Paula J.; Gunter, Jeffrey L.; Ward, Chadwick P.; Whitwell, Jennifer L.; Borowski, Bret J.; Fleisher, Adam S.; Fox, Nick C.; Harvey, Danielle; Kornak, John; Schuff, Norbert; Studholme, Colin; Alexander, Gene E.; Weiner, Michael W.; Thompson, Paul M.

2007-01-01

Measures of brain change can be computed from sequential MRI scans, providing valuable information on disease progression, e.g., for patient monitoring and drug trials. Tensor-based morphometry (TBM) creates maps of these brain changes, visualizing the 3D profile and rates of tissue growth or atrophy, but its sensitivity depends on the contrast and geometric stability of the images. A s part of the Alzheimer’s Disease Neuroimaging Initiative (ADNI), 17 normal elderly subjects were scanned twice (at a 2-week interval) with several 3D 1.5 T MRI pulse sequences: high and low flip angle SPGR/FLASH (from which Synthetic T1 images were generated), MP-RAGE, IR-SPGR (N = 10) and MEDIC (N = 7) scans. For each subject and scan type, a 3D deformation map aligned baseline and follow-up scans, computed with a nonlinear, inverse-consistent elastic registration algorithm. Voxelwise statistics, in ICBM stereotaxic space, visualized the profile of mean absolute change and its cross-subject variance; these maps were then compared using permutation testing. Image stability depended on: (1) the pulse sequence; (2) the transmit/receive coil type (birdcage versus phased array); (3) spatial distortion corrections (using MEDIC sequence information); (4) B1-field intensity inhomogeneity correction (using N3). SPGR/FLASH images acquired using a birdcage coil had least overall deviation. N3 correction reduced coil type and pulse sequence differences and improved scan reproducibility, except for Synthetic T1 images (which were intrinsically corrected for B1-inhomogeneity). No strong evidence favored B0 correction. Although SPGR/FLASH images showed least deviation here, pulse sequence selection for the ADNI project was based on multiple additional image analyses, to be reported elsewhere. PMID:16480900

Protocols for efficient simulations of long-time protein dynamics using coarse-grained CABS model.

PubMed

Jamroz, Michal; Kolinski, Andrzej; Kmiecik, Sebastian

2014-01-01

Coarse-grained (CG) modeling is a well-acknowledged simulation approach for getting insight into long-time scale protein folding events at reasonable computational cost. Depending on the design of a CG model, the simulation protocols vary from highly case-specific-requiring user-defined assumptions about the folding scenario-to more sophisticated blind prediction methods for which only a protein sequence is required. Here we describe the framework protocol for the simulations of long-term dynamics of globular proteins, with the use of the CABS CG protein model and sequence data. The simulations can start from a random or a selected (e.g., native) structure. The described protocol has been validated using experimental data for protein folding model systems-the prediction results agreed well with the experimental results.

High-Resolution Sequence-Function Mapping of Full-Length Proteins

PubMed Central

Kowalsky, Caitlin A.; Klesmith, Justin R.; Stapleton, James A.; Kelly, Vince; Reichkitzer, Nolan; Whitehead, Timothy A.

2015-01-01

Comprehensive sequence-function mapping involves detailing the fitness contribution of every possible single mutation to a gene by comparing the abundance of each library variant before and after selection for the phenotype of interest. Deep sequencing of library DNA allows frequency reconstruction for tens of thousands of variants in a single experiment, yet short read lengths of current sequencers makes it challenging to probe genes encoding full-length proteins. Here we extend the scope of sequence-function maps to entire protein sequences with a modular, universal sequence tiling method. We demonstrate the approach with both growth-based selections and FACS screening, offer parameters and best practices that simplify design of experiments, and present analytical solutions to normalize data across independent selections. Using this protocol, sequence-function maps covering full sequences can be obtained in four to six weeks. Best practices introduced in this manuscript are fully compatible with, and complementary to, other recently published sequence-function mapping protocols. PMID:25790064

The carboxyl-terminus directs TAF(I)48 to the nucleus and nucleolus and associates with multiple nuclear import receptors.

PubMed

Dynes, Joseph L; Xu, Shuping; Bothner, Sarah; Lahti, Jill M; Hori, Roderick T

2004-03-01

The protein complex Selectivity Factor 1, composed of TBP, TAF(I)48, TAF(I)63 and TAF(I)110, is required for rRNA transcription by RNA polymerase I in the nucleolus. The steps involved in targeting Selectivity Factor 1 will be dependent on the transport pathways that are used and the localization signals that direct this trafficking. In order to investigate these issues, we characterized human TAF(I)48, a subunit of Selectivity Factor 1. By domain analysis of TAF(I)48, the carboxyl-terminal 51 residues were found to be required for the localization of TAF(I)48, as well as sufficient to direct Green Fluorescent Protein to the nucleus and nucleolus. The carboxyl-terminus of TAF(I)48 also has the ability to associate with multiple members of the beta-karyopherin family of nuclear import receptors, including importin beta (karyopherin beta1), transportin (karyopherin beta2) and RanBP5 (karyopherin beta3), in a Ran-dependent manner. This property of interacting with multiple beta-karyopherins has been previously reported for the nuclear localization signals of some ribosomal proteins that are likewise directed to the nucleolus. This study identifies the first nuclear import sequence identified within the TBP-Associated Factor subunits of Selectivity Factor 1.

Graphical workstation capability for reliability modeling

NASA Technical Reports Server (NTRS)

Bavuso, Salvatore J.; Koppen, Sandra V.; Haley, Pamela J.

1992-01-01

In addition to computational capabilities, software tools for estimating the reliability of fault-tolerant digital computer systems must also provide a means of interfacing with the user. Described here is the new graphical interface capability of the hybrid automated reliability predictor (HARP), a software package that implements advanced reliability modeling techniques. The graphics oriented (GO) module provides the user with a graphical language for modeling system failure modes through the selection of various fault-tree gates, including sequence-dependency gates, or by a Markov chain. By using this graphical input language, a fault tree becomes a convenient notation for describing a system. In accounting for any sequence dependencies, HARP converts the fault-tree notation to a complex stochastic process that is reduced to a Markov chain, which it can then solve for system reliability. The graphics capability is available for use on an IBM-compatible PC, a Sun, and a VAX workstation. The GO module is written in the C programming language and uses the graphical kernal system (GKS) standard for graphics implementation. The PC, VAX, and Sun versions of the HARP GO module are currently in beta-testing stages.

How do biological systems discriminate among physically similar ions?

PubMed

Diamond, J M

1975-10-01

This paper reviews the history of understanding how biological systems can discriminate so strikingly among physically similar ions, especially alkali cations. Appreciation of qualitative regularities ("permitted sequences") and quantitative regularities ("selectivity isotherms") in ion selectivity grew first from studies of ion exchangers and glass electrodes, then of biological systems such as enzymes and cell membranes, and most recently of lipid bilayers doped with model pores and carriers. Discrimination of ions depends on both electrostatic and steric forces. "Black-box" studies on intact biological membranes have in some cases yielded molecular clues to the structure of the actual biological pores and carriers. Major current problems involve the extraction of these molecules; how to do it, what to do when it is achieved, and how (and if) it is relevant to the central problems of membrane function. Further advances are expected soon from studies of rate barriers within membranes, of voltage-dependent ("excitable") conducting channels, and of increasingly complex model systems and biological membranes.

Signal sequence and keyword trap in silico for selection of full-length human cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries.

PubMed

Otsuki, Tetsuji; Ota, Toshio; Nishikawa, Tetsuo; Hayashi, Koji; Suzuki, Yutaka; Yamamoto, Jun-ichi; Wakamatsu, Ai; Kimura, Kouichi; Sakamoto, Katsuhiko; Hatano, Naoto; Kawai, Yuri; Ishii, Shizuko; Saito, Kaoru; Kojima, Shin-ichi; Sugiyama, Tomoyasu; Ono, Tetsuyoshi; Okano, Kazunori; Yoshikawa, Yoko; Aotsuka, Satoshi; Sasaki, Naokazu; Hattori, Atsushi; Okumura, Koji; Nagai, Keiichi; Sugano, Sumio; Isogai, Takao

2005-01-01

We have developed an in silico method of selection of human full-length cDNAs encoding secretion or membrane proteins from oligo-capped cDNA libraries. Fullness rates were increased to about 80% by combination of the oligo-capping method and ATGpr, software for prediction of translation start point and the coding potential. Then, using 5'-end single-pass sequences, cDNAs having the signal sequence were selected by PSORT ('signal sequence trap'). We also applied 'secretion or membrane protein-related keyword trap' based on the result of BLAST search against the SWISS-PROT database for the cDNAs which could not be selected by PSORT. Using the above procedures, 789 cDNAs were primarily selected and subjected to full-length sequencing, and 334 of these cDNAs were finally selected as novel. Most of the cDNAs (295 cDNAs: 88.3%) were predicted to encode secretion or membrane proteins. In particular, 165(80.5%) of the 205 cDNAs selected by PSORT were predicted to have signal sequences, while 70 (54.2%) of the 129 cDNAs selected by 'keyword trap' preserved the secretion or membrane protein-related keywords. Many important cDNAs were obtained, including transporters, receptors, and ligands, involved in significant cellular functions. Thus, an efficient method of selecting secretion or membrane protein-encoding cDNAs was developed by combining the above four procedures.

A novel light-dependent selection marker system in plants.

PubMed

Koh, Serry; Kim, Hongsup; Kim, Jinwoo; Goo, Eunhye; Kim, Yun-Jung; Choi, Okhee; Jwa, Nam-Soo; Ma, Jun; Nagamatsu, Tomohisa; Moon, Jae Sun; Hwang, Ingyu

2011-04-01

Photosensitizers are common in nature and play diverse roles as defense compounds and pathogenicity determinants and as important molecules in many biological processes. Toxoflavin, a photosensitizer produced by Burkholderia glumae, has been implicated as an essential virulence factor causing bacterial rice grain rot. Toxoflavin produces superoxide and H₂O₂ during redox cycles under oxygen and light, and these reactive oxygen species cause phytotoxic effects. To utilize toxoflavin as a selection agent in plant transformation, we identified a gene, tflA, which encodes a toxoflavin-degrading enzyme in the Paenibacillus polymyxa JH2 strain. TflA was estimated as 24.56 kDa in size based on the amino acid sequence and is similar to a ring-cleavage extradiol dioxygenase in the Exiguobacterium sp. 255-15; however, unlike other extradiol dioxygenases, Mn(2+) and dithiothreitol were required for toxoflavin degradation by TflA. Here, our results suggested toxoflavin is a photosensitizer and its degradation by TflA serves as a light-dependent selection marker system in diverse plant species. We examined the efficiencies of two different plant selection systems, toxoflavin/tflA and hygromycin/hygromycin phosphotransferase (hpt) in both rice and Arabidopsis. The toxoflavin/tflA selection was more remarkable than hygromycin/hpt selection in the high-density screening of transgenic Arabidopsis seeds. Based on these results, we propose the toxoflavin/tflA selection system, which is based on the degradation of the photosensitizer, provides a new robust nonantibiotic selection marker system for diverse plants. © 2010 The Authors. Plant Biotechnology Journal © 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

Clustalnet: the joining of Clustal and CORBA.

PubMed

Campagne, F

2000-07-01

Performing sequence alignment operations from a different program than the original sequence alignment code, and/or through a network connection, is often required. Interactive alignment editors and large-scale biological data analysis are common examples where such a flexibility is important. Interoperability between the alignment engine and the client should be obtained regardless of the architectures and programming languages of the server and client. Clustalnet, a Clustal alignment CORBA server is described, which was developed on the basis of Clustalw. This server brings the robustness of the algorithms and implementations of Clustal to a new level of reuse. A Clustalnet server object can be accessed from a program, transparently through the network. We present interfaces to perform the alignment operations and to control these operations via immutable contexts. The interfaces that select the contexts do not depend on the nature of the operation to be performed, making the design modular. The IDL interfaces presented here are not specific to Clustal and can be implemented on top of different sequence alignment algorithm implementations.

Using the Self-Select Paradigm to Delineate the Nature of Speech Motor Programming

PubMed Central

Wright, David L.; Robin, Don A.; Rhee, Jooyhun; Vaculin, Amber; Jacks, Adam; Guenther, Frank H.; Fox, Peter T.

2015-01-01

Purpose The authors examined the involvement of 2 speech motor programming processes identified by S. T. Klapp (1995, 2003) during the articulation of utterances differing in syllable and sequence complexity. According to S. T. Klapp, 1 process, INT, resolves the demands of the programmed unit, whereas a second process, SEQ, oversees the serial order demands of longer sequences. Method A modified reaction time paradigm was used to assess INT and SEQ demands. Specifically, syllable complexity was dependent on syllable structure, whereas sequence complexity involved either repeated or unique syllabi within an utterance. Results INT execution was slowed when articulating single syllables in the form CCCV compared to simpler CV syllables. Planning unique syllables within a multisyllabic utterance rather than repetitions of the same syllable slowed INT but not SEQ. Conclusions The INT speech motor programming process, important for mental syllabary access, is sensitive to changes in both syllable structure and the number of unique syllables in an utterance. PMID:19474396

The dot{M}-M_* relation of pre-main-sequence stars: a consequence of X-ray driven disc evolution

NASA Astrophysics Data System (ADS)

Ercolano, B.; Mayr, D.; Owen, J. E.; Rosotti, G.; Manara, C. F.

2014-03-01

We analyse current measurements of accretion rates on to pre-main-sequence stars as a function of stellar mass, and conclude that the steep dependence of accretion rates on stellar mass is real and not driven by selection/detection threshold, as has been previously feared. These conclusions are reached by means of statistical tests including a survival analysis which can account for upper limits. The power-law slope of the dot{M}-M_* relation is found to be in the range of 1.6-1.9 for young stars with masses lower than 1 M⊙. The measured slopes and distributions can be easily reproduced by means of a simple disc model which includes viscous accretion and X-ray photoevaporation. We conclude that the dot{M}-M_* relation in pre-main-sequence stars bears the signature of disc dispersal by X-ray photoevaporation, suggesting that the relation is a straightforward consequence of disc physics rather than an imprint of initial conditions.

Sequence-dependent DNA deformability studied using molecular dynamics simulations.

PubMed

Fujii, Satoshi; Kono, Hidetoshi; Takenaka, Shigeori; Go, Nobuhiro; Sarai, Akinori

2007-01-01

Proteins recognize specific DNA sequences not only through direct contact between amino acids and bases, but also indirectly based on the sequence-dependent conformation and deformability of the DNA (indirect readout). We used molecular dynamics simulations to analyze the sequence-dependent DNA conformations of all 136 possible tetrameric sequences sandwiched between CGCG sequences. The deformability of dimeric steps obtained by the simulations is consistent with that by the crystal structures. The simulation results further showed that the conformation and deformability of the tetramers can highly depend on the flanking base pairs. The conformations of xATx tetramers show the most rigidity and are not affected by the flanking base pairs and the xYRx show by contrast the greatest flexibility and change their conformations depending on the base pairs at both ends, suggesting tetramers with the same central dimer can show different deformabilities. These results suggest that analysis of dimeric steps alone may overlook some conformational features of DNA and provide insight into the mechanism of indirect readout during protein-DNA recognition. Moreover, the sequence dependence of DNA conformation and deformability may be used to estimate the contribution of indirect readout to the specificity of protein-DNA recognition as well as nucleosome positioning and large-scale behavior of nucleic acids.

PRISE2: software for designing sequence-selective PCR primers and probes.

PubMed

Huang, Yu-Ting; Yang, Jiue-in; Chrobak, Marek; Borneman, James

2014-09-25

PRISE2 is a new software tool for designing sequence-selective PCR primers and probes. To achieve high level of selectivity, PRISE2 allows the user to specify a collection of target sequences that the primers are supposed to amplify, as well as non-target sequences that should not be amplified. The program emphasizes primer selectivity on the 3' end, which is crucial for selective amplification of conserved sequences such as rRNA genes. In PRISE2, users can specify desired properties of primers, including length, GC content, and others. They can interactively manipulate the list of candidate primers, to choose primer pairs that are best suited for their needs. A similar process is used to add probes to selected primer pairs. More advanced features include, for example, the capability to define a custom mismatch penalty function. PRISE2 is equipped with a graphical, user-friendly interface, and it runs on Windows, Macintosh or Linux machines. PRISE2 has been tested on two very similar strains of the fungus Dactylella oviparasitica, and it was able to create highly selective primers and probes for each of them, demonstrating the ability to create useful sequence-selective assays. PRISE2 is a user-friendly, interactive software package that can be used to design high-quality selective primers for PCR experiments. In addition to choosing primers, users have an option to add a probe to any selected primer pair, enabling design of Taqman and other primer-probe based assays. PRISE2 can also be used to design probes for FISH and other hybridization-based assays.

Rhipicephalus microplus dataset of nonredundant raw sequence reads from 454 GS FLX sequencing of Cot-selected (Cot = 660) genomic DNA

USDA-ARS?s Scientific Manuscript database

A reassociation kinetics-based approach was used to reduce the complexity of genomic DNA from the Deutsch laboratory strain of the cattle tick, Rhipicephalus microplus, to facilitate genome sequencing. Selected genomic DNA (Cot value = 660) was sequenced using 454 GS FLX technology, resulting in 356...

Biological function in the twilight zone of sequence conservation.

PubMed

Ponting, Chris P

2017-08-16

Strong DNA conservation among divergent species is an indicator of enduring functionality. With weaker sequence conservation we enter a vast 'twilight zone' in which sequence subject to transient or lower constraint cannot be distinguished easily from neutrally evolving, non-functional sequence. Twilight zone functional sequence is illuminated instead by principles of selective constraint and positive selection using genomic data acquired from within a species' population. Application of these principles reveals that despite being biochemically active, most twilight zone sequence is not functional.

A 3D sequence-independent representation of the protein data bank.

PubMed

Fischer, D; Tsai, C J; Nussinov, R; Wolfson, H

1995-10-01

Here we address the following questions. How many structurally different entries are there in the Protein Data Bank (PDB)? How do the proteins populate the structural universe? To investigate these questions a structurally non-redundant set of representative entries was selected from the PDB. Construction of such a dataset is not trivial: (i) the considerable size of the PDB requires a large number of comparisons (there were more than 3250 structures of protein chains available in May 1994); (ii) the PDB is highly redundant, containing many structurally similar entries, not necessarily with significant sequence homology, and (iii) there is no clear-cut definition of structural similarity. The latter depend on the criteria and methods used. Here, we analyze structural similarity ignoring protein topology. To date, representative sets have been selected either by hand, by sequence comparison techniques which ignore the three-dimensional (3D) structures of the proteins or by using sequence comparisons followed by linear structural comparison (i.e. the topology, or the sequential order of the chains, is enforced in the structural comparison). Here we describe a 3D sequence-independent automated and efficient method to obtain a representative set of protein molecules from the PDB which contains all unique structures and which is structurally non-redundant. The method has two novel features. The first is the use of strictly structural criteria in the selection process without taking into account the sequence information. To this end we employ a fast structural comparison algorithm which requires on average approximately 2 s per pairwise comparison on a workstation. The second novel feature is the iterative application of a heuristic clustering algorithm that greatly reduces the number of comparisons required. We obtain a representative set of 220 chains with resolution better than 3.0 A, or 268 chains including lower resolution entries, NMR entries and models. The resulting set can serve as a basis for extensive structural classification and studies of 3D recurring motifs and of sequence-structure relationships. The clustering algorithm succeeds in classifying into the same structural family chains with no significant sequence homology, e.g. all the globins in one single group, all the trypsin-like serine proteases in another or all the immunoglobulin-like folds into a third. In addition, unexpected structural similarities of interest have been automatically detected between pairs of chains. A cluster analysis of the representative structures demonstrates the way the "structural universe' is populated.

Evaluation of two main RNA-seq approaches for gene quantification in clinical RNA sequencing: polyA+ selection versus rRNA depletion.

PubMed

Zhao, Shanrong; Zhang, Ying; Gamini, Ramya; Zhang, Baohong; von Schack, David

2018-03-19

To allow efficient transcript/gene detection, highly abundant ribosomal RNAs (rRNA) are generally removed from total RNA either by positive polyA+ selection or by rRNA depletion (negative selection) before sequencing. Comparisons between the two methods have been carried out by various groups, but the assessments have relied largely on non-clinical samples. In this study, we evaluated these two RNA sequencing approaches using human blood and colon tissue samples. Our analyses showed that rRNA depletion captured more unique transcriptome features, whereas polyA+ selection outperformed rRNA depletion with higher exonic coverage and better accuracy of gene quantification. For blood- and colon-derived RNAs, we found that 220% and 50% more reads, respectively, would have to be sequenced to achieve the same level of exonic coverage in the rRNA depletion method compared with the polyA+ selection method. Therefore, in most cases we strongly recommend polyA+ selection over rRNA depletion for gene quantification in clinical RNA sequencing. Our evaluation revealed that a small number of lncRNAs and small RNAs made up a large fraction of the reads in the rRNA depletion RNA sequencing data. Thus, we recommend that these RNAs are specifically depleted to improve the sequencing depth of the remaining RNAs.

Digital RNA sequencing minimizes sequence-dependent bias and amplification noise with optimized single-molecule barcodes

PubMed Central

Shiroguchi, Katsuyuki; Jia, Tony Z.; Sims, Peter A.; Xie, X. Sunney

2012-01-01

RNA sequencing (RNA-Seq) is a powerful tool for transcriptome profiling, but is hampered by sequence-dependent bias and inaccuracy at low copy numbers intrinsic to exponential PCR amplification. We developed a simple strategy for mitigating these complications, allowing truly digital RNA-Seq. Following reverse transcription, a large set of barcode sequences is added in excess, and nearly every cDNA molecule is uniquely labeled by random attachment of barcode sequences to both ends. After PCR, we applied paired-end deep sequencing to read the two barcodes and cDNA sequences. Rather than counting the number of reads, RNA abundance is measured based on the number of unique barcode sequences observed for a given cDNA sequence. We optimized the barcodes to be unambiguously identifiable, even in the presence of multiple sequencing errors. This method allows counting with single-copy resolution despite sequence-dependent bias and PCR-amplification noise, and is analogous to digital PCR but amendable to quantifying a whole transcriptome. We demonstrated transcriptome profiling of Escherichia coli with more accurate and reproducible quantification than conventional RNA-Seq. PMID:22232676

Object tracking using plenoptic image sequences

NASA Astrophysics Data System (ADS)

Kim, Jae Woo; Bae, Seong-Joon; Park, Seongjin; Kim, Do Hyung

2017-05-01

Object tracking is a very important problem in computer vision research. Among the difficulties of object tracking, partial occlusion problem is one of the most serious and challenging problems. To address the problem, we proposed novel approaches to object tracking on plenoptic image sequences. Our approaches take advantage of the refocusing capability that plenoptic images provide. Our approaches input the sequences of focal stacks constructed from plenoptic image sequences. The proposed image selection algorithms select the sequence of optimal images that can maximize the tracking accuracy from the sequence of focal stacks. Focus measure approach and confidence measure approach were proposed for image selection and both of the approaches were validated by the experiments using thirteen plenoptic image sequences that include heavily occluded target objects. The experimental results showed that the proposed approaches were satisfactory comparing to the conventional 2D object tracking algorithms.

Comparison and evaluation of two exome capture kits and sequencing platforms for variant calling.

PubMed

Zhang, Guoqiang; Wang, Jianfeng; Yang, Jin; Li, Wenjie; Deng, Yutian; Li, Jing; Huang, Jun; Hu, Songnian; Zhang, Bing

2015-08-05

To promote the clinical application of next-generation sequencing, it is important to obtain accurate and consistent variants of target genomic regions at low cost. Ion Proton, the latest updated semiconductor-based sequencing instrument from Life Technologies, is designed to provide investigators with an inexpensive platform for human whole exome sequencing that achieves a rapid turnaround time. However, few studies have comprehensively compared and evaluated the accuracy of variant calling between Ion Proton and Illumina sequencing platforms such as HiSeq 2000, which is the most popular sequencing platform for the human genome. The Ion Proton sequencer combined with the Ion TargetSeq Exome Enrichment Kit together make up TargetSeq-Proton, whereas SureSelect-Hiseq is based on the Agilent SureSelect Human All Exon v4 Kit and the HiSeq 2000 sequencer. Here, we sequenced exonic DNA from four human blood samples using both TargetSeq-Proton and SureSelect-HiSeq. We then called variants in the exonic regions that overlapped between the two exome capture kits (33.6 Mb). The rates of shared variant loci called by two sequencing platforms were from 68.0 to 75.3% in four samples, whereas the concordance of co-detected variant loci reached 99%. Sanger sequencing validation revealed that the validated rate of concordant single nucleotide polymorphisms (SNPs) (91.5%) was higher than the SNPs specific to TargetSeq-Proton (60.0%) or specific to SureSelect-HiSeq (88.3%). With regard to 1-bp small insertions and deletions (InDels), the Sanger sequencing validated rates of concordant variants (100.0%) and SureSelect-HiSeq-specific (89.6%) were higher than those of TargetSeq-Proton-specific (15.8%). In the sequencing of exonic regions, a combination of using of two sequencing strategies (SureSelect-HiSeq and TargetSeq-Proton) increased the variant calling specificity for concordant variant loci and the sensitivity for variant loci called by any one platform. However, for the sequencing of platform-specific variants, the accuracy of variant calling by HiSeq 2000 was higher than that of Ion Proton, specifically for the InDel detection. Moreover, the variant calling software also influences the detection of SNPs and, specifically, InDels in Ion Proton exome sequencing.

Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis.

PubMed

Du, Yushen; Wu, Nicholas C; Jiang, Lin; Zhang, Tianhao; Gong, Danyang; Shu, Sara; Wu, Ting-Ting; Sun, Ren

2016-11-01

Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp), we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available. To fully comprehend the diverse functions of a protein, it is essential to understand the functionality of individual residues. Current methods are highly dependent on evolutionary sequence conservation, which is usually limited by sampling size. Sequence conservation-based methods are further confounded by structural constraints and multifunctionality of proteins. Here we present a method that can systematically identify and annotate functional residues of a given protein. We used a high-throughput functional profiling platform to identify essential residues. Coupling it with homologous-structure comparison, we were able to annotate multiple functions of proteins. We demonstrated the method with the PB1 protein of influenza A virus and identified novel functional residues in addition to its canonical function as an RNA-dependent RNA polymerase. Not limited to virology, this method is generally applicable to other proteins that can be functionally selected and about which homologous-structure information is available. Copyright © 2016 Du et al.

Influence of active site location on catalytic activity in de novo-designed zinc metalloenzymes.

PubMed

Zastrow, Melissa L; Pecoraro, Vincent L

2013-04-17

While metalloprotein design has now yielded a number of successful metal-bound and even catalytically active constructs, the question of where to put a metal site along a linear, repetitive sequence has not been thoroughly addressed. Often several possibilities in a given sequence may exist that would appear equivalent but may in fact differ for metal affinity, substrate access, or protein dynamics. We present a systematic variation of active site location for a hydrolytically active ZnHis3O site contained within a de novo-designed three-stranded coiled coil. We find that the maximal rate, substrate access, and metal-binding affinity are dependent on the selected position, while catalytic efficiency for p-nitrophenyl acetate hydrolysis can be retained regardless of the location of the active site. This achievement demonstrates how efficient, tailor-made enzymes which control rate, pKa, substrate and solvent access (and selectivity), and metal-binding affinity may be realized. These findings may be applied to the more advanced de novo design of constructs containing secondary interactions, such as hydrogen-bonding channels. We are now confident that changes to location for accommodating such channels can be achieved without location-dependent loss of catalytic efficiency. These findings bring us closer to our ultimate goal of incorporating the secondary interactions we believe will be necessary in order to improve both active site properties and the catalytic efficiency to be competitive with the native enzyme, carbonic anhydrase.

Genotype-specific signal generation based on digestion of 3-way DNA junctions: application to KRAS variation detection.

PubMed

Amicarelli, Giulia; Adlerstein, Daniel; Shehi, Erlet; Wang, Fengfei; Makrigiorgos, G Mike

2006-10-01

Genotyping methods that reveal single-nucleotide differences are useful for a wide range of applications. We used digestion of 3-way DNA junctions in a novel technology, OneCutEventAmplificatioN (OCEAN) that allows sequence-specific signal generation and amplification. We combined OCEAN with peptide-nucleic-acid (PNA)-based variant enrichment to detect and simultaneously genotype v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) codon 12 sequence variants in human tissue specimens. We analyzed KRAS codon 12 sequence variants in 106 lung cancer surgical specimens. We conducted a PNA-PCR reaction that suppresses wild-type KRAS amplification and genotyped the product with a set of OCEAN reactions carried out in fluorescence microplate format. The isothermal OCEAN assay enabled a 3-way DNA junction to form between the specific target nucleic acid, a fluorescently labeled "amplifier", and an "anchor". The amplifier-anchor contact contains the recognition site for a restriction enzyme. Digestion produces a cleaved amplifier and generation of a fluorescent signal. The cleaved amplifier dissociates from the 3-way DNA junction, allowing a new amplifier to bind and propagate the reaction. The system detected and genotyped KRAS sequence variants down to approximately 0.3% variant-to-wild-type alleles. PNA-PCR/OCEAN had a concordance rate with PNA-PCR/sequencing of 93% to 98%, depending on the exact implementation. Concordance rate with restriction endonuclease-mediated selective-PCR/sequencing was 89%. OCEAN is a practical and low-cost novel technology for sequence-specific signal generation. Reliable analysis of KRAS sequence alterations in human specimens circumvents the requirement for sequencing. Application is expected in genotyping KRAS codon 12 sequence variants in surgical specimens or in bodily fluids, as well as single-base variations and sequence alterations in other genes.

Inflammatory bowel diseases phenotype, C. difficile and NOD2 genotype are associated with shifts in human ileum associated microbial composition.

PubMed

Li, Ellen; Hamm, Christina M; Gulati, Ajay S; Sartor, R Balfour; Chen, Hongyan; Wu, Xiao; Zhang, Tianyi; Rohlf, F James; Zhu, Wei; Gu, Chi; Robertson, Charles E; Pace, Norman R; Boedeker, Edgar C; Harpaz, Noam; Yuan, Jeffrey; Weinstock, George M; Sodergren, Erica; Frank, Daniel N

2012-01-01

We tested the hypothesis that Crohn's disease (CD)-related genetic polymorphisms involved in host innate immunity are associated with shifts in human ileum-associated microbial composition in a cross-sectional analysis of human ileal samples. Sanger sequencing of the bacterial 16S ribosomal RNA (rRNA) gene and 454 sequencing of 16S rRNA gene hypervariable regions (V1-V3 and V3-V5), were conducted on macroscopically disease-unaffected ileal biopsies collected from 52 ileal CD, 58 ulcerative colitis and 60 control patients without inflammatory bowel diseases (IBD) undergoing initial surgical resection. These subjects also were genotyped for the three major NOD2 risk alleles (Leu1007fs, R708W, G908R) and the ATG16L1 risk allele (T300A). The samples were linked to clinical metadata, including body mass index, smoking status and Clostridia difficile infection. The sequences were classified into seven phyla/subphyla categories using the Naïve Bayesian Classifier of the Ribosome Database Project. Centered log ratio transformation of six predominant categories was included as the dependent variable in the permutation based MANCOVA for the overall composition with stepwise variable selection. Polymerase chain reaction (PCR) assays were conducted to measure the relative frequencies of the Clostridium coccoides - Eubacterium rectales group and the Faecalibacterium prausnitzii spp. Empiric logit transformations of the relative frequencies of these two microbial groups were included in permutation-based ANCOVA. Regardless of sequencing method, IBD phenotype, Clostridia difficile and NOD2 genotype were selected as associated (FDR ≤ 0.05) with shifts in overall microbial composition. IBD phenotype and NOD2 genotype were also selected as associated with shifts in the relative frequency of the C. coccoides--E. rectales group. IBD phenotype, smoking and IBD medications were selected as associated with shifts in the relative frequency of F. prausnitzii spp. These results indicate that the effects of genetic and environmental factors on IBD are mediated at least in part by the enteric microbiota.

Inflammatory Bowel Diseases Phenotype, C. difficile and NOD2 Genotype Are Associated with Shifts in Human Ileum Associated Microbial Composition

PubMed Central

Li, Ellen; Hamm, Christina M.; Gulati, Ajay S.; Sartor, R. Balfour; Chen, Hongyan; Wu, Xiao; Zhang, Tianyi; Rohlf, F. James; Zhu, Wei; Gu, Chi; Robertson, Charles E.; Pace, Norman R.; Boedeker, Edgar C.; Harpaz, Noam; Yuan, Jeffrey; Weinstock, George M.; Sodergren, Erica; Frank, Daniel N.

2012-01-01

We tested the hypothesis that Crohn’s disease (CD)-related genetic polymorphisms involved in host innate immunity are associated with shifts in human ileum–associated microbial composition in a cross-sectional analysis of human ileal samples. Sanger sequencing of the bacterial 16S ribosomal RNA (rRNA) gene and 454 sequencing of 16S rRNA gene hypervariable regions (V1–V3 and V3–V5), were conducted on macroscopically disease-unaffected ileal biopsies collected from 52 ileal CD, 58 ulcerative colitis and 60 control patients without inflammatory bowel diseases (IBD) undergoing initial surgical resection. These subjects also were genotyped for the three major NOD2 risk alleles (Leu1007fs, R708W, G908R) and the ATG16L1 risk allele (T300A). The samples were linked to clinical metadata, including body mass index, smoking status and Clostridia difficile infection. The sequences were classified into seven phyla/subphyla categories using the Naïve Bayesian Classifier of the Ribosome Database Project. Centered log ratio transformation of six predominant categories was included as the dependent variable in the permutation based MANCOVA for the overall composition with stepwise variable selection. Polymerase chain reaction (PCR) assays were conducted to measure the relative frequencies of the Clostridium coccoides – Eubacterium rectales group and the Faecalibacterium prausnitzii spp. Empiric logit transformations of the relative frequencies of these two microbial groups were included in permutation-based ANCOVA. Regardless of sequencing method, IBD phenotype, Clostridia difficile and NOD2 genotype were selected as associated (FDR ≤0.05) with shifts in overall microbial composition. IBD phenotype and NOD2 genotype were also selected as associated with shifts in the relative frequency of the C. coccoides – E. rectales group. IBD phenotype, smoking and IBD medications were selected as associated with shifts in the relative frequency of F. prausnitzii spp. These results indicate that the effects of genetic and environmental factors on IBD are mediated at least in part by the enteric microbiota. PMID:22719818

Development of new antiatherosclerotic and antithrombotic drugs utilizing F11 receptor (F11R/JAM-A) peptides.

PubMed

Babinska, A; Clement, C C; Swiatkowska, M; Szymanski, J; Shon, A; Ehrlich, Y H; Kornecki, E; Salifu, M O

2014-07-01

Peptides with enhanced resistance to proteolysis, based on the amino acid sequence of the F11 receptor molecule (F11R, aka JAM-A/Junctional adhesion molecule-A), were designed, prepared, and examined as potential candidates for the development of anti-atherosclerotic and anti-thrombotic therapeutic drugs. A sequence at the N-terminal of F11R together with another sequence located in the first Ig-loop of this protein, were identified to form a steric active-site operating in the F11R-dependent adhesion between cells that express F11R molecules on their external surface. In silico modeling of the complex between two polypeptide chains with the sequences positioned in the active-site was used to generate peptide-candidates designed to inhibit homophilic interactions between surface-located F11R molecules. The two lead F11R peptides were modified with D-Arg and D-Lys at selective sites, for attaining higher stability to proteolysis in vivo. Using molecular docking experiments we tested different conformational states and the putative binding affinity between two selected D-Arg and D-Lys-modified F11R peptides and the proposed binding pocket. The inhibitory effects of the F11R peptide 2HN-(dK)-SVT-(dR)-EDTGTYTC-CONH2 on antibody-induced platelet aggregation and on the adhesion of platelets to cytokine-inflammed endothelial cells are reported in detail, and the results point out the significant potential utilization of F11R peptides for the prevention and treatment of atherosclerotic plaques and associated thrombotic events. © 2014 Wiley Periodicals, Inc.

Analysis and Dynamics of the Chromosomal Complements of Wild Sparkling-Wine Yeast Strains

PubMed Central

Nadal, Dolors; Carro, David; Fernández-Larrea, Juan; Piña, Benjamin

1999-01-01

We isolated Saccharomyces cerevisiae yeast strains that are able to carry out the second fermentation of sparkling wine from spontaneously fermenting musts in El Penedès (Spain) by specifically designed selection protocols. All of them (26 strains) showed one of two very similar mitochondrial DNA (mtDNA) restriction patterns, whereas their karyotypes differed. These strains showed high rates of karyotype instability, which were dependent on both the medium and the strain, during vegetative growth. In all cases, the mtDNA restriction pattern was conserved in strains kept under the same conditions. Analysis of different repetitive sequences in their genomes suggested that ribosomal DNA repeats play an important role in the changes in size observed in chromosome XII, whereas SUC genes or Ty elements did not show amplification or transposition processes that could be related to rearrangements of the chromosomes showing these sequences. Karyotype changes also occurred in monosporidic diploid derivatives. We propose that these changes originated mainly from ectopic recombination between repeated sequences interspersed in the genome. None of the rearranged karyotypes provided a selective advantage strong enough to allow the strains to displace the parental strains. The nature and frequency of these changes suggest that they may play an important role in the establishment and maintenance of the genetic diversity observed in S. cerevisiae wild populations. PMID:10103269

Genome-wide characterization and selection of expressed sequence tag simple sequence repeat primers for optimized marker distribution and reliability in peach

USDA-ARS?s Scientific Manuscript database

Expressed sequence tag (EST) simple sequence repeats (SSRs) in Prunus were mined, and flanking primers designed and used for genome-wide characterization and selection of primers to optimize marker distribution and reliability. A total of 12,618 contigs were assembled from 84,727 ESTs, along with 34...

Bifurcation structures of a cobweb model with memory and competing technologies

NASA Astrophysics Data System (ADS)

Agliari, Anna; Naimzada, Ahmad; Pecora, Nicolò

2018-05-01

In this paper we study a simple model based on the cobweb demand-supply framework with costly innovators and free imitators. The evolutionary selection between technologies depends on a performance measure which is related to the degree of memory. The resulting dynamics is described by a two-dimensional map. The map has a fixed point which may lose stability either via supercritical Neimark-Sacker bifurcation or flip bifurcation and several multistability situations exist. We describe some sequences of global bifurcations involving attracting and repelling closed invariant curves. These bifurcations, characterized by the creation of homoclinic connections or homoclinic tangles, are described through several numerical simulations. Particular bifurcation phenomena are also observed when the parameters are selected inside a periodicity region.

Dynamic properties in the four-state haploid coupled discrete-time mutation-selection model with an infinite population limit

NASA Astrophysics Data System (ADS)

Lee, Kyu Sang; Gill, Wonpyong

2017-11-01

The dynamic properties, such as the crossing time and time-dependence of the relative density of the four-state haploid coupled discrete-time mutation-selection model, were calculated with the assumption that μ ij = μ ji , where μ ij denotes the mutation rate between the sequence elements, i and j. The crossing time for s = 0 and r 23 = r 42 = 1 in the four-state model became saturated at a large fitness parameter when r 12 > 1, was scaled as a power law in the fitness parameter when r 12 = 1, and diverged when the fitness parameter approached the critical fitness parameter when r 12 < 1, where r ij = μ ij / μ 14.

Evolution of X-ray activity of 1-3 Msun late-type stars in early post-main-sequence phases

NASA Astrophysics Data System (ADS)

Pizzolato, N.; Maggio, A.; Sciortino, S.

2000-09-01

We have investigated the variation of coronal X-ray emission during early post-main-sequence phases for a sample of 120 late-type stars within 100 pc, and with estimated masses in the range 1-3 Msun, based on Hipparcos parallaxes and recent evolutionary models. These stars were observed with the ROSAT/PSPC, and the data processed with the Palermo-CfA pipeline, including detection and evaluation of X-ray fluxes (or upper limits) by means of a wavelet transform algorithm. We have studied the evolutionary history of X-ray luminosity and surface flux for stars in selected mass ranges, including stars with inactive A-type progenitors on the main sequence and lower mass solar-type stars. Our stellar sample suggests a trend of increasing X-ray emission level with age for stars with masses M > 1.5 Msun, and a decline for lower-mass stars. A similar behavior holds for the average coronal temperature, which follows a power-law correlation with the X-ray luminosity, independently of their mass and evolutionary state. We have also studied the relationship between X-ray luminosity and surface rotation rate for stars in the same mass ranges, and how this relationships departs from the Lx ~ vrot2 law followed by main-sequence stars. Our results are interpreted in terms of a magnetic dynamo whose efficiency depends on the stellar evolutionary state through the mass-dependent changes of the stellar internal structure, including the properties of envelope convection and the internal rotation profile.

Quantitative trait loci markers derived from whole genome sequence data increases the reliability of genomic prediction.

PubMed

Brøndum, R F; Su, G; Janss, L; Sahana, G; Guldbrandtsen, B; Boichard, D; Lund, M S

2015-06-01

This study investigated the effect on the reliability of genomic prediction when a small number of significant variants from single marker analysis based on whole genome sequence data were added to the regular 54k single nucleotide polymorphism (SNP) array data. The extra markers were selected with the aim of augmenting the custom low-density Illumina BovineLD SNP chip (San Diego, CA) used in the Nordic countries. The single-marker analysis was done breed-wise on all 16 index traits included in the breeding goals for Nordic Holstein, Danish Jersey, and Nordic Red cattle plus the total merit index itself. Depending on the trait's economic weight, 15, 10, or 5 quantitative trait loci (QTL) were selected per trait per breed and 3 to 5 markers were selected to tag each QTL. After removing duplicate markers (same marker selected for more than one trait or breed) and filtering for high pairwise linkage disequilibrium and assaying performance on the array, a total of 1,623 QTL markers were selected for inclusion on the custom chip. Genomic prediction analyses were performed for Nordic and French Holstein and Nordic Red animals using either a genomic BLUP or a Bayesian variable selection model. When using the genomic BLUP model including the QTL markers in the analysis, reliability was increased by up to 4 percentage points for production traits in Nordic Holstein animals, up to 3 percentage points for Nordic Reds, and up to 5 percentage points for French Holstein. Smaller gains of up to 1 percentage point was observed for mastitis, but only a 0.5 percentage point increase was seen for fertility. When using a Bayesian model accuracies were generally higher with only 54k data compared with the genomic BLUP approach, but increases in reliability were relatively smaller when QTL markers were included. Results from this study indicate that the reliability of genomic prediction can be increased by including markers significant in genome-wide association studies on whole genome sequence data alongside the 54k SNP set. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Structure prediction of Fe(II) 2-oxoglutarate dioxygenase from a psychrophilic yeast Glaciozyma antarctica PI12

NASA Astrophysics Data System (ADS)

Yusof, Nik Yusnoraini; Bakar, Farah Diba Abu; Mahadi, Nor Muhammad; Raih, Mohd Firdaus; Murad, Abdul Munir Abdul

2015-09-01

A cDNA encoding Fe(II) 2-oxoglutarate (2OG) dependent dioxygenases was isolated from psychrophilic yeast, Glaciozyma antarctica PI12. We have successfully amplified 1,029 bp cDNA sequence that encodes 342 amino acid with predicted molecular weight 38 kDa. The prediction protein was analysed using various bioinformatics tools to explore the properties of the protein. Based on a BLAST search analysis, the Fe2OX amino acid sequence showed 61% identity to the sequence of oxoglutarate/iron-dependent oxygenase from Rhodosporidium toruloides NP11. SignalP prediction showed that the Fe2OX protein contains no putative signal peptide, which suggests that this enzyme most probably localised intracellularly.The structure of Fe2OX was predicted by homology modelling using MODELLER9v11. The model with the lowest objective function was selected from hundred models generated using MODELLER9v11. Analysis of the structure revealed the longer loop at Fe2OX from G.antarctica that might be responsible for the flexibility of the structure, which contributes to its adaptation to low temperatures. Fe2OX hold a highly conserved Fe(II) binding HXD/E…H triad motif. The binding site for 2-oxoglutarate was found conserved for Arg280 among reported studies, however the Phe268 was found to be different in Fe2OX.

Selectivity in the Use of Gi/o Proteins Is Determined by the DRF Motif in CXCR6 and Is Cell-Type Specific.

PubMed

Singh, Satya P; Foley, John F; Zhang, Hongwei H; Hurt, Darrell E; Richards, Jennifer L; Smith, Craig S; Liao, Fang; Farber, Joshua M

2015-11-01

CXCR6, the receptor for CXCL16, is expressed on multiple cell types and can be a coreceptor for human immunodeficiency virus 1. Except for CXCR6, all human chemokine receptors contain the D(3.49)R(3.50)Y(3.51) sequence, and all but two contain A(3.53) at the cytoplasmic terminus of the third transmembrane helix (H3C), a region within class A G protein-coupled receptors that contacts G proteins. In CXCR6, H3C contains D(3.49)R(3.50)F(3.51)I(3.52)V(3.53) at positions 126-130. We investigated the importance and interdependence of the canonical D126 and the noncanonical F128 and V130 in CXCR6 by mutating D126 to Y, F128 to Y, and V130 to A singly and in combination. For comparison, we mutated the analogous positions D142, Y144, and A146 to Y, F, and V, respectively, in CCR6, a related receptor containing the canonical sequences. Mutants were analyzed in both human embryonic kidney 293T and Jurkat E6-1 cells. Our data show that for CXCR6 and/or CCR6, mutations in H3C can affect both receptor signaling and chemokine binding; noncanonical H3C sequences are functionally linked, with dual changes mitigating the effects of single mutations; mutations in H3C that compromise receptor activity show selective defects in the use of individual Gi/o proteins; and the effects of mutations in H3C on receptor function and selectivity in Gi/o protein use can be cell-type specific. Our findings indicate that the ability of CXCR6 to make promiscuous use of the available Gi/o proteins is exquisitely dependent on sequences within the H3C and suggest that the native sequence allows for preservation of this function across different cellular environments. U.S. Government work not protected by U.S. copyright.

Selectivity in the Use of Gi/o Proteins Is Determined by the DRF Motif in CXCR6 and Is Cell-Type Specific

PubMed Central

Singh, Satya P.; Foley, John F.; Zhang, Hongwei H.; Hurt, Darrell E.; Richards, Jennifer L.; Smith, Craig S.; Liao, Fang

2015-01-01

CXCR6, the receptor for CXCL16, is expressed on multiple cell types and can be a coreceptor for human immunodeficiency virus 1. Except for CXCR6, all human chemokine receptors contain the D3.49R3.50Y3.51 sequence, and all but two contain A3.53 at the cytoplasmic terminus of the third transmembrane helix (H3C), a region within class A G protein–coupled receptors that contacts G proteins. In CXCR6, H3C contains D3.49R3.50F3.51I3.52V3.53 at positions 126–130. We investigated the importance and interdependence of the canonical D126 and the noncanonical F128 and V130 in CXCR6 by mutating D126 to Y, F128 to Y, and V130 to A singly and in combination. For comparison, we mutated the analogous positions D142, Y144, and A146 to Y, F, and V, respectively, in CCR6, a related receptor containing the canonical sequences. Mutants were analyzed in both human embryonic kidney 293T and Jurkat E6-1 cells. Our data show that for CXCR6 and/or CCR6, mutations in H3C can affect both receptor signaling and chemokine binding; noncanonical H3C sequences are functionally linked, with dual changes mitigating the effects of single mutations; mutations in H3C that compromise receptor activity show selective defects in the use of individual Gi/o proteins; and the effects of mutations in H3C on receptor function and selectivity in Gi/o protein use can be cell-type specific. Our findings indicate that the ability of CXCR6 to make promiscuous use of the available Gi/o proteins is exquisitely dependent on sequences within the H3C and suggest that the native sequence allows for preservation of this function across different cellular environments. PMID:26316539

Toward a model for lexical access based on acoustic landmarks and distinctive features

NASA Astrophysics Data System (ADS)

Stevens, Kenneth N.

2002-04-01

This article describes a model in which the acoustic speech signal is processed to yield a discrete representation of the speech stream in terms of a sequence of segments, each of which is described by a set (or bundle) of binary distinctive features. These distinctive features specify the phonemic contrasts that are used in the language, such that a change in the value of a feature can potentially generate a new word. This model is a part of a more general model that derives a word sequence from this feature representation, the words being represented in a lexicon by sequences of feature bundles. The processing of the signal proceeds in three steps: (1) Detection of peaks, valleys, and discontinuities in particular frequency ranges of the signal leads to identification of acoustic landmarks. The type of landmark provides evidence for a subset of distinctive features called articulator-free features (e.g., [vowel], [consonant], [continuant]). (2) Acoustic parameters are derived from the signal near the landmarks to provide evidence for the actions of particular articulators, and acoustic cues are extracted by sampling selected attributes of these parameters in these regions. The selection of cues that are extracted depends on the type of landmark and on the environment in which it occurs. (3) The cues obtained in step (2) are combined, taking context into account, to provide estimates of ``articulator-bound'' features associated with each landmark (e.g., [lips], [high], [nasal]). These articulator-bound features, combined with the articulator-free features in (1), constitute the sequence of feature bundles that forms the output of the model. Examples of cues that are used, and justification for this selection, are given, as well as examples of the process of inferring the underlying features for a segment when there is variability in the signal due to enhancement gestures (recruited by a speaker to make a contrast more salient) or due to overlap of gestures from neighboring segments.

Design and characterization of a 52K SNP chip for goats.

PubMed

Tosser-Klopp, Gwenola; Bardou, Philippe; Bouchez, Olivier; Cabau, Cédric; Crooijmans, Richard; Dong, Yang; Donnadieu-Tonon, Cécile; Eggen, André; Heuven, Henri C M; Jamli, Saadiah; Jiken, Abdullah Johari; Klopp, Christophe; Lawley, Cynthia T; McEwan, John; Martin, Patrice; Moreno, Carole R; Mulsant, Philippe; Nabihoudine, Ibouniyamine; Pailhoux, Eric; Palhière, Isabelle; Rupp, Rachel; Sarry, Julien; Sayre, Brian L; Tircazes, Aurélie; Jun Wang; Wang, Wen; Zhang, Wenguang

2014-01-01

The success of Genome Wide Association Studies in the discovery of sequence variation linked to complex traits in humans has increased interest in high throughput SNP genotyping assays in livestock species. Primary goals are QTL detection and genomic selection. The purpose here was design of a 50-60,000 SNP chip for goats. The success of a moderate density SNP assay depends on reliable bioinformatic SNP detection procedures, the technological success rate of the SNP design, even spacing of SNPs on the genome and selection of Minor Allele Frequencies (MAF) suitable to use in diverse breeds. Through the federation of three SNP discovery projects consolidated as the International Goat Genome Consortium, we have identified approximately twelve million high quality SNP variants in the goat genome stored in a database together with their biological and technical characteristics. These SNPs were identified within and between six breeds (meat, milk and mixed): Alpine, Boer, Creole, Katjang, Saanen and Savanna, comprising a total of 97 animals. Whole genome and Reduced Representation Library sequences were aligned on >10 kb scaffolds of the de novo goat genome assembly. The 60,000 selected SNPs, evenly spaced on the goat genome, were submitted for oligo manufacturing (Illumina, Inc) and published in dbSNP along with flanking sequences and map position on goat assemblies (i.e. scaffolds and pseudo-chromosomes), sheep genome V2 and cattle UMD3.1 assembly. Ten breeds were then used to validate the SNP content and 52,295 loci could be successfully genotyped and used to generate a final cluster file. The combined strategy of using mainly whole genome Next Generation Sequencing and mapping on a contig genome assembly, complemented with Illumina design tools proved to be efficient in producing this GoatSNP50 chip. Advances in use of molecular markers are expected to accelerate goat genomic studies in coming years.

Design and Characterization of a 52K SNP Chip for Goats

PubMed Central

Tosser-Klopp, Gwenola; Bardou, Philippe; Bouchez, Olivier; Cabau, Cédric; Crooijmans, Richard; Dong, Yang; Donnadieu-Tonon, Cécile; Eggen, André; Heuven, Henri C. M.; Jamli, Saadiah; Jiken, Abdullah Johari; Klopp, Christophe; Lawley, Cynthia T.; McEwan, John; Martin, Patrice; Moreno, Carole R.; Mulsant, Philippe; Nabihoudine, Ibouniyamine; Pailhoux, Eric; Palhière, Isabelle; Rupp, Rachel; Sarry, Julien; Sayre, Brian L.; Tircazes, Aurélie; Jun Wang; Wang, Wen; Zhang, Wenguang

2014-01-01

The success of Genome Wide Association Studies in the discovery of sequence variation linked to complex traits in humans has increased interest in high throughput SNP genotyping assays in livestock species. Primary goals are QTL detection and genomic selection. The purpose here was design of a 50–60,000 SNP chip for goats. The success of a moderate density SNP assay depends on reliable bioinformatic SNP detection procedures, the technological success rate of the SNP design, even spacing of SNPs on the genome and selection of Minor Allele Frequencies (MAF) suitable to use in diverse breeds. Through the federation of three SNP discovery projects consolidated as the International Goat Genome Consortium, we have identified approximately twelve million high quality SNP variants in the goat genome stored in a database together with their biological and technical characteristics. These SNPs were identified within and between six breeds (meat, milk and mixed): Alpine, Boer, Creole, Katjang, Saanen and Savanna, comprising a total of 97 animals. Whole genome and Reduced Representation Library sequences were aligned on >10 kb scaffolds of the de novo goat genome assembly. The 60,000 selected SNPs, evenly spaced on the goat genome, were submitted for oligo manufacturing (Illumina, Inc) and published in dbSNP along with flanking sequences and map position on goat assemblies (i.e. scaffolds and pseudo-chromosomes), sheep genome V2 and cattle UMD3.1 assembly. Ten breeds were then used to validate the SNP content and 52,295 loci could be successfully genotyped and used to generate a final cluster file. The combined strategy of using mainly whole genome Next Generation Sequencing and mapping on a contig genome assembly, complemented with Illumina design tools proved to be efficient in producing this GoatSNP50 chip. Advances in use of molecular markers are expected to accelerate goat genomic studies in coming years. PMID:24465974

Analyzing the relationship between sequence divergence and nodal support using Bayesian phylogenetic analyses.

PubMed

Makowsky, Robert; Cox, Christian L; Roelke, Corey; Chippindale, Paul T

2010-11-01

Determining the appropriate gene for phylogeny reconstruction can be a difficult process. Rapidly evolving genes tend to resolve recent relationships, but suffer from alignment issues and increased homoplasy among distantly related species. Conversely, slowly evolving genes generally perform best for deeper relationships, but lack sufficient variation to resolve recent relationships. We determine the relationship between sequence divergence and Bayesian phylogenetic reconstruction ability using both natural and simulated datasets. The natural data are based on 28 well-supported relationships within the subphylum Vertebrata. Sequences of 12 genes were acquired and Bayesian analyses were used to determine phylogenetic support for correct relationships. Simulated datasets were designed to determine whether an optimal range of sequence divergence exists across extreme phylogenetic conditions. Across all genes we found that an optimal range of divergence for resolving the correct relationships does exist, although this level of divergence expectedly depends on the distance metric. Simulated datasets show that an optimal range of sequence divergence exists across diverse topologies and models of evolution. We determine that a simple to measure property of genetic sequences (genetic distance) is related to phylogenic reconstruction ability in Bayesian analyses. This information should be useful for selecting the most informative gene to resolve any relationships, especially those that are difficult to resolve, as well as minimizing both cost and confounding information during project design. Copyright © 2010. Published by Elsevier Inc.

Community detection in sequence similarity networks based on attribute clustering

DOE PAGES

Chowdhary, Janamejaya; Loeffler, Frank E.; Smith, Jeremy C.

2017-07-24

Networks are powerful tools for the presentation and analysis of interactions in multi-component systems. A commonly studied mesoscopic feature of networks is their community structure, which arises from grouping together similar nodes into one community and dissimilar nodes into separate communities. Here in this paper, the community structure of protein sequence similarity networks is determined with a new method: Attribute Clustering Dependent Communities (ACDC). Sequence similarity has hitherto typically been quantified by the alignment score or its expectation value. However, pair alignments with the same score or expectation value cannot thus be differentiated. To overcome this deficiency, the method constructs,more » for pair alignments, an extended alignment metric, the link attribute vector, which includes the score and other alignment characteristics. Rescaling components of the attribute vectors qualitatively identifies a systematic variation of sequence similarity within protein superfamilies. The problem of community detection is then mapped to clustering the link attribute vectors, selection of an optimal subset of links and community structure refinement based on the partition density of the network. ACDC-predicted communities are found to be in good agreement with gold standard sequence databases for which the "ground truth" community structures (or families) are known. ACDC is therefore a community detection method for sequence similarity networks based entirely on pair similarity information. A serial implementation of ACDC is available from https://cmb.ornl.gov/resources/developments« less

Community detection in sequence similarity networks based on attribute clustering

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chowdhary, Janamejaya; Loeffler, Frank E.; Smith, Jeremy C.

Networks are powerful tools for the presentation and analysis of interactions in multi-component systems. A commonly studied mesoscopic feature of networks is their community structure, which arises from grouping together similar nodes into one community and dissimilar nodes into separate communities. Here in this paper, the community structure of protein sequence similarity networks is determined with a new method: Attribute Clustering Dependent Communities (ACDC). Sequence similarity has hitherto typically been quantified by the alignment score or its expectation value. However, pair alignments with the same score or expectation value cannot thus be differentiated. To overcome this deficiency, the method constructs,more » for pair alignments, an extended alignment metric, the link attribute vector, which includes the score and other alignment characteristics. Rescaling components of the attribute vectors qualitatively identifies a systematic variation of sequence similarity within protein superfamilies. The problem of community detection is then mapped to clustering the link attribute vectors, selection of an optimal subset of links and community structure refinement based on the partition density of the network. ACDC-predicted communities are found to be in good agreement with gold standard sequence databases for which the "ground truth" community structures (or families) are known. ACDC is therefore a community detection method for sequence similarity networks based entirely on pair similarity information. A serial implementation of ACDC is available from https://cmb.ornl.gov/resources/developments« less

Primer-Free Aptamer Selection Using A Random DNA Library

PubMed Central

Pan, Weihua; Xin, Ping; Patrick, Susan; Dean, Stacey; Keating, Christine; Clawson, Gary

2010-01-01

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified through an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) to recognize a wide variety of targets, from small molecules to proteins and other macromolecules 2-4. Aptamers have properties that are well suited for in vivo diagnostic and/or therapeutic applications: Besides good specificity and affinity, they are easily synthesized, survive more rigorous processing conditions, they are poorly immunogenic, and their relatively small size can result in facile penetration of tissues. Aptamers that are identified through the standard SELEX process usually comprise ~80 nucleotides (nt), since they are typically selected from nucleic acid libraries with ~40 nt long randomized regions plus fixed primer sites of ~20 nt on each side. The fixed primer sequences thus can comprise nearly ~50% of the library sequences, and therefore may positively or negatively compromise identification of aptamers in the selection process 3, although bioinformatics approaches suggest that the fixed sequences do not contribute significantly to aptamer structure after selection 5. To address these potential problems, primer sequences have been blocked by complementary oligonucleotides or switched to different sequences midway during the rounds of SELEX 6, or they have been trimmed to 6-9 nt 7, 8. Wen and Gray 9 designed a primer-free genomic SELEX method, in which the primer sequences were completely removed from the library before selection and were then regenerated to allow amplification of the selected genomic fragments. However, to employ the technique, a unique genomic library has to be constructed, which possesses limited diversity, and regeneration after rounds of selection relies on a linear reamplification step. Alternatively, efforts to circumvent problems caused by fixed primer sequences using high efficiency partitioning are met with problems regarding PCR amplification 10. We have developed a primer-free (PF) selection method that significantly simplifies SELEX procedures and effectively eliminates primer-interference problems 11, 12. The protocols work in a straightforward manner. The central random region of the library is purified without extraneous flanking sequences and is bound to a suitable target (for example to a purified protein or complex mixtures such as cell lines). Then the bound sequences are obtained, reunited with flanking sequences, and re-amplified to generate selected sub-libraries. As an example, here we selected aptamers to S100B, a protein marker for melanoma. Binding assays showed Kd s in the 10-7 - 10-8 M range after a few rounds of selection, and we demonstrate that the aptamers function effectively in a sandwich binding format. PMID:20689511

Consolidating the effects of waking and sleep on motor-sequence learning.

PubMed

Brawn, Timothy P; Fenn, Kimberly M; Nusbaum, Howard C; Margoliash, Daniel

2010-10-20

Sleep is widely believed to play a critical role in memory consolidation. Sleep-dependent consolidation has been studied extensively in humans using an explicit motor-sequence learning paradigm. In this task, performance has been reported to remain stable across wakefulness and improve significantly after sleep, making motor-sequence learning the definitive example of sleep-dependent enhancement. Recent work, however, has shown that enhancement disappears when the task is modified to reduce task-related inhibition that develops over a training session, thus questioning whether sleep actively consolidates motor learning. Here we use the same motor-sequence task to demonstrate sleep-dependent consolidation for motor-sequence learning and explain the discrepancies in results across studies. We show that when training begins in the morning, motor-sequence performance deteriorates across wakefulness and recovers after sleep, whereas performance remains stable across both sleep and subsequent waking with evening training. This pattern of results challenges an influential model of memory consolidation defined by a time-dependent stabilization phase and a sleep-dependent enhancement phase. Moreover, the present results support a new account of the behavioral effects of waking and sleep on explicit motor-sequence learning that is consistent across a wide range of tasks. These observations indicate that current theories of memory consolidation that have been formulated to explain sleep-dependent performance enhancements are insufficient to explain the range of behavioral changes associated with sleep.

Identification of Bacterial Species in Kuwaiti Waters Through DNA Sequencing

NASA Astrophysics Data System (ADS)

Chen, K.

2017-01-01

With an objective of identifying the bacterial diversity associated with ecosystem of various Kuwaiti Seas, bacteria were cultured and isolated from 3 water samples. Due to the difficulties for cultured and isolated fecal coliforms on the selective agar plates, bacterial isolates from marine agar plates were selected for molecular identification. 16S rRNA genes were successfully amplified from the genome of the selected isolates using Universal Eubacterial 16S rRNA primers. The resulted amplification products were subjected to automated DNA sequencing. Partial 16S rDNA sequences obtained were compared directly with sequences in the NCBI database using BLAST as well as with the sequences available with Ribosomal Database Project (RDP).

A 20-Amino Acid Module of Protein Kinase Cϵ Involved in Translocation and Selective Targeting at Cell-Cell Contacts*

PubMed Central

Diouf, Barthélémy; Collazos, Alejandra; Labesse, Gilles; Macari, Françoise; Choquet, Armelle; Clair, Philippe; Gauthier-Rouvière, Cécile; Guérineau, Nathalie C.; Jay, Philippe; Hollande, Frédéric; Joubert, Dominique

2009-01-01

In the pituitary gland, activated protein kinase C (PKC) isoforms accumulate either selectively at the cell-cell contact (α and ϵ) or at the entire plasma membrane (β1 and δ). The molecular mechanisms underlying these various subcellular locations are not known. Here, we demonstrate the existence within PKCϵ of a cell-cell contact targeting sequence (3CTS) that, upon stimulation, is capable of targeting PKCδ, chimerin-α1, and the PKCϵ C1 domain to the cell-cell contact. We show that this selective targeting of PKCϵ is lost upon overexpression of 3CTS fused to a (R-Ahx-R)4 (where Ahx is 6-aminohexanoic acid) vectorization peptide, reflecting a dominant-negative effect of the overexpressed 3CTS on targeting selectivity. 3CTS contains a putative amphipathic α-helix, a 14-3-3-binding site, and the Glu-374 amino acid, involved in targeting selectivity. We show that the integrity of the α-helix is important for translocation but that 14-3-3 is not involved in targeting selectivity. However, PKCϵ translocation is increased when PKCϵ/14-3-3 interaction is abolished, suggesting that phorbol 12-myristate 13-acetate activation may initiate two sets of PKCϵ functions, those depending on 14-3-3 and those depending on translocation to cell-cell contacts. Thus, 3CTS is involved in the modulation of translocation via its 14-3-3-binding site, in cytoplasmic desequestration via the α-helix, and in selective PKCϵ targeting at the cell-cell contact via Glu-374. PMID:19429675

A general framework for optimization of probes for gene expression microarray and its application to the fungus Podospora anserina

PubMed Central

2010-01-01

Background The development of new microarray technologies makes custom long oligonucleotide arrays affordable for many experimental applications, notably gene expression analyses. Reliable results depend on probe design quality and selection. Probe design strategy should cope with the limited accuracy of de novo gene prediction programs, and annotation up-dating. We present a novel in silico procedure which addresses these issues and includes experimental screening, as an empirical approach is the best strategy to identify optimal probes in the in silico outcome. Findings We used four criteria for in silico probe selection: cross-hybridization, hairpin stability, probe location relative to coding sequence end and intron position. This latter criterion is critical when exon-intron gene structure predictions for intron-rich genes are inaccurate. For each coding sequence (CDS), we selected a sub-set of four probes. These probes were included in a test microarray, which was used to evaluate the hybridization behavior of each probe. The best probe for each CDS was selected according to three experimental criteria: signal-to-noise ratio, signal reproducibility, and representative signal intensities. This procedure was applied for the development of a gene expression Agilent platform for the filamentous fungus Podospora anserina and the selection of a single 60-mer probe for each of the 10,556 P. anserina CDS. Conclusions A reliable gene expression microarray version based on the Agilent 44K platform was developed with four spot replicates of each probe to increase statistical significance of analysis. PMID:20565839

A computational proposal for designing structured RNA pools for in vitro selection of RNAs.

PubMed

Kim, Namhee; Gan, Hin Hark; Schlick, Tamar

2007-04-01

Although in vitro selection technology is a versatile experimental tool for discovering novel synthetic RNA molecules, finding complex RNA molecules is difficult because most RNAs identified from random sequence pools are simple motifs, consistent with recent computational analysis of such sequence pools. Thus, enriching in vitro selection pools with complex structures could increase the probability of discovering novel RNAs. Here we develop an approach for engineering sequence pools that links RNA sequence space regions with corresponding structural distributions via a "mixing matrix" approach combined with a graph theory analysis. We define five classes of mixing matrices motivated by covariance mutations in RNA; these constructs define nucleotide transition rates and are applied to chosen starting sequences to yield specific nonrandom pools. We examine the coverage of sequence space as a function of the mixing matrix and starting sequence via clustering analysis. We show that, in contrast to random sequences, which are associated only with a local region of sequence space, our designed pools, including a structured pool for GTP aptamers, can target specific motifs. It follows that experimental synthesis of designed pools can benefit from using optimized starting sequences, mixing matrices, and pool fractions associated with each of our constructed pools as a guide. Automation of our approach could provide practical tools for pool design applications for in vitro selection of RNAs and related problems.

Processing multiple non-adjacent dependencies: evidence from sequence learning

PubMed Central

de Vries, Meinou H.; Petersson, Karl Magnus; Geukes, Sebastian; Zwitserlood, Pienie; Christiansen, Morten H.

2012-01-01

Processing non-adjacent dependencies is considered to be one of the hallmarks of human language. Assuming that sequence-learning tasks provide a useful way to tap natural-language-processing mechanisms, we cross-modally combined serial reaction time and artificial-grammar learning paradigms to investigate the processing of multiple nested (A1A2A3B3B2B1) and crossed dependencies (A1A2A3B1B2B3), containing either three or two dependencies. Both reaction times and prediction errors highlighted problems with processing the middle dependency in nested structures (A1A2A3B3_B1), reminiscent of the ‘missing-verb effect’ observed in English and French, but not with crossed structures (A1A2A3B1_B3). Prior linguistic experience did not play a major role: native speakers of German and Dutch—which permit nested and crossed dependencies, respectively—showed a similar pattern of results for sequences with three dependencies. As for sequences with two dependencies, reaction times and prediction errors were similar for both nested and crossed dependencies. The results suggest that constraints on the processing of multiple non-adjacent dependencies are determined by the specific ordering of the non-adjacent dependencies (i.e. nested or crossed), as well as the number of non-adjacent dependencies to be resolved (i.e. two or three). Furthermore, these constraints may not be specific to language but instead derive from limitations on structured sequence learning. PMID:22688641

Draft Genome Sequences of Several Fungal Strains Selected for Exposure to Microgravity at the International Space Station

DOE PAGES

Singh, Nitin K.; Blachowicz, Adriana; Romsdahl, Jillian; ...

2017-04-13

Presented here are the whole-genome sequences of eight fungal strains that were selected for exposure to microgravity at the International Space Station. These baseline sequences will help to understand the observed production of novel bioactive compounds.

Draft Genome Sequences of Several Fungal Strains Selected for Exposure to Microgravity at the International Space Station

DOE Office of Scientific and Technical Information (OSTI.GOV)

Singh, Nitin K.; Blachowicz, Adriana; Romsdahl, Jillian

Presented here are the whole-genome sequences of eight fungal strains that were selected for exposure to microgravity at the International Space Station. These baseline sequences will help to understand the observed production of novel bioactive compounds.

RNA-ID, a highly sensitive and robust method to identify cis-regulatory sequences using superfolder GFP and a fluorescence-based assay.

PubMed

Dean, Kimberly M; Grayhack, Elizabeth J

2012-12-01

We have developed a robust and sensitive method, called RNA-ID, to screen for cis-regulatory sequences in RNA using fluorescence-activated cell sorting (FACS) of yeast cells bearing a reporter in which expression of both superfolder green fluorescent protein (GFP) and yeast codon-optimized mCherry red fluorescent protein (RFP) is driven by the bidirectional GAL1,10 promoter. This method recapitulates previously reported progressive inhibition of translation mediated by increasing numbers of CGA codon pairs, and restoration of expression by introduction of a tRNA with an anticodon that base pairs exactly with the CGA codon. This method also reproduces effects of paromomycin and context on stop codon read-through. Five key features of this method contribute to its effectiveness as a selection for regulatory sequences: The system exhibits greater than a 250-fold dynamic range, a quantitative and dose-dependent response to known inhibitory sequences, exquisite resolution that allows nearly complete physical separation of distinct populations, and a reproducible signal between different cells transformed with the identical reporter, all of which are coupled with simple methods involving ligation-independent cloning, to create large libraries. Moreover, we provide evidence that there are sequences within a 9-nt library that cause reduced GFP fluorescence, suggesting that there are novel cis-regulatory sequences to be found even in this short sequence space. This method is widely applicable to the study of both RNA-mediated and codon-mediated effects on expression.

Noise-robust speech recognition through auditory feature detection and spike sequence decoding.

PubMed

Schafer, Phillip B; Jin, Dezhe Z

2014-03-01

Speech recognition in noisy conditions is a major challenge for computer systems, but the human brain performs it routinely and accurately. Automatic speech recognition (ASR) systems that are inspired by neuroscience can potentially bridge the performance gap between humans and machines. We present a system for noise-robust isolated word recognition that works by decoding sequences of spikes from a population of simulated auditory feature-detecting neurons. Each neuron is trained to respond selectively to a brief spectrotemporal pattern, or feature, drawn from the simulated auditory nerve response to speech. The neural population conveys the time-dependent structure of a sound by its sequence of spikes. We compare two methods for decoding the spike sequences--one using a hidden Markov model-based recognizer, the other using a novel template-based recognition scheme. In the latter case, words are recognized by comparing their spike sequences to template sequences obtained from clean training data, using a similarity measure based on the length of the longest common sub-sequence. Using isolated spoken digits from the AURORA-2 database, we show that our combined system outperforms a state-of-the-art robust speech recognizer at low signal-to-noise ratios. Both the spike-based encoding scheme and the template-based decoding offer gains in noise robustness over traditional speech recognition methods. Our system highlights potential advantages of spike-based acoustic coding and provides a biologically motivated framework for robust ASR development.

A new feedback image encryption scheme based on perturbation with dynamical compound chaotic sequence cipher generator

NASA Astrophysics Data System (ADS)

Tong, Xiaojun; Cui, Minggen; Wang, Zhu

2009-07-01

The design of the new compound two-dimensional chaotic function is presented by exploiting two one-dimensional chaotic functions which switch randomly, and the design is used as a chaotic sequence generator which is proved by Devaney's definition proof of chaos. The properties of compound chaotic functions are also proved rigorously. In order to improve the robustness against difference cryptanalysis and produce avalanche effect, a new feedback image encryption scheme is proposed using the new compound chaos by selecting one of the two one-dimensional chaotic functions randomly and a new image pixels method of permutation and substitution is designed in detail by array row and column random controlling based on the compound chaos. The results from entropy analysis, difference analysis, statistical analysis, sequence randomness analysis, cipher sensitivity analysis depending on key and plaintext have proven that the compound chaotic sequence cipher can resist cryptanalytic, statistical and brute-force attacks, and especially it accelerates encryption speed, and achieves higher level of security. By the dynamical compound chaos and perturbation technology, the paper solves the problem of computer low precision of one-dimensional chaotic function.

Influence of time and length size feature selections for human activity sequences recognition.

PubMed

Fang, Hongqing; Chen, Long; Srinivasan, Raghavendiran

2014-01-01

In this paper, Viterbi algorithm based on a hidden Markov model is applied to recognize activity sequences from observed sensors events. Alternative features selections of time feature values of sensors events and activity length size feature values are tested, respectively, and then the results of activity sequences recognition performances of Viterbi algorithm are evaluated. The results show that the selection of larger time feature values of sensor events and/or smaller activity length size feature values will generate relatively better results on the activity sequences recognition performances. © 2013 ISA Published by ISA All rights reserved.

New radio detections of early-type pre-main-sequence stars

NASA Technical Reports Server (NTRS)

Skinner, Stephen L.; Brown, Alexander; Linsky, Jeffrey L.

1990-01-01

Results of VLA radio continuum observations of 13 early-type pre-main-sequence stars selected from the 1984 catalog of Finkenzeller and Mundt are presented. The stars HD 259431 and MWC 1080 were detected at 3.6 cm, while HD 200775 and TY CrA were detected at both 3.6 and 6 cm. The flux density of HD 200775 has a frequency dependence consistent with the behavior expected for free-free emission originating in a fully ionized wind. However, an observation in A configuration suggests that the source geometry may not be spherically symmetric. In contrast, the spectral index of TY CrA is negative with a flux behavior implying nonthermal emission. The physical mechanism responsible for the nonthermal emission has not yet been identified, although gyrosynchrotron and synchrotron processes cannot be ruled out.

TCL1A, a Novel Transcription Factor and a Coregulator of Nuclear Factor κB p65: Single Nucleotide Polymorphism and Estrogen Dependence.

PubMed

Ho, Ming-Fen; Lummertz da Rocha, Edroaldo; Zhang, Cheng; Ingle, James N; Goss, Paul E; Shepherd, Lois E; Kubo, Michiaki; Wang, Liewei; Li, Hu; Weinshilboum, Richard M

2018-06-01

T-cell leukemia 1A ( TCL1A ) single-nucleotide polymorphisms (SNPs) have been associated with aromatase inhibitor-induced musculoskeletal adverse events. We previously demonstrated that TCL1A is inducible by estradiol (E 2 ) and plays a critical role in the regulation of cytokines, chemokines, and Toll-like receptors in a TCL1A SNP genotype and estrogen-dependent fashion. Furthermore, TCLIA SNP-dependent expression phenotypes can be "reversed" by exposure to selective estrogen receptor modulators such as 4-hydroxytamoxifen (4OH-TAM). The present study was designed to comprehensively characterize the role of TCL1A in transcriptional regulation across the genome by performing RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) assays with lymphoblastoid cell lines. RNA-seq identified 357 genes that were regulated in a TCL1A SNP- and E 2 -dependent fashion with expression patterns that were 4OH-TAM reversible. ChIP-seq for the same cells identified 57 TCL1A binding sites that could be regulated by E 2 in a SNP-dependent fashion. Even more striking, nuclear factor- κ B (NF- κ B) p65 bound to those same DNA regions. In summary, TCL1A is a novel transcription factor with expression that is regulated in a SNP- and E 2 -dependent fashion-a pattern of expression that can be reversed by 4OH-TAM. Integrated RNA-seq and ChIP-seq results suggest that TCL1A also acts as a transcriptional coregulator with NF- κ B p65, an important immune system transcription factor. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

cgDNAweb: a web interface to the cgDNA sequence-dependent coarse-grain model of double-stranded DNA.

PubMed

De Bruin, Lennart; Maddocks, John H

2018-06-14

The sequence-dependent statistical mechanical properties of fragments of double-stranded DNA is believed to be pertinent to its biological function at length scales from a few base pairs (or bp) to a few hundreds of bp, e.g. indirect read-out protein binding sites, nucleosome positioning sequences, phased A-tracts, etc. In turn, the equilibrium statistical mechanics behaviour of DNA depends upon its ground state configuration, or minimum free energy shape, as well as on its fluctuations as governed by its stiffness (in an appropriate sense). We here present cgDNAweb, which provides browser-based interactive visualization of the sequence-dependent ground states of double-stranded DNA molecules, as predicted by the underlying cgDNA coarse-grain rigid-base model of fragments with arbitrary sequence. The cgDNAweb interface is specifically designed to facilitate comparison between ground state shapes of different sequences. The server is freely available at cgDNAweb.epfl.ch with no login requirement.

Adaptive Learning Resources Sequencing in Educational Hypermedia Systems

ERIC Educational Resources Information Center

Karampiperis, Pythagoras; Sampson, Demetrios

2005-01-01

Adaptive learning resources selection and sequencing is recognized as among the most interesting research questions in adaptive educational hypermedia systems (AEHS). In order to adaptively select and sequence learning resources in AEHS, the definition of adaptation rules contained in the Adaptation Model, is required. Although, some efforts have…

Draft Genome Sequences of Several Fungal Strains Selected for Exposure to Microgravity at the International Space Station

PubMed Central

Singh, Nitin K.; Blachowicz, Adriana; Romsdahl, Jillian; Wang, Clay; Torok, Tamas

2017-01-01

ABSTRACT The whole-genome sequences of eight fungal strains that were selected for exposure to microgravity at the International Space Station are presented here. These baseline sequences will help to understand the observed production of novel bioactive compounds. PMID:28408692

Sequence-similar, structure-dissimilar protein pairs in the PDB.

PubMed

Kosloff, Mickey; Kolodny, Rachel

2008-05-01

It is often assumed that in the Protein Data Bank (PDB), two proteins with similar sequences will also have similar structures. Accordingly, it has proved useful to develop subsets of the PDB from which "redundant" structures have been removed, based on a sequence-based criterion for similarity. Similarly, when predicting protein structure using homology modeling, if a template structure for modeling a target sequence is selected by sequence alone, this implicitly assumes that all sequence-similar templates are equivalent. Here, we show that this assumption is often not correct and that standard approaches to create subsets of the PDB can lead to the loss of structurally and functionally important information. We have carried out sequence-based structural superpositions and geometry-based structural alignments of a large number of protein pairs to determine the extent to which sequence similarity ensures structural similarity. We find many examples where two proteins that are similar in sequence have structures that differ significantly from one another. The source of the structural differences usually has a functional basis. The number of such proteins pairs that are identified and the magnitude of the dissimilarity depend on the approach that is used to calculate the differences; in particular sequence-based structure superpositioning will identify a larger number of structurally dissimilar pairs than geometry-based structural alignments. When two sequences can be aligned in a statistically meaningful way, sequence-based structural superpositioning provides a meaningful measure of structural differences. This approach and geometry-based structure alignments reveal somewhat different information and one or the other might be preferable in a given application. Our results suggest that in some cases, notably homology modeling, the common use of nonredundant datasets, culled from the PDB based on sequence, may mask important structural and functional information. We have established a data base of sequence-similar, structurally dissimilar protein pairs that will help address this problem (http://luna.bioc.columbia.edu/rachel/seqsimstrdiff.htm).

Development and validation of a D-loop mtDNA SNP assay for the screening of specimens in forensic casework.

PubMed

Chemale, Gustavo; Paneto, Greiciane Gaburro; Menezes, Meiga Aurea Mendes; de Freitas, Jorge Marcelo; Jacques, Guilherme Silveira; Cicarelli, Regina Maria Barretto; Fagundes, Paulo Roberto

2013-05-01

Mitochondrial DNA (mtDNA) analysis is usually a last resort in routine forensic DNA casework. However, it has become a powerful tool for the analysis of highly degraded samples or samples containing too little or no nuclear DNA, such as old bones and hair shafts. The gold standard methodology still constitutes the direct sequencing of polymerase chain reaction (PCR) products or cloned amplicons from the HVS-1 and HVS-2 (hypervariable segment) control region segments. Identifications using mtDNA are time consuming, expensive and can be very complex, depending on the amount and nature of the material being tested. The main goal of this work is to develop a less labour-intensive and less expensive screening method for mtDNA analysis, in order to aid in the exclusion of non-matching samples and as a presumptive test prior to final confirmatory DNA sequencing. We have selected 14 highly discriminatory single nucleotide polymorphisms (SNPs) based on simulations performed by Salas and Amigo (2010) to be typed using SNaPShot(TM) (Applied Biosystems, Foster City, CA, USA). The assay was validated by typing more than 100 HVS-1/HVS-2 sequenced samples. No differences were observed between the SNP typing and DNA sequencing when results were compared, with the exception of allelic dropouts observed in a few haplotypes. Haplotype diversity simulations were performed using 172 mtDNA sequences representative of the Brazilian population and a score of 0.9794 was obtained when the 14 SNPs were used, showing that the theoretical prediction approach for the selection of highly discriminatory SNPs suggested by Salas and Amigo (2010) was confirmed in the population studied. As the main goal of the work is to develop a screening assay to skip the sequencing of all samples in a particular case, a pair-wise comparison of the sequences was done using the selected SNPs. When both HVS-1/HVS-2 SNPs were used for simulations, at least two differences were observed in 93.2% of the comparisons performed. The assay was validated with casework samples. Results show that the method is straightforward and can be used for exclusionary purposes, saving time and laboratory resources. The assay confirms the theoretic prediction suggested by Salas and Amigo (2010). All forensic advantages, such as high sensitivity and power of discrimination, as also the disadvantages, such as the occurrence of allele dropouts, are discussed throughout the article. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Polymorphism and selection in the major histocompatibility complex DRA and DQA genes in the family Equidae.

PubMed

Janova, Eva; Matiasovic, Jan; Vahala, Jiri; Vodicka, Roman; Van Dyk, Enette; Horin, Petr

2009-07-01

The major histocompatibility complex genes coding for antigen binding and presenting molecules are the most polymorphic genes in the vertebrate genome. We studied the DRA and DQA gene polymorphism of the family Equidae. In addition to 11 previously reported DRA and 24 DQA alleles, six new DRA sequences and 13 new DQA alleles were identified in the genus Equus. Phylogenetic analysis of both DRA and DQA sequences provided evidence for trans-species polymorphism in the family Equidae. The phylogenetic trees differed from species relationships defined by standard taxonomy of Equidae and from trees based on mitochondrial or neutral gene sequence data. Analysis of selection showed differences between the less variable DRA and more variable DQA genes. DRA alleles were more often shared by more species. The DQA sequences analysed showed strong amongst-species positive selection; the selected amino acid positions mostly corresponded to selected positions in rodent and human DQA genes.

EVIDENCE FOR THE UNIVERSALITY OF PROPERTIES OF RED-SEQUENCE GALAXIES IN X-RAY- AND RED-SEQUENCE-SELECTED CLUSTERS AT z ∼ 1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Foltz, R.; Wilson, G.; DeGroot, A.

We study the slope, intercept, and scatter of the color–magnitude and color–mass relations for a sample of 10 infrared red-sequence-selected clusters at z ∼ 1. The quiescent galaxies in these clusters formed the bulk of their stars above z ≳ 3 with an age spread Δt ≳ 1 Gyr. We compare UVJ color–color and spectroscopic-based galaxy selection techniques, and find a 15% difference in the galaxy populations classified as quiescent by these methods. We compare the color–magnitude relations from our red-sequence selected sample with X-ray- and photometric-redshift-selected cluster samples of similar mass and redshift. Within uncertainties, we are unable tomore » detect any difference in the ages and star formation histories of quiescent cluster members in clusters selected by different methods, suggesting that the dominant quenching mechanism is insensitive to cluster baryon partitioning at z ∼ 1.« less

Branchpoint selection in the splicing of U12-dependent introns in vitro.

PubMed

McConnell, Timothy S; Cho, Soo-Jin; Frilander, Mikko J; Steitz, Joan A

2002-05-01

In metazoans, splicing of introns from pre-mRNAs can occur by two pathways: the major U2-dependent or the minor U12-dependent pathways. Whereas the U2-dependent pathway has been well characterized, much about the U12-dependent pathway remains to be discovered. Most of the information regarding U12-type introns has come from in vitro studies of a very few known introns of this class. To expand our understanding of U12-type splicing, especially to test the hypothesis that the simple base-pairing mechanism between the intron and U12 snRNA defines the branchpoint of U12-dependent introns, additional in vitro splicing substrates were created from three putative U12-type introns: the third intron of the Xenopus RPL1 a gene (XRP), the sixth intron of the Xenopus TFIIS.oA gene (XTF), and the first intron of the human Sm E gene (SME). In vitro splicing in HeLa nuclear extract confirmed U12-dependent splicing of each of these introns. Surprisingly, branchpoint mapping of the XRP splicing intermediate shows use of the upstream rather than the downstream of two consecutive adenosines within the branchpoint sequence (BPS), contrary to the prediction based on alignment with the sixth intron of human P120, a U12-dependent intron whose branch site was previously determined. Also, in the SME intron, the position of the branchpoint A residue within the region base paired with U12 differs from that in P120 and XTF. Analysis of these three additional introns therefore rules out simple models for branchpoint selection by the U12-type spliceosome.

Branchpoint selection in the splicing of U12-dependent introns in vitro.

PubMed Central

McConnell, Timothy S; Cho, Soo-Jin; Frilander, Mikko J; Steitz, Joan A

2002-01-01

In metazoans, splicing of introns from pre-mRNAs can occur by two pathways: the major U2-dependent or the minor U12-dependent pathways. Whereas the U2-dependent pathway has been well characterized, much about the U12-dependent pathway remains to be discovered. Most of the information regarding U12-type introns has come from in vitro studies of a very few known introns of this class. To expand our understanding of U12-type splicing, especially to test the hypothesis that the simple base-pairing mechanism between the intron and U12 snRNA defines the branchpoint of U12-dependent introns, additional in vitro splicing substrates were created from three putative U12-type introns: the third intron of the Xenopus RPL1 a gene (XRP), the sixth intron of the Xenopus TFIIS.oA gene (XTF), and the first intron of the human Sm E gene (SME). In vitro splicing in HeLa nuclear extract confirmed U12-dependent splicing of each of these introns. Surprisingly, branchpoint mapping of the XRP splicing intermediate shows use of the upstream rather than the downstream of two consecutive adenosines within the branchpoint sequence (BPS), contrary to the prediction based on alignment with the sixth intron of human P120, a U12-dependent intron whose branch site was previously determined. Also, in the SME intron, the position of the branchpoint A residue within the region base paired with U12 differs from that in P120 and XTF. Analysis of these three additional introns therefore rules out simple models for branchpoint selection by the U12-type spliceosome. PMID:12022225

The functional importance of sequence versus expression variability of MHC alleles in parasite resistance.

PubMed

Axtner, Jan; Sommer, Simone

2012-12-01

Understanding selection processes driving the pronounced allelic polymorphism of the major histocompatibility complex (MHC) genes and its functional associations to parasite load have been the focus of many recent wildlife studies. Two main selection scenarios are currently debated which explain the susceptibility or resistance to parasite infections either by the effects of (1) specific MHC alleles which are selected frequency-dependent in space and time or (2) a heterozygote or divergent allele advantage. So far, most studies have focused only on structural variance in co-evolutionary processes although this might not be the only trait subject to natural selection. In the present study, we analysed structural variance stretching from exon1 through exon3 of MHC class II DRB genes as well as genotypic expression variance in relation to the gastrointestinal helminth prevalence and infection intensity in wild yellow-necked mice (Apodemus flavicollis). We found support for the functional importance of specific alleles both on the sequence and expression level. By resampling a previously investigated study population we identified specific MHC alleles affected by temporal shifts in parasite pressure and recorded associated changes in allele frequencies. The allele Apfl-DRB*23 was associated with resistance to infections by the oxyurid nematode Syphacia stroma and at the same time with susceptibility to cestode infection intensity. In line with our expectation, MHC mRNA transcript levels tended to be higher in cestode-infected animals carrying the allele Apfl-DRB*23. However, no support for a heterozygote or divergent allele advantage on the sequence or expression level was detected. The individual amino acid distance of genotypes did not explain individual differences in parasite loads and the genetic distance had no effect on MHC genotype expression. For ongoing studies on the functional importance of expression variance in parasite resistance, allele-specific expression data would be preferable.

Characterization of host-dependent mutations of apple fruit crinkle viroid replicating in newly identified experimental hosts suggests maintenance of stem-loop structures in the left-hand half of the molecule is important for replication.

PubMed

Suzuki, Takahiro; Fujibayashi, Misato; Hataya, Tatsuji; Taneda, Akito; He, Ying-Hong; Tsushima, Taro; Duraisamy, Ganesh Selvaraj; Siglová, Kristyna; Matoušek, Jaroslav; Sano, Teruo

2017-03-01

Apple fruit crinkle viroid (AFCVd) is a tentative member of the genus Apscaviroid, family Pospiviroidae. AFCVd has a narrow host range and is known to infect apple, hop and persimmon as natural hosts. In this study, tomato, cucumber and wild hop have been identified as new experimental herbaceous hosts. Foliar symptoms were very mild or virtually undetectable, but fruits of infected tomato were small, cracked and distorted. These symptoms resemble those observed on some AFCVd-sensitive apple cultivars. After transfer to tomato, cucumber and wild hop, sequence changes were detected in a natural AFCVd isolate from hop, and major variants in tomato, cucumber and wild hop differed in 10, 8 or 2 nucleotides, respectively, from the predominant one in the inoculum. The major variants in tomato and cucumber were almost identical, and the one in wild hop was very similar to the one in cultivated hop. Detailed analyses of the host-dependent sequence changes that appear in a naturally occurring AFCVd isolate from hop after transfer to tomato using small RNA deep sequence data and infectivity studies with dimeric RNA transcripts followed by progeny analysis indicate that the major AFCVd variant in tomato emerged by selection of a minor variant present in the inoculum (i.e. hop) followed by one to two host-dependent de novo mutations. Comparison of the secondary structures of major variants in hop, tomato and persimmon after transfer to tomato suggested that maintenance of stem-loop structures in the left-hand half of the molecule is critical for infection.

Prevalence of MLH1 constitutional epimutations as a cause of Lynch syndrome in unselected versus selected consecutive series of patients with colorectal cancer.

PubMed

Castillejo, Adela; Hernández-Illán, Eva; Rodriguez-Soler, María; Pérez-Carbonell, Lucía; Egoavil, Cecilia; Barberá, Victor M; Castillejo, María-Isabel; Guarinos, Carla; Martínez-de-Dueñas, Eduardo; Juan, María-Jose; Sánchez-Heras, Ana-Beatriz; García-Casado, Zaida; Ruiz-Ponte, Clara; Brea-Fernández, Alejandro; Juárez, Miriam; Bujanda, Luis; Clofent, Juan; Llor, Xavier; Andreu, Montserrat; Castells, Antoni; Carracedo, Angel; Alenda, Cristina; Payá, Artemio; Jover, Rodrigo; Soto, José-Luis

2015-07-01

The prevalence of MLH1 constitutional epimutations in the general population is unknown. We sought to analyse the prevalence of MLH1 constitutional epimutations in unselected and selected series of patients with colorectal cancer (CRC). Patients with diagnoses of CRC (n=2123) were included in the unselected group. For comparison, a group of 847 selected patients with CRC who fulfilled the revised Bethesda guidelines (rBG) were also included. Somatic and constitutional MLH1 methylation was assayed via methylation-specific multiplex ligation-dependent probe amplification of cases lacking MLH1 expression. Germline alterations in mismatch-repair (MMR) genes were assessed via Sanger sequencing and methylation-specific multiplex ligation-dependent probe amplification. Loss of MLH1 expression occurred in 5.5% of the unselected series and 12.5% of the selected series (p<0.0001). No constitutional epimutations in MLH1 were detected in the unselected population (0/62); five cases from the selected series were positive for MLH1 epimutations (15.6%, 5/32; p=0.004). Our results suggest a negligible prevalence of MLH1 constitutional epimutations in unselected cases of CRC. Therefore, MLH1 constitutional epimutation analysis should be conducted only for patients who fulfil the rBG and who lack MLH1 expression with methylated MLH1. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

Reduced TCR signaling potential impairs negative selection but does not result in autoimmune disease

PubMed Central

Hwang, SuJin; Song, Ki-Duk; Lesourne, Renaud; Lee, Jan; Pinkhasov, Julia; Li, LiQi; El-Khoury, Dalal

2012-01-01

Negative selection and regulatory T (T reg) cell development are two thymus-dependent processes necessary for the enforcement of self-tolerance, and both require high-affinity interactions between the T cell receptor (TCR) and self-ligands. However, it remains unclear if they are similarly impacted by alterations in TCR signaling potential. We generated a knock-in allele (6F) of the TCR ζ chain gene encoding a mutant protein lacking signaling capability whose expression is controlled by endogenous ζ regulatory sequences. Although negative selection was defective in 6F/6F mice, leading to the survival of autoreactive T cells, 6F/6F mice did not develop autoimmune disease. We found that 6F/6F mice generated increased numbers of thymus-derived T reg cells. We show that attenuation of TCR signaling potential selectively impacts downstream signaling responses and that this differential effect favors Foxp3 expression and T reg cell lineage commitment. These results identify a potential compensatory pathway for the enforcement of immune tolerance in response to defective negative selection caused by reduced TCR signaling capability. PMID:22945921

Rhipicephalus (Boophilus) microplus strain Deutsch, whole genome shotgun sequencing project first submission of genome sequence

USDA-ARS?s Scientific Manuscript database

The size and repetitive nature of the Rhipicephalus microplus genome makes obtaining a full genome sequence difficult. Cot filtration/selection techniques were used to reduce the repetitive fraction of the tick genome and enrich for the fraction of DNA with gene-containing regions. The Cot-selected ...

Processing Dynamic Image Sequences from a Moving Sensor.

DTIC Science & Technology

1984-02-01

65 Roadsign Image Sequence ..... ................ ... 70 Roadsign Sequence with Redundant Features .. ........ . 79 Roadsign Subimage...Selected Feature Error Values .. ........ 66 2c. Industrial Image Selected Feature Local Search Values. .. .... 67 3ab. Roadsign Image Error Values...72 3c. Roadsign Image Local Search Values ............. 73 4ab. Roadsign Redundant Feature Error Values. ............ 8 4c. Roadsign

Draft Genome Sequences of Several Fungal Strains Selected for Exposure to Microgravity at the International Space Station.

PubMed

Singh, Nitin K; Blachowicz, Adriana; Romsdahl, Jillian; Wang, Clay; Torok, Tamas; Venkateswaran, Kasthuri

2017-04-13

The whole-genome sequences of eight fungal strains that were selected for exposure to microgravity at the International Space Station are presented here. These baseline sequences will help to understand the observed production of novel bioactive compounds. Copyright © 2017 Singh et al.

Identifying functionally informative evolutionary sequence profiles.

PubMed

Gil, Nelson; Fiser, Andras

2018-04-15

Multiple sequence alignments (MSAs) can provide essential input to many bioinformatics applications, including protein structure prediction and functional annotation. However, the optimal selection of sequences to obtain biologically informative MSAs for such purposes is poorly explored, and has traditionally been performed manually. We present Selection of Alignment by Maximal Mutual Information (SAMMI), an automated, sequence-based approach to objectively select an optimal MSA from a large set of alternatives sampled from a general sequence database search. The hypothesis of this approach is that the mutual information among MSA columns will be maximal for those MSAs that contain the most diverse set possible of the most structurally and functionally homogeneous protein sequences. SAMMI was tested to select MSAs for functional site residue prediction by analysis of conservation patterns on a set of 435 proteins obtained from protein-ligand (peptides, nucleic acids and small substrates) and protein-protein interaction databases. Availability and implementation: A freely accessible program, including source code, implementing SAMMI is available at https://github.com/nelsongil92/SAMMI.git. andras.fiser@einstein.yu.edu. Supplementary data are available at Bioinformatics online.

Neutrality and evolvability of designed protein sequences

NASA Astrophysics Data System (ADS)

Bhattacherjee, Arnab; Biswas, Parbati

2010-07-01

The effect of foldability on protein’s evolvability is analyzed by a two-prong approach consisting of a self-consistent mean-field theory and Monte Carlo simulations. Theory and simulation models representing protein sequences with binary patterning of amino acid residues compatible with a particular foldability criteria are used. This generalized foldability criterion is derived using the high temperature cumulant expansion approximating the free energy of folding. The effect of cumulative point mutations on these designed proteins is studied under neutral condition. The robustness, protein’s ability to tolerate random point mutations is determined with a selective pressure of stability (ΔΔG) for the theory designed sequences, which are found to be more robust than that of Monte Carlo and mean-field-biased Monte Carlo generated sequences. The results show that this foldability criterion selects viable protein sequences more effectively compared to the Monte Carlo method, which has a marked effect on how the selective pressure shapes the evolutionary sequence space. These observations may impact de novo sequence design and its applications in protein engineering.

Studies on DNA-binding selectivity of WRKY transcription factors lend structural clues into WRKY-domain function.

PubMed

Ciolkowski, Ingo; Wanke, Dierk; Birkenbihl, Rainer P; Somssich, Imre E

2008-09-01

WRKY transcription factors have been shown to play a major role in regulating, both positively and negatively, the plant defense transcriptome. Nearly all studied WRKY factors appear to have a stereotypic binding preference to one DNA element termed the W-box. How specificity for certain promoters is accomplished therefore remains completely unknown. In this study, we tested five distinct Arabidopsis WRKY transcription factor subfamily members for their DNA binding selectivity towards variants of the W-box embedded in neighboring DNA sequences. These studies revealed for the first time differences in their binding site preferences, which are partly dependent on additional adjacent DNA sequences outside of the TTGACY-core motif. A consensus WRKY binding site derived from these studies was used for in silico analysis to identify potential target genes within the Arabidopsis genome. Furthermore, we show that even subtle amino acid substitutions within the DNA binding region of AtWRKY11 strongly impinge on its binding activity. Additionally, all five factors were found localized exclusively to the plant cell nucleus and to be capable of trans-activating expression of a reporter gene construct in vivo.

Self-assembled bionanostructures: proteins following the lead of DNA nanostructures

PubMed Central

2014-01-01

Natural polymers are able to self-assemble into versatile nanostructures based on the information encoded into their primary structure. The structural richness of biopolymer-based nanostructures depends on the information content of building blocks and the available biological machinery to assemble and decode polymers with a defined sequence. Natural polypeptides comprise 20 amino acids with very different properties in comparison to only 4 structurally similar nucleotides, building elements of nucleic acids. Nevertheless the ease of synthesizing polynucleotides with selected sequence and the ability to encode the nanostructural assembly based on the two specific nucleotide pairs underlay the development of techniques to self-assemble almost any selected three-dimensional nanostructure from polynucleotides. Despite more complex design rules, peptides were successfully used to assemble symmetric nanostructures, such as fibrils and spheres. While earlier designed protein-based nanostructures used linked natural oligomerizing domains, recent design of new oligomerizing interaction surfaces and introduction of the platform for topologically designed protein fold may enable polypeptide-based design to follow the track of DNA nanostructures. The advantages of protein-based nanostructures, such as the functional versatility and cost effective and sustainable production methods provide strong incentive for further development in this direction. PMID:24491139

Improved Model for Predicting the Free Energy Contribution of Dinucleotide Bulges to RNA Duplex Stability.

PubMed

Tomcho, Jeremy C; Tillman, Magdalena R; Znosko, Brent M

2015-09-01

Predicting the secondary structure of RNA is an intermediate in predicting RNA three-dimensional structure. Commonly, determining RNA secondary structure from sequence uses free energy minimization and nearest neighbor parameters. Current algorithms utilize a sequence-independent model to predict free energy contributions of dinucleotide bulges. To determine if a sequence-dependent model would be more accurate, short RNA duplexes containing dinucleotide bulges with different sequences and nearest neighbor combinations were optically melted to derive thermodynamic parameters. These data suggested energy contributions of dinucleotide bulges were sequence-dependent, and a sequence-dependent model was derived. This model assigns free energy penalties based on the identity of nucleotides in the bulge (3.06 kcal/mol for two purines, 2.93 kcal/mol for two pyrimidines, 2.71 kcal/mol for 5'-purine-pyrimidine-3', and 2.41 kcal/mol for 5'-pyrimidine-purine-3'). The predictive model also includes a 0.45 kcal/mol penalty for an A-U pair adjacent to the bulge and a -0.28 kcal/mol bonus for a G-U pair adjacent to the bulge. The new sequence-dependent model results in predicted values within, on average, 0.17 kcal/mol of experimental values, a significant improvement over the sequence-independent model. This model and new experimental values can be incorporated into algorithms that predict RNA stability and secondary structure from sequence.

Hybrid selection for sequencing pathogen genomes from clinical samples

PubMed Central

2011-01-01

We have adapted a solution hybrid selection protocol to enrich pathogen DNA in clinical samples dominated by human genetic material. Using mock mixtures of human and Plasmodium falciparum malaria parasite DNA as well as clinical samples from infected patients, we demonstrate an average of approximately 40-fold enrichment of parasite DNA after hybrid selection. This approach will enable efficient genome sequencing of pathogens from clinical samples, as well as sequencing of endosymbiotic organisms such as Wolbachia that live inside diverse metazoan phyla. PMID:21835008

Nucleic acid constructs containing orthogonal site selective recombinases (OSSRs)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gilmore, Joshua M.; Anderson, J. Christopher; Dueber, John E.

The present invention provides for a recombinant nucleic acid comprising a nucleotide sequence comprising a plurality of constructs, wherein each construct independently comprises a nucleotide sequence of interest flanked by a pair of recombinase recognition sequences. Each pair of recombinase recognition sequences is recognized by a distinct recombinase. Optionally, each construct can, independently, further comprise one or more genes encoding a recombinase capable of recognizing the pair of recombinase recognition sequences of the construct. The recombinase can be an orthogonal (non-cross reacting), site-selective recombinase (OSSR).

Molecular signatures of selection on reproductive character displacement of flower color in Phlox drummondii.

PubMed

Hopkins, Robin; Levin, Donald A; Rausher, Mark D

2012-02-01

Character displacement, which arises when species diverge in sympatry to decrease competition for resources or reproductive interference, has been observed in a wide variety of plants and animals. A classic example of reproductive character displacement, presumed to be caused by reinforcing selection, is flower-color variation in the native Texas wildflower Phlox drummondii. Here, we use population genetic analyses to investigate molecular signatures of selection on flower-color variation in this species. First, we quantify patterns of neutral genetic variation across the range of P. drummondii to demonstrate that restricted gene flow and genetic drift cannot explain the pattern of flower-color divergence in this species. There is evidence of extensive gene flow across populations with different flower colors, suggesting selection caused flower-color divergence. Second, analysis of sequence variation in the genes underlying this divergence reveals a signature of a selective sweep in one of the two genes, further indicating selection is responsible for divergence in sympatry. The lack of a signature of selection at the second locus does not necessarily indicate a lack of selection on this locus but instead brings attention to the uncertainty in depending on molecular signatures to identify selection. © 2011 The Author(s). Evolution© 2011 The Society for the Study of Evolution.

Relative stability of major types of beta-turns as a function of amino acid composition: a study based on Ab initio energetic and natural abundance data.

PubMed

Perczel, András; Jákli, Imre; McAllister, Michael A; Csizmadia, Imre G

2003-06-06

Folding properties of small globular proteins are determined by their amino acid sequence (primary structure). This holds both for local (secondary structure) and for global conformational features of linear polypeptides and proteins composed from natural amino acid derivatives. It thus provides the rational basis of structure prediction algorithms. The shortest secondary structure element, the beta-turn, most typically adopts either a type I or a type II form, depending on the amino acid composition. Herein we investigate the sequence-dependent folding stability of both major types of beta-turns using simple dipeptide models (-Xxx-Yyy-). Gas-phase ab initio properties of 16 carefully selected and suitably protected dipeptide models (for example Val-Ser, Ala-Gly, Ser-Ser) were studied. For each backbone fold most probable side-chain conformers were considered. Fully optimized 321G RHF molecular structures were employed in medium level [B3LYP/6-311++G(d,p)//RHF/3-21G] energy calculations to estimate relative populations of the different backbone conformers. Our results show that the preference for beta-turn forms as calculated by quantum mechanics and observed in Xray determined proteins correlates significantly.

The Function and Catalysis of 2-Oxoglutarate-Dependent Oxygenases Involved in Plant Flavonoid Biosynthesis

PubMed Central

Cheng, Ai-Xia; Han, Xiao-Juan; Wu, Yi-Feng; Lou, Hong-Xiang

2014-01-01

Flavonoids are secondary metabolites derived from phenylalanine and acetate metabolism. They fulfil a variety of functions in plants and have health benefits for humans. During the synthesis of the tricyclic flavonoid natural products in plants, oxidative modifications to the central C ring are catalyzed by four of FeII and 2-oxoglutarate dependent (2-ODD) oxygenases, namely flavone synthase I (FNS I), flavonol synthase (FLS), anthocyanidin synthase (ANS) and flavanone 3β-hydroxylase (FHT). FNS I, FLS and ANS are involved in desaturation of C2–C3 of flavonoids and FHT in hydroxylation of C3. FNS I, which is restricted to the Apiaceae species and in rice, is predicted to have evolved from FHT by duplication. Due to their sequence similarity and substrate specificity, FLS and ANS, which interact with the α surface of the substrate, belong to a group of dioxygenases having a broad substrate specificity, while FNS I and FHT are more selective, and interact with the naringenin β surface. Here, we summarize recent findings regarding the function of the four 2-ODD oxygenases and the relationship between their catalytic activity, their polypeptide sequence and their tertiary structure. PMID:24434621

Fault trees and sequence dependencies

NASA Technical Reports Server (NTRS)

Dugan, Joanne Bechta; Boyd, Mark A.; Bavuso, Salvatore J.

1990-01-01

One of the frequently cited shortcomings of fault-tree models, their inability to model so-called sequence dependencies, is discussed. Several sources of such sequence dependencies are discussed, and new fault-tree gates to capture this behavior are defined. These complex behaviors can be included in present fault-tree models because they utilize a Markov solution. The utility of the new gates is demonstrated by presenting several models of the fault-tolerant parallel processor, which include both hot and cold spares.

Algorithms for optimizing cross-overs in DNA shuffling.

PubMed

He, Lu; Friedman, Alan M; Bailey-Kellogg, Chris

2012-03-21

DNA shuffling generates combinatorial libraries of chimeric genes by stochastically recombining parent genes. The resulting libraries are subjected to large-scale genetic selection or screening to identify those chimeras with favorable properties (e.g., enhanced stability or enzymatic activity). While DNA shuffling has been applied quite successfully, it is limited by its homology-dependent, stochastic nature. Consequently, it is used only with parents of sufficient overall sequence identity, and provides no control over the resulting chimeric library. This paper presents efficient methods to extend the scope of DNA shuffling to handle significantly more diverse parents and to generate more predictable, optimized libraries. Our CODNS (cross-over optimization for DNA shuffling) approach employs polynomial-time dynamic programming algorithms to select codons for the parental amino acids, allowing for zero or a fixed number of conservative substitutions. We first present efficient algorithms to optimize the local sequence identity or the nearest-neighbor approximation of the change in free energy upon annealing, objectives that were previously optimized by computationally-expensive integer programming methods. We then present efficient algorithms for more powerful objectives that seek to localize and enhance the frequency of recombination by producing "runs" of common nucleotides either overall or according to the sequence diversity of the resulting chimeras. We demonstrate the effectiveness of CODNS in choosing codons and allocating substitutions to promote recombination between parents targeted in earlier studies: two GAR transformylases (41% amino acid sequence identity), two very distantly related DNA polymerases, Pol X and β (15%), and beta-lactamases of varying identity (26-47%). Our methods provide the protein engineer with a new approach to DNA shuffling that supports substantially more diverse parents, is more deterministic, and generates more predictable and more diverse chimeric libraries.

MYO7A and USH2A gene sequence variants in Italian patients with Usher syndrome.

PubMed

Sodi, Andrea; Mariottini, Alessandro; Passerini, Ilaria; Murro, Vittoria; Tachyla, Iryna; Bianchi, Benedetta; Menchini, Ugo; Torricelli, Francesca

2014-01-01

To analyze the spectrum of sequence variants in the MYO7A and USH2A genes in a group of Italian patients affected by Usher syndrome (USH). Thirty-six Italian patients with a diagnosis of USH were recruited. They received a standard ophthalmologic examination, visual field testing, optical coherence tomography (OCT) scan, and electrophysiological tests. Fluorescein angiography and fundus autofluorescence imaging were performed in selected cases. All the patients underwent an audiologic examination for the 0.25-8,000 Hz frequencies. Vestibular function was evaluated with specific tests. DNA samples were analyzed for sequence variants of the MYO7A gene (for USH1) and the USH2A gene (for USH2) with direct sequencing techniques. A few patients were analyzed for both genes. In the MYO7A gene, ten missense variants were found; three patients were compound heterozygous, and two were homozygous. Thirty-four USH2A gene variants were detected, including eight missense variants, nine nonsense variants, six splicing variants, and 11 duplications/deletions; 19 patients were compound heterozygous, and three were homozygous. Four MYO7A and 17 USH2A variants have already been described in the literature. Among the novel mutations there are four USH2A large deletions, detected with multiplex ligation dependent probe amplification (MLPA) technology. Two potentially pathogenic variants were found in 27 patients (75%). Affected patients showed variable clinical pictures without a clear genotype-phenotype correlation. Ten variants in the MYO7A gene and 34 variants in the USH2A gene were detected in Italian patients with USH at a high detection rate. A selective analysis of these genes may be valuable for molecular analysis, combining diagnostic efficiency with little time wastage and less resource consumption.

Experimental rugged fitness landscape in protein sequence space.

PubMed

Hayashi, Yuuki; Aita, Takuyo; Toyota, Hitoshi; Husimi, Yuzuru; Urabe, Itaru; Yomo, Tetsuya

2006-12-20

The fitness landscape in sequence space determines the process of biomolecular evolution. To plot the fitness landscape of protein function, we carried out in vitro molecular evolution beginning with a defective fd phage carrying a random polypeptide of 139 amino acids in place of the g3p minor coat protein D2 domain, which is essential for phage infection. After 20 cycles of random substitution at sites 12-130 of the initial random polypeptide and selection for infectivity, the selected phage showed a 1.7x10(4)-fold increase in infectivity, defined as the number of infected cells per ml of phage suspension. Fitness was defined as the logarithm of infectivity, and we analyzed (1) the dependence of stationary fitness on library size, which increased gradually, and (2) the time course of changes in fitness in transitional phases, based on an original theory regarding the evolutionary dynamics in Kauffman's n-k fitness landscape model. In the landscape model, single mutations at single sites among n sites affect the contribution of k other sites to fitness. Based on the results of these analyses, k was estimated to be 18-24. According to the estimated parameters, the landscape was plotted as a smooth surface up to a relative fitness of 0.4 of the global peak, whereas the landscape had a highly rugged surface with many local peaks above this relative fitness value. Based on the landscapes of these two different surfaces, it appears possible for adaptive walks with only random substitutions to climb with relative ease up to the middle region of the fitness landscape from any primordial or random sequence, whereas an enormous range of sequence diversity is required to climb further up the rugged surface above the middle region.

Experimental Rugged Fitness Landscape in Protein Sequence Space

PubMed Central

Hayashi, Yuuki; Aita, Takuyo; Toyota, Hitoshi; Husimi, Yuzuru; Urabe, Itaru; Yomo, Tetsuya

2006-01-01

The fitness landscape in sequence space determines the process of biomolecular evolution. To plot the fitness landscape of protein function, we carried out in vitro molecular evolution beginning with a defective fd phage carrying a random polypeptide of 139 amino acids in place of the g3p minor coat protein D2 domain, which is essential for phage infection. After 20 cycles of random substitution at sites 12–130 of the initial random polypeptide and selection for infectivity, the selected phage showed a 1.7×104-fold increase in infectivity, defined as the number of infected cells per ml of phage suspension. Fitness was defined as the logarithm of infectivity, and we analyzed (1) the dependence of stationary fitness on library size, which increased gradually, and (2) the time course of changes in fitness in transitional phases, based on an original theory regarding the evolutionary dynamics in Kauffman's n-k fitness landscape model. In the landscape model, single mutations at single sites among n sites affect the contribution of k other sites to fitness. Based on the results of these analyses, k was estimated to be 18–24. According to the estimated parameters, the landscape was plotted as a smooth surface up to a relative fitness of 0.4 of the global peak, whereas the landscape had a highly rugged surface with many local peaks above this relative fitness value. Based on the landscapes of these two different surfaces, it appears possible for adaptive walks with only random substitutions to climb with relative ease up to the middle region of the fitness landscape from any primordial or random sequence, whereas an enormous range of sequence diversity is required to climb further up the rugged surface above the middle region. PMID:17183728

Effects of methylation-sensitive enzymes on the enrichment of genic SNPs and the degree of genome complexity reduction in a two-enzyme genotyping-by-sequencing (GBS) approach: a case study in oil palm (Elaeis guineensis).

PubMed

Pootakham, Wirulda; Sonthirod, Chutima; Naktang, Chaiwat; Jomchai, Nukoon; Sangsrakru, Duangjai; Tangphatsornruang, Sithichoke

2016-01-01

Advances in next generation sequencing have facilitated a large-scale single nucleotide polymorphism (SNP) discovery in many crop species. Genotyping-by-sequencing (GBS) approach couples next generation sequencing with genome complexity reduction techniques to simultaneously identify and genotype SNPs. Choice of enzymes used in GBS library preparation depends on several factors including the number of markers required, the desired level of multiplexing, and whether the enrichment of genic SNP is preferred. We evaluated various combinations of methylation-sensitive ( Aat II, Pst I, Msp I) and methylation-insensitive ( Sph I, Mse I) enzymes for their effectiveness in genome complexity reduction and enrichment of genic SNPs. We discovered that the use of two methylation-sensitive enzymes effectively reduced genome complexity and did not require a size selection step. On the contrary, the genome coverage of libraries constructed with methylation-insensitive enzymes was quite high, and the additional size selection step may be required to increase the overall read depth. We also demonstrated the effectiveness of methylation-sensitive enzymes in enriching for SNPs located in genic regions. When two methylation-insensitive enzymes were used, only 16% of SNPs identified were located in genes and 18% in the vicinity (± 5 kb) of the genic regions, while most SNPs resided in the intergenic regions. In contrast, a remarkable degree of enrichment was observed when two methylation-sensitive enzymes were employed. Almost two thirds of the SNPs were located either inside (32-36%) or in the vicinity (28-31%) of the genic regions. These results provide useful information to help researchers choose appropriate GBS enzymes in oil palm and other crop species.

Immune Selection In Vitro Reveals Human Immunodeficiency Virus Type 1 Nef Sequence Motifs Important for Its Immune Evasion Function In Vivo

PubMed Central

Lee, Patricia; Ng, Hwee L.; Yang, Otto O.

2012-01-01

Human immunodeficiency virus type 1 (HIV-1) Nef downregulates major histocompatibility complex class I (MHC-I), impairing the clearance of infected cells by CD8+ cytotoxic T lymphocytes (CTLs). While sequence motifs mediating this function have been determined by in vitro mutagenesis studies of laboratory-adapted HIV-1 molecular clones, it is unclear whether the highly variable Nef sequences of primary isolates in vivo rely on the same sequence motifs. To address this issue, nef quasispecies from nine chronically HIV-1-infected persons were examined for sequence evolution and altered MHC-I downregulatory function under Gag-specific CTL immune pressure in vitro. This selection resulted in decreased nef diversity and strong purifying selection. Site-by-site analysis identified 13 codons undergoing purifying selection and 1 undergoing positive selection. Of the former, only 6 have been reported to have roles in Nef function, including 4 associated with MHC-I downregulation. Functional testing of naturally occurring in vivo polymorphisms at the 7 sites with no previously known functional role revealed 3 mutations (A84D, Y135F, and G140R) that ablated MHC-I downregulation and 3 (N52A, S169I, and V180E) that partially impaired MHC-I downregulation. Globally, the CTL pressure in vitro selected functional Nef from the in vivo quasispecies mixtures that predominately lacked MHC-I downregulatory function at the baseline. Overall, these data demonstrate that CTL pressure exerts a strong purifying selective pressure for MHC-I downregulation and identifies novel functional motifs present in Nef sequences in vivo. PMID:22553319

PHASTpep: Analysis Software for Discovery of Cell-Selective Peptides via Phage Display and Next-Generation Sequencing

PubMed Central

Dasa, Siva Sai Krishna; Kelly, Kimberly A.

2016-01-01

Next-generation sequencing has enhanced the phage display process, allowing for the quantification of millions of sequences resulting from the biopanning process. In response, many valuable analysis programs focused on specificity and finding targeted motifs or consensus sequences were developed. For targeted drug delivery and molecular imaging, it is also necessary to find peptides that are selective—targeting only the cell type or tissue of interest. We present a new analysis strategy and accompanying software, PHage Analysis for Selective Targeted PEPtides (PHASTpep), which identifies highly specific and selective peptides. Using this process, we discovered and validated, both in vitro and in vivo in mice, two sequences (HTTIPKV and APPIMSV) targeted to pancreatic cancer-associated fibroblasts that escaped identification using previously existing software. Our selectivity analysis makes it possible to discover peptides that target a specific cell type and avoid other cell types, enhancing clinical translatability by circumventing complications with systemic use. PMID:27186887

Emergence of a replicating species from an in vitro RNA evolution reaction

NASA Technical Reports Server (NTRS)

Breaker, R. R.; Joyce, G. F.

1994-01-01

The technique of self-sustained sequence replication allows isothermal amplification of DNA and RNA molecules in vitro. This method relies on the activities of a reverse transcriptase and a DNA-dependent RNA polymerase to amplify specific nucleic acid sequences. We have modified this protocol to allow selective amplification of RNAs that catalyze a particular chemical reaction. During an in vitro RNA evolution experiment employing this modified system, a unique class of "selfish" RNAs emerged and replicated to the exclusion of the intended RNAs. Members of this class of selfish molecules, termed RNA Z, amplify efficiently despite their inability to catalyze the target chemical reaction. Their amplification requires the action of both reverse transcriptase and RNA polymerase and involves the synthesis of both DNA and RNA replication intermediates. The proposed amplification mechanism for RNA Z involves the formation of a DNA hairpin that functions as a template for transcription by RNA polymerase. This arrangement links the two strands of the DNA, resulting in the production of RNA transcripts that contain an embedded RNA polymerase promoter sequence.

Ages of intermediate-age Magellanic Cloud star clusters

NASA Technical Reports Server (NTRS)

Flower, P. J.

1984-01-01

Ages of intermediate-age Large Magellanic Cloud star clusters have been estimated without locating the faint, unevolved portion of cluster main sequences. Six clusters with established color-magnitude diagrams were selected for study: SL 868, NGC 1783, NGC 1868, NGC 2121, NGC 2209, and NGC 2231. Since red giant photometry is more accurate than the necessarily fainter main-sequence photometry, the distributions of red giants on the cluster color-magnitude diagrams were compared to a grid of 33 stellar evolutionary tracks, evolved from the main sequence through core-helium exhaustion, spanning the expected mass and metallicity range for Magellanic Cloud cluster red giants. The time-dependent behavior of the luminosity of the model red giants was used to estimate cluster ages from the observed cluster red giant luminosities. Except for the possibility of SL 868 being an old globular cluster, all clusters studied were found to have ages less than 10 to the 9th yr. It is concluded that there is currently no substantial evidence for a major cluster population of large, populous clusters greater than 10 to the 9th yr old in the Large Magellanic Cloud.

Probing the Structures of Viral RNA Regulatory Elements with SHAPE and Related Methodologies

PubMed Central

Rausch, Jason W.; Sztuba-Solinska, Joanna; Le Grice, Stuart F. J.

2018-01-01

Viral RNAs were selected by evolution to possess maximum functionality in a minimal sequence. Depending on the classification of the virus and the type of RNA in question, viral RNAs must alternately be replicated, spliced, transcribed, transported from the nucleus into the cytoplasm, translated and/or packaged into nascent virions, and in most cases, provide the sequence and structural determinants to facilitate these processes. One consequence of this compact multifunctionality is that viral RNA structures can be exquisitely complex, often involving intermolecular interactions with RNA or protein, intramolecular interactions between sequence segments separated by several thousands of nucleotides, or specialized motifs such as pseudoknots or kissing loops. The fluidity of viral RNA structure can also present a challenge when attempting to characterize it, as genomic RNAs especially are likely to sample numerous conformations at various stages of the virus life cycle. Here we review advances in chemoenzymatic structure probing that have made it possible to address such challenges with respect to cis-acting elements, full-length viral genomes and long non-coding RNAs that play a major role in regulating viral gene expression. PMID:29375504

microRNA-122 target sites in the hepatitis C virus RNA NS5B coding region and 3' untranslated region: function in replication and influence of RNA secondary structure.

PubMed

Gerresheim, Gesche K; Dünnes, Nadia; Nieder-Röhrmann, Anika; Shalamova, Lyudmila A; Fricke, Markus; Hofacker, Ivo; Höner Zu Siederdissen, Christian; Marz, Manja; Niepmann, Michael

2017-02-01

We have analyzed the binding of the liver-specific microRNA-122 (miR-122) to three conserved target sites of hepatitis C virus (HCV) RNA, two in the non-structural protein 5B (NS5B) coding region and one in the 3' untranslated region (3'UTR). miR-122 binding efficiency strongly depends on target site accessibility under conditions when the range of flanking sequences available for the formation of local RNA secondary structures changes. Our results indicate that the particular sequence feature that contributes most to the correlation between target site accessibility and binding strength varies between different target sites. This suggests that the dynamics of miRNA/Ago2 binding not only depends on the target site itself but also on flanking sequence context to a considerable extent, in particular in a small viral genome in which strong selection constraints act on coding sequence and overlapping cis-signals and model the accessibility of cis-signals. In full-length genomes, single and combination mutations in the miR-122 target sites reveal that site 5B.2 is positively involved in regulating overall genome replication efficiency, whereas mutation of site 5B.3 showed a weaker effect. Mutation of the 3'UTR site and double or triple mutants showed no significant overall effect on genome replication, whereas in a translation reporter RNA, the 3'UTR target site inhibits translation directed by the HCV 5'UTR. Thus, the miR-122 target sites in the 3'-region of the HCV genome are involved in a complex interplay in regulating different steps of the HCV replication cycle.

Molecular selection in a unified evolutionary sequence

NASA Technical Reports Server (NTRS)

Fox, S. W.

1986-01-01

With guidance from experiments and observations that indicate internally limited phenomena, an outline of unified evolutionary sequence is inferred. Such unification is not visible for a context of random matrix and random mutation. The sequence proceeds from Big Bang through prebiotic matter, protocells, through the evolving cell via molecular and natural selection, to mind, behavior, and society.

How to Select a Good Training-data Subset for Transcription: Submodular Active Selection for Sequences

DTIC Science & Technology

2009-01-01

selection and uncertainty sampling signif- icantly. Index Terms: Transcription, labeling, submodularity, submod- ular selection, active learning , sequence...name of batch active learning , where a subset of data that is most informative and represen- tative of the whole is selected for labeling. Often...representative subset. Note that our Fisher ker- nel is over an unsupervised generative model, which enables us to bootstrap our active learning approach

Translating working memory into action: behavioral and neural evidence for using motor representations in encoding visuo-spatial sequences.

PubMed

Langner, Robert; Sternkopf, Melanie A; Kellermann, Tanja S; Grefkes, Christian; Kurth, Florian; Schneider, Frank; Zilles, Karl; Eickhoff, Simon B

2014-07-01

The neurobiological organization of action-oriented working memory is not well understood. To elucidate the neural correlates of translating visuo-spatial stimulus sequences into delayed (memory-guided) sequential actions, we measured brain activity using functional magnetic resonance imaging while participants encoded sequences of four to seven dots appearing on fingers of a left or right schematic hand. After variable delays, sequences were to be reproduced with the corresponding fingers. Recall became less accurate with longer sequences and was initiated faster after long delays. Across both hands, encoding and recall activated bilateral prefrontal, premotor, superior and inferior parietal regions as well as the basal ganglia, whereas hand-specific activity was found (albeit to a lesser degree during encoding) in contralateral premotor, sensorimotor, and superior parietal cortex. Activation differences after long versus short delays were restricted to motor-related regions, indicating that rehearsal during long delays might have facilitated the conversion of the memorandum into concrete motor programs at recall. Furthermore, basal ganglia activity during encoding selectively predicted correct recall. Taken together, the results suggest that to-be-reproduced visuo-spatial sequences are encoded as prospective action representations (motor intentions), possibly in addition to retrospective sensory codes. Overall, our study supports and extends multi-component models of working memory, highlighting the notion that sensory input can be coded in multiple ways depending on what the memorandum is to be used for. Copyright © 2013 Wiley Periodicals, Inc.

Evaluation and Selection of Best Priority Sequencing Rule in Job Shop Scheduling using Hybrid MCDM Technique

NASA Astrophysics Data System (ADS)

Kiran Kumar, Kalla; Nagaraju, Dega; Gayathri, S.; Narayanan, S.

2017-05-01

Priority Sequencing Rules provide the guidance for the order in which the jobs are to be processed at a workstation. The application of different priority rules in job shop scheduling gives different order of scheduling. More experimentation needs to be conducted before a final choice is made to know the best priority sequencing rule. Hence, a comprehensive method of selecting the right choice is essential in managerial decision making perspective. This paper considers seven different priority sequencing rules in job shop scheduling. For evaluation and selection of the best priority sequencing rule, a set of eight criteria are considered. The aim of this work is to demonstrate the methodology of evaluating and selecting the best priority sequencing rule by using hybrid multi criteria decision making technique (MCDM), i.e., analytical hierarchy process (AHP) with technique for order preference by similarity to ideal solution (TOPSIS). The criteria weights are calculated by using AHP whereas the relative closeness values of all priority sequencing rules are computed based on TOPSIS with the help of data acquired from the shop floor of a manufacturing firm. Finally, from the findings of this work, the priority sequencing rules are ranked from most important to least important. The comprehensive methodology presented in this paper is very much essential for the management of a workstation to choose the best priority sequencing rule among the available alternatives for processing the jobs with maximum benefit.

How to infer relative fitness from a sample of genomic sequences.

PubMed

Dayarian, Adel; Shraiman, Boris I

2014-07-01

Mounting evidence suggests that natural populations can harbor extensive fitness diversity with numerous genomic loci under selection. It is also known that genealogical trees for populations under selection are quantifiably different from those expected under neutral evolution and described statistically by Kingman's coalescent. While differences in the statistical structure of genealogies have long been used as a test for the presence of selection, the full extent of the information that they contain has not been exploited. Here we demonstrate that the shape of the reconstructed genealogical tree for a moderately large number of random genomic samples taken from a fitness diverse, but otherwise unstructured, asexual population can be used to predict the relative fitness of individuals within the sample. To achieve this we define a heuristic algorithm, which we test in silico, using simulations of a Wright-Fisher model for a realistic range of mutation rates and selection strength. Our inferred fitness ranking is based on a linear discriminator that identifies rapidly coalescing lineages in the reconstructed tree. Inferred fitness ranking correlates strongly with actual fitness, with a genome in the top 10% ranked being in the top 20% fittest with false discovery rate of 0.1-0.3, depending on the mutation/selection parameters. The ranking also enables us to predict the genotypes that future populations inherit from the present one. While the inference accuracy increases monotonically with sample size, samples of 200 nearly saturate the performance. We propose that our approach can be used for inferring relative fitness of genomes obtained in single-cell sequencing of tumors and in monitoring viral outbreaks. Copyright © 2014 by the Genetics Society of America.

The structured ancestral selection graph and the many-demes limit.

PubMed

Slade, Paul F; Wakeley, John

2005-02-01

We show that the unstructured ancestral selection graph applies to part of the history of a sample from a population structured by restricted migration among subpopulations, or demes. The result holds in the limit as the number of demes tends to infinity with proportionately weak selection, and we have also made the assumptions of island-type migration and that demes are equivalent in size. After an instantaneous sample-size adjustment, this structured ancestral selection graph converges to an unstructured ancestral selection graph with a mutation parameter that depends inversely on the migration rate. In contrast, the selection parameter for the population is independent of the migration rate and is identical to the selection parameter in an unstructured population. We show analytically that estimators of the migration rate, based on pairwise sequence differences, derived under the assumption of neutrality should perform equally well in the presence of weak selection. We also modify an algorithm for simulating genealogies conditional on the frequencies of two selected alleles in a sample. This permits efficient simulation of stronger selection than was previously possible. Using this new algorithm, we simulate gene genealogies under the many-demes ancestral selection graph and identify some situations in which migration has a strong effect on the time to the most recent common ancestor of the sample. We find that a similar effect also increases the sensitivity of the genealogy to selection.

Tuning Selectivity of Fluorescent Carbon Nanotube-Based Neurotransmitter Sensors.

PubMed

Mann, Florian A; Herrmann, Niklas; Meyer, Daniel; Kruss, Sebastian

2017-06-28

Detection of neurotransmitters is an analytical challenge and essential to understand neuronal networks in the brain and associated diseases. However, most methods do not provide sufficient spatial, temporal, or chemical resolution. Near-infrared (NIR) fluorescent single-walled carbon nanotubes (SWCNTs) have been used as building blocks for sensors/probes that detect catecholamine neurotransmitters, including dopamine. This approach provides a high spatial and temporal resolution, but it is not understood if these sensors are able to distinguish dopamine from similar catecholamine neurotransmitters, such as epinephrine or norepinephrine. In this work, the organic phase (DNA sequence) around SWCNTs was varied to create sensors with different selectivity and sensitivity for catecholamine neurotransmitters. Most DNA-functionalized SWCNTs responded to catecholamine neurotransmitters, but both dissociation constants ( K d ) and limits of detection were highly dependent on functionalization (sequence). K d values span a range of 2.3 nM (SWCNT-(GC) 15 + norepinephrine) to 9.4 μM (SWCNT-(AT) 15 + dopamine) and limits of detection are mostly in the single-digit nM regime. Additionally, sensors of different SWCNT chirality show different fluorescence increases. Moreover, certain sensors (e.g., SWCNT-(GT) 10 ) distinguish between different catecholamines, such as dopamine and norepinephrine at low concentrations (50 nM). These results show that SWCNTs functionalized with certain DNA sequences are able to discriminate between catecholamine neurotransmitters or to detect them in the presence of interfering substances of similar structure. Such sensors will be useful to measure and study neurotransmitter signaling in complex biological settings.

In vitro selection using a dual RNA library that allows primerless selection

PubMed Central

Jarosch, Florian; Buchner, Klaus; Klussmann, Sven

2006-01-01

High affinity target-binding aptamers are identified from random oligonucleotide libraries by an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX). Since the SELEX process includes a PCR amplification step the randomized region of the oligonucleotide libraries need to be flanked by two fixed primer binding sequences. These primer binding sites are often difficult to truncate because they may be necessary to maintain the structure of the aptamer or may even be part of the target binding motif. We designed a novel type of RNA library that carries fixed sequences which constrain the oligonucleotides into a partly double-stranded structure, thereby minimizing the risk that the primer binding sequences become part of the target-binding motif. Moreover, the specific design of the library including the use of tandem RNA Polymerase promoters allows the selection of oligonucleotides without any primer binding sequences. The library was used to select aptamers to the mirror-image peptide of ghrelin. Ghrelin is a potent stimulator of growth-hormone release and food intake. After selection, the identified aptamer sequences were directly synthesized in their mirror-image configuration. The final 44 nt-Spiegelmer, named NOX-B11-3, blocks ghrelin action in a cell culture assay displaying an IC50 of 4.5 nM at 37°C. PMID:16855281

Bacterial diversity and composition of an alkaline uranium mine tailings-water interface.

PubMed

Khan, Nurul H; Bondici, Viorica F; Medihala, Prabhakara G; Lawrence, John R; Wolfaardt, Gideon M; Warner, Jeff; Korber, Darren R

2013-10-01

The microbial diversity and biogeochemical potential associated with a northern Saskatchewan uranium mine water-tailings interface was examined using culture-dependent and -independent techniques. Morphologically-distinct colonies from uranium mine water-tailings and a reference lake (MC) obtained using selective and non-selective media were selected for 16S rRNA gene sequencing and identification, revealing that culturable organisms from the uranium tailings interface were dominated by Firmicutes and Betaproteobacteria; whereas, MC organisms mainly consisted of Bacteroidetes and Gammaproteobacteria. Ion Torrent (IT) 16S rRNA metagenomic analysis carried out on extracted DNA from tailings and MC interfaces demonstrated the dominance of Firmicutes in both of the systems. Overall, the tailings-water interface environment harbored a distinct bacterial community relative to the MC, reflective of the ambient conditions (i.e., total dissolved solids, pH, salinity, conductivity, heavy metals) dominating the uranium tailings system. Significant correlations among the physicochemical data and the major bacterial groups present in the tailings and MC were also observed. Presence of sulfate reducing bacteria demonstrated by culture-dependent analyses and the dominance of Desulfosporosinus spp. indicated by Ion Torrent analyses within the tailings-water interface suggests the existence of anaerobic microenvironments along with the potential for reductive metabolic processes.

Refining the Results of a Classical SELEX Experiment by Expanding the Sequence Data Set of an Aptamer Pool Selected for Protein A

PubMed Central

2018-01-01

New, as yet undiscovered aptamers for Protein A were identified by applying next generation sequencing (NGS) to a previously selected aptamer pool. This pool was obtained in a classical SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiment using the FluMag-SELEX procedure followed by cloning and Sanger sequencing. PA#2/8 was identified as the only Protein A-binding aptamer from the Sanger sequence pool, and was shown to be able to bind intact cells of Staphylococcus aureus. In this study, we show the extension of the SELEX results by re-sequencing of the same aptamer pool using a medium throughput NGS approach and data analysis. Both data pools were compared. They confirm the selection of a highly complex and heterogeneous oligonucleotide pool and show consistently a high content of orphans as well as a similar relative frequency of certain sequence groups. But in contrast to the Sanger data pool, the NGS pool was clearly dominated by one sequence group containing the known Protein A-binding aptamer PA#2/8 as the most frequent sequence in this group. In addition, we found two new sequence groups in the NGS pool represented by PA-C10 and PA-C8, respectively, which also have high specificity for Protein A. Comparative affinity studies reveal differences between the aptamers and confirm that PA#2/8 remains the most potent sequence within the selected aptamer pool reaching affinities in the low nanomolar range of KD = 20 ± 1 nM. PMID:29495282

Refining the Results of a Classical SELEX Experiment by Expanding the Sequence Data Set of an Aptamer Pool Selected for Protein A.

PubMed

Stoltenburg, Regina; Strehlitz, Beate

2018-02-24

New, as yet undiscovered aptamers for Protein A were identified by applying next generation sequencing (NGS) to a previously selected aptamer pool. This pool was obtained in a classical SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiment using the FluMag-SELEX procedure followed by cloning and Sanger sequencing. PA#2/8 was identified as the only Protein A-binding aptamer from the Sanger sequence pool, and was shown to be able to bind intact cells of Staphylococcus aureus . In this study, we show the extension of the SELEX results by re-sequencing of the same aptamer pool using a medium throughput NGS approach and data analysis. Both data pools were compared. They confirm the selection of a highly complex and heterogeneous oligonucleotide pool and show consistently a high content of orphans as well as a similar relative frequency of certain sequence groups. But in contrast to the Sanger data pool, the NGS pool was clearly dominated by one sequence group containing the known Protein A-binding aptamer PA#2/8 as the most frequent sequence in this group. In addition, we found two new sequence groups in the NGS pool represented by PA-C10 and PA-C8, respectively, which also have high specificity for Protein A. Comparative affinity studies reveal differences between the aptamers and confirm that PA#2/8 remains the most potent sequence within the selected aptamer pool reaching affinities in the low nanomolar range of K D = 20 ± 1 nM.

DeepGO: predicting protein functions from sequence and interactions using a deep ontology-aware classifier.

PubMed

Kulmanov, Maxat; Khan, Mohammed Asif; Hoehndorf, Robert; Wren, Jonathan

2018-02-15

A large number of protein sequences are becoming available through the application of novel high-throughput sequencing technologies. Experimental functional characterization of these proteins is time-consuming and expensive, and is often only done rigorously for few selected model organisms. Computational function prediction approaches have been suggested to fill this gap. The functions of proteins are classified using the Gene Ontology (GO), which contains over 40 000 classes. Additionally, proteins have multiple functions, making function prediction a large-scale, multi-class, multi-label problem. We have developed a novel method to predict protein function from sequence. We use deep learning to learn features from protein sequences as well as a cross-species protein-protein interaction network. Our approach specifically outputs information in the structure of the GO and utilizes the dependencies between GO classes as background information to construct a deep learning model. We evaluate our method using the standards established by the Computational Assessment of Function Annotation (CAFA) and demonstrate a significant improvement over baseline methods such as BLAST, in particular for predicting cellular locations. Web server: http://deepgo.bio2vec.net, Source code: https://github.com/bio-ontology-research-group/deepgo. robert.hoehndorf@kaust.edu.sa. Supplementary data are available at Bioinformatics online. © The Author(s) 2017. Published by Oxford University Press.

Phenotypic and genotypic expression of self-incompatibility haplotypes in Arabidopsis lyrata suggests unique origin of alleles in different dominance classes.

PubMed

Prigoda, Nadia L; Nassuth, Annette; Mable, Barbara K

2005-07-01

The highly divergent alleles of the SRK gene in outcrossing Arabidopsis lyrata have provided important insights into the evolutionary history of self-incompatibility (SI) alleles and serve as an ideal model for studies of the evolutionary and molecular interactions between alleles in cell-cell recognition systems in general. One tantalizing question is how new specificities arise in systems that require coordination between male and female components. Allelic recruitment via gene conversion has been proposed as one possibility, based on the division of DNA sequences at the SRK locus into two distinctive groups: (1) sequences whose relationships are not well resolved and display the long branch lengths expected for a gene under balancing selection (Class A); and (2) sequences falling into a well-supported group with shorter branch lengths (Class B) that are closely related to an unlinked paralogous locus. The purpose of this study was to determine if differences in phenotype (site of expression assayed using allele-specific reverse transcription-polymerase chain reaction) or function (dominance relationships assayed through controlled pollinations) accompany the sequence-based classification. Expression of Class A alleles was restricted to floral tissues, as predicted for genes involved in the SI response. In contrast, Class B alleles, despite being tightly linked to the SI phenotype, were unexpectedly expressed in both leaves and floral tissues; the same pattern found for a related unlinked paralogous sequence. Whereas Class A included haplotypes in three different dominance classes, all Class B haplotypes were found to be recessive to all except one Class A haplotype. In addition, mapping of expression and dominance patterns onto an S-domain-based genealogy suggested that allelic dominance may be determined more by evolutionary history than by frequency-dependent selection for lowered dominance as some theories suggest. The possibility that interlocus gene conversion might have contributed to allelic diversity is discussed.

Volume interpolated 3D-spoiled gradient echo sequence is better than dynamic contrast spin echo sequence for MRI detection of corticotropin secreting pituitary microadenomas.

PubMed

Kasaliwal, Rajeev; Sankhe, Shilpa S; Lila, Anurag R; Budyal, Sweta R; Jagtap, Varsha S; Sarathi, Vijaya; Kakade, Harshal; Bandgar, Tushar; Menon, Padmavathy S; Shah, Nalini S

2013-06-01

Various techniques have been attempted to increase the yield of magnetic resonance imaging (MRI) for localization of pituitary microadenomas in corticotropin (ACTH)-dependent Cushing's syndrome (CS). To compare the performance of dynamic contrast spin echo (DC-SE) and volume interpolated 3D-spoiled gradient echo (VI-SGE) MR sequences in the diagnostic evaluation of ACTH-dependent CS. Data was analysed retrospectively from a series of ACTH-dependent CS patients treated over 2-year period at a tertiary care referral centre (2009-2011). Thirty-six patients (24 female and 12 male) were diagnosed to have ACTH-dependent CS during the study period. All patients underwent MRI by both sequences during a single examination. Cases with negative and equivocal pituitary MR imaging underwent corticotropin-releasing hormone (CRH) stimulated bilateral inferior petrosal sinus sampling (BIPSS) to confirm pituitary origin of ACTH excess state. Thirty patients were finally diagnosed to have Cushing's disease (CD) [based on histopathology proof of adenoma and/or remission (partial/complete) of hypercortisolism postsurgery]. Six patients were diagnosed to have histopathologically proven ectopic CS. Of 30 patients with CD, 24 patients had microadenomas and 6 patients had macroadenomas. DC-SE MRI sequence was able to identify microadenomas in 16 of 24 patients, whereas postcontrast VI-SGE sequence was able to identify microadenomas in 21 of 24 patients. All six patients of ectopic CS had negative pituitary MR imaging by both techniques (specificity: 100%). VI-SGE MR sequence was better for localization of pituitary microadenomas particularly when DC-SE MR sequence is negative or equivocal and should be used in addition to DC-SE MR sequence for the evaluation of ACTH-dependent CS. © 2012 John Wiley & Sons Ltd.

U-Groove aluminum weld strength improvement

NASA Technical Reports Server (NTRS)

Verderaime, V.; Vaughan, R.

1996-01-01

Though butt-welds are among the most preferred joining methods in aerostructures, their strength dependence on inelastic mechanics is generally the least understood. This study investigated experimental strain distributions across a thick aluminum U-grooved weld and identified two weld process considerations for improving the multipass weld strength. The extreme thermal expansion and contraction gradient of the fusion heat input across the groove tab thickness produces severe peaking, which induces bending under uniaxial loading. The filler strain-hardening decreased with increasing filler pass sequence, producing the weakest welds on the last pass side. Current welding schedules unknowingly compound these effects which reduce the weld strength. A depeaking index model was developed to select filler pass thicknesses, pass numbers, and sequences to improve depeaking in the welding process. The intent is to combine the strongest weld pass side with the peaking induced bending tension to provide a more uniform stress and stronger weld under axial tensile loading.

IgA Function in Relation to the Intestinal Microbiota.

PubMed

Macpherson, Andrew J; Yilmaz, Bahtiyar; Limenitakis, Julien P; Ganal-Vonarburg, Stephanie C

2018-04-26

IgA is the dominant immunoglobulin isotype produced in mammals, largely secreted across the intestinal mucosal surface. Although induction of IgA has been a hallmark feature of microbiota colonization following colonization in germ-free animals, until recently appreciation of the function of IgA in host-microbial mutualism has depended mainly on indirect evidence of alterations in microbiota composition or penetration of microbes in the absence of somatic mutations in IgA (or compensatory IgM). Highly parallel sequencing techniques that enable high-resolution analysis of either microbial consortia or IgA sequence diversity are now giving us new perspectives on selective targeting of microbial taxa and the trajectory of IgA diversification according to induction mechanisms, between different individuals and over time. The prospects are to link the range of diversified IgA clonotypes to specific antigenic functions in modulating the microbiota composition, position and metabolism to ensure host mutualism.

Optimization of rotamers prior to template minimization improves stability predictions made by computational protein design.

PubMed

Davey, James A; Chica, Roberto A

2015-04-01

Computational protein design (CPD) predictions are highly dependent on the structure of the input template used. However, it is unclear how small differences in template geometry translate to large differences in stability prediction accuracy. Herein, we explored how structural changes to the input template affect the outcome of stability predictions by CPD. To do this, we prepared alternate templates by Rotamer Optimization followed by energy Minimization (ROM) and used them to recapitulate the stability of 84 protein G domain β1 mutant sequences. In the ROM process, side-chain rotamers for wild-type (WT) or mutant sequences are optimized on crystal or nuclear magnetic resonance (NMR) structures prior to template minimization, resulting in alternate structures termed ROM templates. We show that use of ROM templates prepared from sequences known to be stable results predominantly in improved prediction accuracy compared to using the minimized crystal or NMR structures. Conversely, ROM templates prepared from sequences that are less stable than the WT reduce prediction accuracy by increasing the number of false positives. These observed changes in prediction outcomes are attributed to differences in side-chain contacts made by rotamers in ROM templates. Finally, we show that ROM templates prepared from sequences that are unfolded or that adopt a nonnative fold result in the selective enrichment of sequences that are also unfolded or that adopt a nonnative fold, respectively. Our results demonstrate the existence of a rotamer bias caused by the input template that can be harnessed to skew predictions toward sequences displaying desired characteristics. © 2014 The Protein Society.

The utility of DNA sequences of an intron from the beta-fibrinogen gene in phylogenetic analysis of woodpeckers (Aves: Picidae).

PubMed

Prychitko, T M; Moore, W S

1997-10-01

Estimating phylogenies from DNA sequence data has become the major methodology of molecular phylogenetics. To date, molecular phylogenetics of the vertebrates has been very dependent on mtDNA, but studies involving mtDNA are limited because the several genes comprising the mt-genome are inherited as a single linkage group. The only apparent solution to this problem is to sequence additional genes, each representing a distinct linkage group, so that the resultant gene trees provide independent estimates of the species tree. There exists the need to find novel gene sequences which contain enough phylogenetic information to resolve relationships between closely related species. A possible source is the nuclear-encoded introns, because they evolve more rapidly than exons. We designed primers to amplify and sequence the 7 intron from the beta-fibrinogen gene for a recently evolved group, the woodpeckers. We sequenced the entire intron for 10 specimens representing five species. Nucleotide substitutions are randomly distributed along the length of the intron, suggesting selective neutrality. A preliminary analysis indicates that the phylogenetic signal in the intron is as strong as that in the mitochondrial encoded cytochrome b (cyt b) gene. The topology of the beta-fibrinogen tree is identical to that of the cyt b tree. This analysis demonstrates the ability of the 7 intron of beta-fibrinogen to provide well resolved, independent gene trees for recently evolved groups and establishes it as a source of sequences to be used in other phylogenetic studies. Copyright 1997 Academic Press

Viral morphogenesis is the dominant source of sequence censorship in M13 combinatorial peptide phage display.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rodi, D. J.; Soares, A. S.; Makowski, L.

Novel statistical methods have been developed and used to quantitate and annotate the sequence diversity within combinatorial peptide libraries on the basis of small numbers (1-200) of sequences selected at random from commercially available M13 p3-based phage display libraries. These libraries behave statistically as though they correspond to populations containing roughly 4.0{+-}1.6% of the random dodecapeptides and 7.9{+-}2.6% of the random constrained heptapeptides that are theoretically possible within the phage populations. Analysis of amino acid residue occurrence patterns shows no demonstrable influence on sequence censorship by Escherichia coli tRNA isoacceptor profiles or either overall codon or Class II codon usagemore » patterns, suggesting no metabolic constraints on recombinant p3 synthesis. There is an overall depression in the occurrence of cysteine, arginine and glycine residues and an overabundance of proline, threonine and histidine residues. The majority of position-dependent amino acid sequence bias is clustered at three positions within the inserted peptides of the dodecapeptide library, +1, +3 and +12 downstream from the signal peptidase cleavage site. Conformational tendency measures of the peptides indicate a significant preference for inserts favoring a {beta}-turn conformation. The observed protein sequence limitations can primarily be attributed to genetic codon degeneracy and signal peptidase cleavage preferences. These data suggest that for applications in which maximal sequence diversity is essential, such as epitope mapping or novel receptor identification, combinatorial peptide libraries should be constructed using codon-corrected trinucleotide cassettes within vector-host systems designed to minimize morphogenesis-related censorship.« less

PET/MR Synchronization by Detection of Switching Gradients

NASA Astrophysics Data System (ADS)

Weissler, Bjoern; Gebhardt, Pierre; Lerche, Christoph W.; Soultanidis, Georgios M.; Wehner, Jakob; Heberling, Dirk; Schulz, Volkmar

2015-06-01

The full potential of simultaneous Positron Emission Tomography (PET) and Magnetic Resonance Imaging (MRI) acquisition, such as dynamic studies or motion compensation, can only be explored if the data of both modalities is temporally synchronized. As such hybrid imaging systems are commonly realized as custom-made PET inserts for commercially available MRI scanner, a synchronization solution has to be implemented (depending on the vendor of the MRI system). In contrast, we demonstrate a simple method for temporal synchronization, which does not require a connection to the MRI. It uses the normally undesired effect of induced voltages on the PET electronics from switching MRI gradients. The electronic circuit needs very few components and the gradient pick-up coils are made from PCB traces and vias on the PET detector boards. Neither programming the MRI nor any physical connection to the MR scanner is needed, thus avoiding electromagnetic compatibility problems. This method works inherently with most MRI sequences and is a vendor- independent solution. A characterization of the sensors in an MRI scanner showed that the MRI gradients are detected with a precision of 120 μs (with the current implementation). Using different trigger thresholds, it is possible to trigger selectively on certain MRI sequences, depending on their gradient slew rate settings. Timings and pulse diagrams of MRI sequences can be recognized from the generated data. The method was successfully used for temporal alignment between PET and MRI in an MRI-based PET-motion-compensation application.

Genetic Rearrangements Can Modify Chromatin Features at Epialleles

PubMed Central

Foerster, Andrea M.; Dinh, Huy Q.; Sedman, Laura; Wohlrab, Bonnie; Mittelsten Scheid, Ortrun

2011-01-01

Analogous to genetically distinct alleles, epialleles represent heritable states of different gene expression from sequence-identical genes. Alleles and epialleles both contribute to phenotypic heterogeneity. While alleles originate from mutation and recombination, the source of epialleles is less well understood. We analyze active and inactive epialleles that were found at a transgenic insert with a selectable marker gene in Arabidopsis. Both converse expression states are stably transmitted to progeny. The silent epiallele was previously shown to change its state upon loss-of-function of trans-acting regulators and drug treatments. We analyzed the composition of the epialleles, their chromatin features, their nuclear localization, transcripts, and homologous small RNA. After mutagenesis by T-DNA transformation of plants carrying the silent epiallele, we found new active alleles. These switches were associated with different, larger or smaller, and non-overlapping deletions or rearrangements in the 3′ regions of the epiallele. These cis-mutations caused different degrees of gene expression stability depending on the nature of the sequence alteration, the consequences for transcription and transcripts, and the resulting chromatin organization upstream. This illustrates a tight dependence of epigenetic regulation on local structures and indicates that sequence alterations can cause epigenetic changes at some distance in regions not directly affected by the mutation. Similar effects may also be involved in gene expression and chromatin changes in the vicinity of transposon insertions or excisions, recombination events, or DNA repair processes and could contribute to the origin of new epialleles. PMID:22028669

Phylogeny and evolution of Newcastle disease virus genotypes isolated in Asia during 2008-2011.

PubMed

Ebrahimi, Mohammad Majid; Shahsavandi, Shahla; Moazenijula, Gholamreza; Shamsara, Mahdi

2012-08-01

The full-length fusion (F) genes of 51 Newcastle disease (ND) strains isolated from chickens in Asia during the period 2008-2011 were genetically analyzed. Phylogenetic analysis showed that genotype VII of NDV still predominant in the domestic poultry of Asia. The sub-genotype VIIb circulated in the Iran and Indian sub-continent countries, whereas VIId sub-genotype existed in Far East countries. The non-synonymous to synonymous substitutions ratio was calculated 0.27 for VIId sub-genotype and 0.51 for VIIb sub-genotype indicates purifying/stabilizing selection which resulted in a low evolution rate in F gene of VIIb sub-genotype. There is evidence of localized positive selection when comparing these sub-genotypes protein sequences. Five codons in F gene of ND viruses had a posterior probability >90% using the Bayesian method, indicating these sites were under positive selection. To identify sites under positive selection; amino acid substitution classified depends on their radicalism and neutrality. The results indicate that although most positions were under purifying selection and can be eliminated, a few positions located in sub-genotype specific regions were subject to positive selection.

MRI-Guided Selection of Patients for Acute Ischemic Stroke Treatment

PubMed Central

Leigh, Richard; Krakauer, John W.

2014-01-01

Purpose of review To summarize what is known about the use of MRI in acute stroke treatments (predominantly thrombolysis), to examine the assumptions and theories behind the interpretation of MR images of acute stroke and how they are used to select patients for therapies, and to suggest directions for future research. Recent findings Recent studies have been contradictory about the usefulness of MRI in selecting patients for treatment. New MRI models for selecting patients have emerged that focus not only on the ischemic penumbra but also the core infarct. Fixed time-window selection parameters are being replaced by individualized MRI features. New ways to interpret traditional MRI sequences are emerging. Summary Although the efficacy of acute stroke treatment is time dependent, the use of fixed time-windows does not account for individual differences in infarct evolution, which could be detected with MRI. While MRI shows promise for identifying patients who should be treated, as well as exclude patients who should not be treated, definitive evidence is still lacking. Future research should focus on validating the use of MRI to select patients for IV therapies in extended time windows. PMID:24978637

Modular prediction of protein structural classes from sequences of twilight-zone identity with predicting sequences.

PubMed

Mizianty, Marcin J; Kurgan, Lukasz

2009-12-13

Knowledge of structural class is used by numerous methods for identification of structural/functional characteristics of proteins and could be used for the detection of remote homologues, particularly for chains that share twilight-zone similarity. In contrast to existing sequence-based structural class predictors, which target four major classes and which are designed for high identity sequences, we predict seven classes from sequences that share twilight-zone identity with the training sequences. The proposed MODular Approach to Structural class prediction (MODAS) method is unique as it allows for selection of any subset of the classes. MODAS is also the first to utilize a novel, custom-built feature-based sequence representation that combines evolutionary profiles and predicted secondary structure. The features quantify information relevant to the definition of the classes including conservation of residues and arrangement and number of helix/strand segments. Our comprehensive design considers 8 feature selection methods and 4 classifiers to develop Support Vector Machine-based classifiers that are tailored for each of the seven classes. Tests on 5 twilight-zone and 1 high-similarity benchmark datasets and comparison with over two dozens of modern competing predictors show that MODAS provides the best overall accuracy that ranges between 80% and 96.7% (83.5% for the twilight-zone datasets), depending on the dataset. This translates into 19% and 8% error rate reduction when compared against the best performing competing method on two largest datasets. The proposed predictor provides accurate predictions at 58% accuracy for membrane proteins class, which is not considered by majority of existing methods, in spite that this class accounts for only 2% of the data. Our predictive model is analyzed to demonstrate how and why the input features are associated with the corresponding classes. The improved predictions stem from the novel features that express collocation of the secondary structure segments in the protein sequence and that combine evolutionary and secondary structure information. Our work demonstrates that conservation and arrangement of the secondary structure segments predicted along the protein chain can successfully predict structural classes which are defined based on the spatial arrangement of the secondary structures. A web server is available at http://biomine.ece.ualberta.ca/MODAS/.

Modular prediction of protein structural classes from sequences of twilight-zone identity with predicting sequences

PubMed Central

2009-01-01

Background Knowledge of structural class is used by numerous methods for identification of structural/functional characteristics of proteins and could be used for the detection of remote homologues, particularly for chains that share twilight-zone similarity. In contrast to existing sequence-based structural class predictors, which target four major classes and which are designed for high identity sequences, we predict seven classes from sequences that share twilight-zone identity with the training sequences. Results The proposed MODular Approach to Structural class prediction (MODAS) method is unique as it allows for selection of any subset of the classes. MODAS is also the first to utilize a novel, custom-built feature-based sequence representation that combines evolutionary profiles and predicted secondary structure. The features quantify information relevant to the definition of the classes including conservation of residues and arrangement and number of helix/strand segments. Our comprehensive design considers 8 feature selection methods and 4 classifiers to develop Support Vector Machine-based classifiers that are tailored for each of the seven classes. Tests on 5 twilight-zone and 1 high-similarity benchmark datasets and comparison with over two dozens of modern competing predictors show that MODAS provides the best overall accuracy that ranges between 80% and 96.7% (83.5% for the twilight-zone datasets), depending on the dataset. This translates into 19% and 8% error rate reduction when compared against the best performing competing method on two largest datasets. The proposed predictor provides accurate predictions at 58% accuracy for membrane proteins class, which is not considered by majority of existing methods, in spite that this class accounts for only 2% of the data. Our predictive model is analyzed to demonstrate how and why the input features are associated with the corresponding classes. Conclusions The improved predictions stem from the novel features that express collocation of the secondary structure segments in the protein sequence and that combine evolutionary and secondary structure information. Our work demonstrates that conservation and arrangement of the secondary structure segments predicted along the protein chain can successfully predict structural classes which are defined based on the spatial arrangement of the secondary structures. A web server is available at http://biomine.ece.ualberta.ca/MODAS/. PMID:20003388

Automated frame selection process for high-resolution microendoscopy

NASA Astrophysics Data System (ADS)

Ishijima, Ayumu; Schwarz, Richard A.; Shin, Dongsuk; Mondrik, Sharon; Vigneswaran, Nadarajah; Gillenwater, Ann M.; Anandasabapathy, Sharmila; Richards-Kortum, Rebecca

2015-04-01

We developed an automated frame selection algorithm for high-resolution microendoscopy video sequences. The algorithm rapidly selects a representative frame with minimal motion artifact from a short video sequence, enabling fully automated image analysis at the point-of-care. The algorithm was evaluated by quantitative comparison of diagnostically relevant image features and diagnostic classification results obtained using automated frame selection versus manual frame selection. A data set consisting of video sequences collected in vivo from 100 oral sites and 167 esophageal sites was used in the analysis. The area under the receiver operating characteristic curve was 0.78 (automated selection) versus 0.82 (manual selection) for oral sites, and 0.93 (automated selection) versus 0.92 (manual selection) for esophageal sites. The implementation of fully automated high-resolution microendoscopy at the point-of-care has the potential to reduce the number of biopsies needed for accurate diagnosis of precancer and cancer in low-resource settings where there may be limited infrastructure and personnel for standard histologic analysis.

X-ray versus infrared selection of distant galaxy clusters: a case study using the XMM-LSS and SpARCS cluster samples

NASA Astrophysics Data System (ADS)

Willis, J. P.; Ramos-Ceja, M. E.; Muzzin, A.; Pacaud, F.; Yee, H. K. C.; Wilson, G.

2018-07-01

We present a comparison of two samples of z> 0.8 galaxy clusters selected using different wavelength-dependent techniques and examine the physical differences between them. We consider 18 clusters from the X-ray-selected XMM Large Scale Structure (LSS) distant cluster survey and 92 clusters from the optical-mid-infrared (MIR)-selected Spitzer Adaptation of the Red Sequence Cluster survey (SpARCS) cluster survey. Both samples are selected from the same approximately 9 sq deg sky area and we examine them using common XMM-Newton, Spitizer Wide-Area Infrared Extra-galactic (SWIRE) survey, and Canada-France-Hawaii Telescope Legacy Survey data. Clusters from each sample are compared employing aperture measures of X-ray and MIR emission. We divide the SpARCS distant cluster sample into three sub-samples: (i) X-ray bright, (ii) X-ray faint, MIR bright, and (iii) X-ray faint, MIR faint clusters. We determine that X-ray- and MIR-selected clusters display very similar surface brightness distributions of galaxy MIR light. In addition, the average location and amplitude of the galaxy red sequence as measured from stacked colour histograms is very similar in the X-ray- and MIR-selected samples. The sub-sample of X-ray faint, MIR bright clusters displays a distribution of brightest cluster galaxy-barycentre position offsets which extends to higher values than all other samples. This observation indicates that such clusters may exist in a more disturbed state compared to the majority of the distant cluster population sampled by XMM-LSS and SpARCS. This conclusion is supported by stacked X-ray images for the X-ray faint, MIR bright cluster sub-sample that display weak, centrally concentrated X-ray emission, consistent with a population of growing clusters accreting from an extended envelope of material.

Alu elements shape the primate transcriptome by cis-regulation of RNA editing

PubMed Central

2014-01-01

Background RNA editing by adenosine to inosine deamination is a widespread phenomenon, particularly frequent in the human transcriptome, largely due to the presence of inverted Alu repeats and their ability to form double-stranded structures – a requisite for ADAR editing. While several hundred thousand editing sites have been identified within these primate-specific repeats, the function of Alu-editing has yet to be elucidated. Results We show that inverted Alu repeats, expressed in the primate brain, can induce site-selective editing in cis on sites located several hundred nucleotides from the Alu elements. Furthermore, a computational analysis, based on available RNA-seq data, finds that site-selective editing occurs significantly closer to edited Alu elements than expected. These targets are poorly edited upon deletion of the editing inducers, as well as in homologous transcripts from organisms lacking Alus. Sequences surrounding sites near edited Alus in UTRs, have been subjected to a lesser extent of evolutionary selection than those far from edited Alus, indicating that their editing generally depends on cis-acting Alus. Interestingly, we find an enrichment of primate-specific editing within encoded sequence or the UTRs of zinc finger-containing transcription factors. Conclusions We propose a model whereby primate-specific editing is induced by adjacent Alu elements that function as recruitment elements for the ADAR editing enzymes. The enrichment of site-selective editing with potentially functional consequences on the expression of transcription factors indicates that editing contributes more profoundly to the transcriptomic regulation and repertoire in primates than previously thought. PMID:24485196

Immunologic and Virologic Mechanisms for Partial Protection from Intravenous Challenge by an Integration-Defective SIV Vaccine †

PubMed Central

Wang, Chu; Jiang, Chunlai; Gao, Nan; Zhang, Kaikai; Liu, Donglai; Wang, Wei; Cong, Zhe; Qin, Chuan; Ganusov, Vitaly V.; Ferrari, Guido; LaBranche, Celia; Montefiori, David C.; Kong, Wei; Yu, Xianghui; Gao, Feng

2017-01-01

The suppression of viral loads and identification of selection signatures in non-human primates after challenge are indicators for effective human immunodeficiency virus (HIV)/simian immunodeficiency virus (SIV) vaccines. To mimic the protective immunity elicited by attenuated SIV vaccines, we developed an integration-defective SIV (idSIV) vaccine by inactivating integrase, mutating sequence motifs critical for integration, and inserting the cytomegalovirus (CMV) promoter for more efficient expression in the SIVmac239 genome. Chinese rhesus macaques were immunized with idSIV DNA and idSIV particles, and the cellular and humoral immune responses were measured. After the intravenous SIVmac239 challenge, viral loads were monitored and selection signatures in viral genomes from vaccinated monkeys were identified by single genome sequencing. T cell responses, heterologous neutralization against tier-1 viruses, and antibody-dependent cellular cytotoxicity (ADCC) were detected in idSIV-vaccinated macaques post immunization. After challenge, the median peak viral load in the vaccine group was significantly lower than that in the control group. However, this initial viral control did not last as viral set-points were similar between vaccinated and control animals. Selection signatures were identified in Nef, Gag, and Env proteins in vaccinated and control macaques, but these signatures were different, suggesting selection pressure on viruses from vaccine-induced immunity in the vaccinated animals. Our results showed that the idSIV vaccine exerted some pressure on the virus population early during the infection but future modifications are needed in order to induce more potent immune responses. PMID:28574482

Evidence for photometric activity cycles in 3203 Kepler stars

NASA Astrophysics Data System (ADS)

Reinhold, Timo; Cameron, Robert H.; Gizon, Laurent

2017-07-01

Context. In recent years it has been claimed that the length of stellar activity cycles is determined by the stellar rotation rate. It has been observed that the cycle period increases with rotation period along two distinct sequences, known as the active and inactive sequences. In this picture the Sun occupies a solitary position between the two sequences. Whether the Sun might undergo a transitional evolutionary stage is currently under debate. Aims: Our goal is to measure cyclic variations of the stellar light curve amplitude and the rotation period using four years of Kepler data. Periodic changes in the light curve amplitude or the stellar rotation period are associated with an underlying activity cycle. Methods: Using a recent sample of active stars we compute the rotation period and the variability amplitude for each individual Kepler quarter and search for periodic variations of both time series. To test for periodicity in each stellar time series we consider Lomb-Scargle periodograms and use a selection based on a false alarm probability (FAP). Results: We detect amplitude periodicities in 3203 stars between 0.5 < Pcyc < 6 yr covering rotation periods between 1 < Prot < 40 days. Given our sample size of 23 601 stars and our selection criteria that the FAP is less than 5%, this number is almost three times higher than that expected from pure noise. We do not detect periodicities in the rotation period beyond those expected from noise. Our measurements reveal that the cycle period shows a weak dependence on rotation rate, slightly increasing for longer rotation periods. We further show that the shape of the variability deviates from a pure sine curve, consistent with observations of the solar cycle. The cycle shape does not show a statistically significant dependence on effective temperature. Conclusions: We detect activity cycles in more than 13% of our final sample with a FAP of 5% (calculated by randomly shuffling the measured 90-day variability measurements for each star). Our measurements do not support the existence of distinct sequences in the Prot-Pcyc plane, although there is some evidence for the inactive sequence for rotation periods between 5-25 days. Unfortunately, the total observing time is too short to draw sound conclusions on activity cycles with similar lengths to that of the solar cycle. A table containing all cycle periods and time series is only available in electronic form at the CDS via anonymous ftp to http://cdsarc.u-strasbg.fr (130.79.128.5) or via http://cdsweb.u-strasbg.fr/cgi-bin/qcat?J/A+A/603/A52

Comparative functional pan-genome analyses to build connections between genomic dynamics and phenotypic evolution in polycyclic aromatic hydrocarbon metabolism in the genus Mycobacterium.

PubMed

Kweon, Ohgew; Kim, Seong-Jae; Blom, Jochen; Kim, Sung-Kwan; Kim, Bong-Soo; Baek, Dong-Heon; Park, Su Inn; Sutherland, John B; Cerniglia, Carl E

2015-02-14

The bacterial genus Mycobacterium is of great interest in the medical and biotechnological fields. Despite a flood of genome sequencing and functional genomics data, significant gaps in knowledge between genome and phenome seriously hinder efforts toward the treatment of mycobacterial diseases and practical biotechnological applications. In this study, we propose the use of systematic, comparative functional pan-genomic analysis to build connections between genomic dynamics and phenotypic evolution in polycyclic aromatic hydrocarbon (PAH) metabolism in the genus Mycobacterium. Phylogenetic, phenotypic, and genomic information for 27 completely genome-sequenced mycobacteria was systematically integrated to reconstruct a mycobacterial phenotype network (MPN) with a pan-genomic concept at a network level. In the MPN, mycobacterial phenotypes show typical scale-free relationships. PAH degradation is an isolated phenotype with the lowest connection degree, consistent with phylogenetic and environmental isolation of PAH degraders. A series of functional pan-genomic analyses provide conserved and unique types of genomic evidence for strong epistatic and pleiotropic impacts on evolutionary trajectories of the PAH-degrading phenotype. Under strong natural selection, the detailed gene gain/loss patterns from horizontal gene transfer (HGT)/deletion events hypothesize a plausible evolutionary path, an epistasis-based birth and pleiotropy-dependent death, for PAH metabolism in the genus Mycobacterium. This study generated a practical mycobacterial compendium of phenotypic and genomic changes, focusing on the PAH-degrading phenotype, with a pan-genomic perspective of the evolutionary events and the environmental challenges. Our findings suggest that when selection acts on PAH metabolism, only a small fraction of possible trajectories is likely to be observed, owing mainly to a combination of the ambiguous phenotypic effects of PAHs and the corresponding pleiotropy- and epistasis-dependent evolutionary adaptation. Evolutionary constraints on the selection of trajectories, like those seen in PAH-degrading phenotypes, are likely to apply to the evolution of other phenotypes in the genus Mycobacterium.

Positive evolutionary selection of an HD motif on Alzheimer precursor protein orthologues suggests a functional role.

PubMed

Miklós, István; Zádori, Zoltán

2012-02-01

HD amino acid duplex has been found in the active center of many different enzymes. The dyad plays remarkably different roles in their catalytic processes that usually involve metal coordination. An HD motif is positioned directly on the amyloid beta fragment (Aβ) and on the carboxy-terminal region of the extracellular domain (CAED) of the human amyloid precursor protein (APP) and a taxonomically well defined group of APP orthologues (APPOs). In human Aβ HD is part of a presumed, RGD-like integrin-binding motif RHD; however, neither RHD nor RXD demonstrates reasonable conservation in APPOs. The sequences of CAEDs and the position of the HD are not particularly conserved either, yet we show with a novel statistical method using evolutionary modeling that the presence of HD on CAEDs cannot be the result of neutral evolutionary forces (p<0.0001). The motif is positively selected along the evolutionary process in the majority of APPOs, despite the fact that HD motif is underrepresented in the proteomes of all species of the animal kingdom. Position migration can be explained by high probability occurrence of multiple copies of HD on intermediate sequences, from which only one is kept by selective evolutionary forces, in a similar way as in the case of the "transcription binding site turnover." CAED of all APP orthologues and homologues are predicted to bind metal ions including Amyloid-like protein 1 (APLP1) and Amyloid-like protein 2 (APLP2). Our results suggest that HDs on the CAEDs are most probably key components of metal-binding domains, which facilitate and/or regulate inter- or intra-molecular interactions in a metal ion-dependent or metal ion concentration-dependent manner. The involvement of naturally occurring mutations of HD (Tottori (D7N) and English (H6R) mutations) in early onset Alzheimer's disease gives additional support to our finding that HD has an evolutionary preserved function on APPOs.

Positive Evolutionary Selection of an HD Motif on Alzheimer Precursor Protein Orthologues Suggests a Functional Role

PubMed Central

Miklós, István; Zádori, Zoltán

2012-01-01

HD amino acid duplex has been found in the active center of many different enzymes. The dyad plays remarkably different roles in their catalytic processes that usually involve metal coordination. An HD motif is positioned directly on the amyloid beta fragment (Aβ) and on the carboxy-terminal region of the extracellular domain (CAED) of the human amyloid precursor protein (APP) and a taxonomically well defined group of APP orthologues (APPOs). In human Aβ HD is part of a presumed, RGD-like integrin-binding motif RHD; however, neither RHD nor RXD demonstrates reasonable conservation in APPOs. The sequences of CAEDs and the position of the HD are not particularly conserved either, yet we show with a novel statistical method using evolutionary modeling that the presence of HD on CAEDs cannot be the result of neutral evolutionary forces (p<0.0001). The motif is positively selected along the evolutionary process in the majority of APPOs, despite the fact that HD motif is underrepresented in the proteomes of all species of the animal kingdom. Position migration can be explained by high probability occurrence of multiple copies of HD on intermediate sequences, from which only one is kept by selective evolutionary forces, in a similar way as in the case of the “transcription binding site turnover.” CAED of all APP orthologues and homologues are predicted to bind metal ions including Amyloid-like protein 1 (APLP1) and Amyloid-like protein 2 (APLP2). Our results suggest that HDs on the CAEDs are most probably key components of metal-binding domains, which facilitate and/or regulate inter- or intra-molecular interactions in a metal ion-dependent or metal ion concentration-dependent manner. The involvement of naturally occurring mutations of HD (Tottori (D7N) and English (H6R) mutations) in early onset Alzheimer's disease gives additional support to our finding that HD has an evolutionary preserved function on APPOs. PMID:22319430

Conservation and diversification of Msx protein in metazoan evolution.

PubMed

Takahashi, Hirokazu; Kamiya, Akiko; Ishiguro, Akira; Suzuki, Atsushi C; Saitou, Naruya; Toyoda, Atsushi; Aruga, Jun

2008-01-01

Msx (/msh) family genes encode homeodomain (HD) proteins that control ontogeny in many animal species. We compared the structures of Msx genes from a wide range of Metazoa (Porifera, Cnidaria, Nematoda, Arthropoda, Tardigrada, Platyhelminthes, Mollusca, Brachiopoda, Annelida, Echiura, Echinodermata, Hemichordata, and Chordata) to gain an understanding of the role of these genes in phylogeny. Exon-intron boundary analysis suggested that the position of the intron located N-terminally to the HDs was widely conserved in all the genes examined, including those of cnidarians. Amino acid (aa) sequence comparison revealed 3 new evolutionarily conserved domains, as well as very strong conservation of the HDs. Two of the three domains were associated with Groucho-like protein binding in both a vertebrate and a cnidarian Msx homolog, suggesting that the interaction between Groucho-like proteins and Msx proteins was established in eumetazoan ancestors. Pairwise comparison among the collected HDs and their C-flanking aa sequences revealed that the degree of sequence conservation varied depending on the animal taxa from which the sequences were derived. Highly conserved Msx genes were identified in the Vertebrata, Cephalochordata, Hemichordata, Echinodermata, Mollusca, Brachiopoda, and Anthozoa. The wide distribution of the conserved sequences in the animal phylogenetic tree suggested that metazoan ancestors had already acquired a set of conserved domains of the current Msx family genes. Interestingly, although strongly conserved sequences were recovered from the Vertebrata, Cephalochordata, and Anthozoa, the sequences from the Urochordata and Hydrozoa showed weak conservation. Because the Vertebrata-Cephalochordata-Urochordata and Anthozoa-Hydrozoa represent sister groups in the Chordata and Cnidaria, respectively, Msx sequence diversification may have occurred differentially in the course of evolution. We speculate that selective loss of the conserved domains in Msx family proteins contributed to the diversification of animal body organization.

Measurement of MMP-9 and -12 degraded elastin (ELM) provides unique information on lung tissue degradation

PubMed Central

2012-01-01

Background Elastin is an essential component of selected connective tissues that provides a unique physiological elasticity. Elastin may be considered a signature protein of lungs where matrix metalloprotease (MMP) -9-and -12, may be considered the signature proteases of the macrophages, which in part are responsible for tissue damage during disease progression. Thus, we hypothesized that a MMP-9/-12 generated fragment of elastin may be a relevant biochemical maker for lung diseases. Methods Elastin fragments were identified by mass-spectrometry and one sequence, generated by MMP-9 and -12 (ELN-441), was selected for monoclonal antibody generation and used in the development of an ELISA. Soluble and insoluble elastin from lung was cleaved in vitro and the time-dependent release of fragments was assessed in the ELN-441 assay. The release of ELN-441 in human serum from patients with chronic obstructive pulmonary disease (COPD) (n = 10) and idiopathic pulmonary fibrosis (IPF) (n = 29) were compared to healthy matched controls (n = 11). Results The sequence ELN-441 was exclusively generated by MMP-9 and -12 and was time-dependently released from soluble lung elastin. ELN-441 levels were 287% higher in patients diagnosed with COPD (p < 0.001) and 124% higher in IPF patients (p < 0.0001) compared with controls. ELN-441 had better diagnostic value in COPD patients (AUC 97%, p = 0.001) than in IPF patients (AUC 90%, p = 0.0001). The odds ratios for differentiating controls from COPD or IPF were 24 [2.06–280] for COPD and 50 [2.64–934] for IPF. Conclusions MMP-9 and -12 time-dependently released the ELN-441 epitope from elastin. This fragment was elevated in serum from patients with the lung diseases IPF and COPD, however these data needs to be validated in larger clinical settings. PMID:22818364

Simultaneous fluorescence light-up and selective multicolor nucleobase recognition based on sequence-dependent strong binding of berberine to DNA abasic site.

PubMed

Wu, Fei; Shao, Yong; Ma, Kun; Cui, Qinghua; Liu, Guiying; Xu, Shujuan

2012-04-28

Label-free DNA nucleobase recognition by fluorescent small molecules has received much attention due to its simplicity in mutation identification and drug screening. However, sequence-dependent fluorescence light-up nucleobase recognition and multicolor emission with individual emission energy for individual nucleobases have been seldom realized. Herein, an abasic site (AP site) in a DNA duplex was employed as a binding field for berberine, one of isoquinoline alkaloids. Unlike weak binding of berberine to the fully matched DNAs without the AP site, strong binding of berberine to the AP site occurs and the berberine's fluorescence light-up behaviors are highly dependent on the target nucleobases opposite the AP site in which the targets thymine and cytosine produce dual emission bands, while the targets guanine and adenine only give a single emission band. Furthermore, more intense emissions are observed for the target pyrimidines than purines. The flanking bases of the AP site also produce some modifications of the berberine's emission behavior. The binding selectivity of berberine at the AP site is also confirmed by measurements of fluorescence resonance energy transfer, excited-state lifetime, DNA melting and fluorescence quenching by ferrocyanide and sodium chloride. It is expected that the target pyrimidines cause berberine to be stacked well within DNA base pairs near the AP site, which results in a strong resonance coupling of the electronic transitions to the particular vibration mode to produce the dual emissions. The fluorescent signal-on and emission energy-modulated sensing for nucleobases based on this fluorophore is substantially advantageous over the previously used fluorophores. We expect that this approach will be developed as a practical device for differentiating pyrimidines from purines by positioning an AP site toward a target that is available for readout by this alkaloid probe. This journal is © The Royal Society of Chemistry 2012

Domain Specificity of MAP3K Family Members, MLK and Tak1, for JNK Signaling in Drosophila

PubMed Central

Stronach, Beth; Lennox, Ashley L.; Garlena, Rebecca A.

2014-01-01

A highly diverse set of protein kinases functions as early responders in the mitogen- and stress-activated protein kinase (MAPK/SAPK) signaling pathways. For instance, humans possess 14 MAPK kinase kinases (MAP3Ks) that activate Jun kinase (JNK) signaling downstream. A major challenge is to decipher the selective and redundant functions of these upstream MAP3Ks. Taking advantage of the relative simplicity of Drosophila melanogaster as a model system, we assessed MAP3K signaling specificity in several JNK-dependent processes during development and stress response. Our approach was to generate molecular chimeras between two MAP3K family members, the mixed lineage kinase, Slpr, and the TGF-β activated kinase, Tak1, which share 32% amino acid identity across the kinase domain but otherwise differ in sequence and domain structure, and then test the contributions of various domains for protein localization, complementation of mutants, and activation of signaling. We found that overexpression of the wild-type kinases stimulated JNK signaling in alternate contexts, so cells were capable of responding to both MAP3Ks, but with distinct outcomes. Relative to wild-type, the catalytic domain swaps compensated weakly or not at all, despite having a shared substrate, the JNK kinase Hep. Tak1 C-terminal domain-containing constructs were inhibitory in Tak1 signaling contexts, including tumor necrosis factor-dependent cell death and innate immune signaling; however, depressing antimicrobial gene expression did not necessarily cause phenotypic susceptibility to infection. These same constructs were neutral in the context of Slpr-dependent developmental signaling, reflecting differential subcellular protein localization and by inference, point of activation. Altogether, our findings suggest that the selective deployment of a particular MAP3K can be attributed in part to its inherent sequence differences, cellular localization, and binding partner availability. PMID:24429281

Supplementation of Nucleosides During Selection can Reduce Sequence Variant Levels in CHO Cells Using GS/MSX Selection System.

PubMed

Tang, Danming; Lam, Cynthia; Louie, Salina; Hoi, Kam Hon; Shaw, David; Yim, Mandy; Snedecor, Brad; Misaghi, Shahram

2018-01-01

In the process of generating stable monoclonal antibody (mAb) producing cell lines, reagents such as methotrexate (MTX) or methionine sulfoximine (MSX) are often used. However, using such selection reagent(s) increases the possibility of having higher occurrence of sequence variants in the expressed antibody molecules due to the effects of MTX or MSX on de novo nucleotide synthesis. Since MSX inhibits glutamine synthase (GS) and results in both amino acid and nucleoside starvation, it is questioned whether supplementing nucleosides into the media could lower sequence variant levels without affecting titer. The results show that the supplementation of nucleosides to the media during MSX selection decreased genomic DNA mutagenesis rates in the selected cells, probably by reducing nucleotide mis-incorporation into the DNA. Furthermore, addition of nucleosides enhance clone recovery post selection and does not affect antibody expression. It is further observed that nucleoside supplements lowered DNA mutagenesis rates only at the initial stage of the clone selection and do not have any effect on DNA mutagenesis rates after stable cell lines are established. Therefore, the data suggests that addition of nucleosides during early stages of MSX selection can lower sequence variant levels without affecting titer or clone stability in antibody expression. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sequence-dependent modelling of local DNA bending phenomena: curvature prediction and vibrational analysis.

PubMed

Vlahovicek, K; Munteanu, M G; Pongor, S

1999-01-01

Bending is a local conformational micropolymorphism of DNA in which the original B-DNA structure is only distorted but not extensively modified. Bending can be predicted by simple static geometry models as well as by a recently developed elastic model that incorporate sequence dependent anisotropic bendability (SDAB). The SDAB model qualitatively explains phenomena including affinity of protein binding, kinking, as well as sequence-dependent vibrational properties of DNA. The vibrational properties of DNA segments can be studied by finite element analysis of a model subjected to an initial bending moment. The frequency spectrum is obtained by applying Fourier analysis to the displacement values in the time domain. This analysis shows that the spectrum of the bending vibrations quite sensitively depends on the sequence, for example the spectrum of a curved sequence is characteristically different from the spectrum of straight sequence motifs of identical basepair composition. Curvature distributions are genome-specific, and pronounced differences are found between protein-coding and regulatory regions, respectively, that is, sites of extreme curvature and/or bendability are less frequent in protein-coding regions. A WWW server is set up for the prediction of curvature and generation of 3D models from DNA sequences (http:@www.icgeb.trieste.it/dna).

Positive Selection Underlies Faster-Z Evolution of Gene Expression in Birds

PubMed Central

Dean, Rebecca; Harrison, Peter W.; Wright, Alison E.; Zimmer, Fabian; Mank, Judith E.

2015-01-01

The elevated rate of evolution for genes on sex chromosomes compared with autosomes (Fast-X or Fast-Z evolution) can result either from positive selection in the heterogametic sex or from nonadaptive consequences of reduced relative effective population size. Recent work in birds suggests that Fast-Z of coding sequence is primarily due to relaxed purifying selection resulting from reduced relative effective population size. However, gene sequence and gene expression are often subject to distinct evolutionary pressures; therefore, we tested for Fast-Z in gene expression using next-generation RNA-sequencing data from multiple avian species. Similar to studies of Fast-Z in coding sequence, we recover clear signatures of Fast-Z in gene expression; however, in contrast to coding sequence, our data indicate that Fast-Z in expression is due to positive selection acting primarily in females. In the soma, where gene expression is highly correlated between the sexes, we detected Fast-Z in both sexes, although at a higher rate in females, suggesting that many positively selected expression changes in females are also expressed in males. In the gonad, where intersexual correlations in expression are much lower, we detected Fast-Z for female gene expression, but crucially, not males. This suggests that a large amount of expression variation is sex-specific in its effects within the gonad. Taken together, our results indicate that Fast-Z evolution of gene expression is the product of positive selection acting on recessive beneficial alleles in the heterogametic sex. More broadly, our analysis suggests that the adaptive potential of Z chromosome gene expression may be much greater than that of gene sequence, results which have important implications for the role of sex chromosomes in speciation and sexual selection. PMID:26067773

Delineation of the KIAA2022 mutation phenotype: two patients with X-linked intellectual disability and distinctive features.

PubMed

Kuroda, Yukiko; Ohashi, Ikuko; Naruto, Takuya; Ida, Kazumi; Enomoto, Yumi; Saito, Toshiyuki; Nagai, Jun-Ichi; Wada, Takahito; Kurosawa, Kenji

2015-06-01

Next-generation sequencing has enabled the screening for a causative mutation in X-linked intellectual disability (XLID). We identified KIAA2022 mutations in two unrelated male patients by targeted sequencing. We selected 13 Japanese male patients with severe intellectual disability (ID), including four sibling patients and nine sporadic patients. Two of thirteen had a KIAA2022 mutation. Patient 1 was a 3-year-old boy. He had severe ID with autistic behavior and hypotonia. Patient 2 was a 5-year-old boy. He also had severe ID with autistic behavior, hypotonia, central hypothyroidism, and steroid-dependent nephrotic syndrome. Both patients revealed consistent distinctive features, including upswept hair, narrow forehead, downslanting eyebrows, wide palpebral fissures, long nose, hypoplastic alae nasi, open mouth, and large ears. De novo KIAA2022 mutations (p.Q705X in Patient 1, p.R322X in Patient 2) were detected by targeted sequencing and confirmed by Sanger sequencing. KIAA2022 mutations and alterations have been reported in only four families with nonsyndromic ID and epilepsy. KIAA2022 is highly expressed in the fetal and adult brain and plays a crucial role in neuronal development. These additional patients support the evidence that KIAA2022 is a causative gene for XLID. © 2015 Wiley Periodicals, Inc.

Disoxaril mutants of Coxsackievirus B1: phenotypic characteristics and analysis of the target VP1 gene.

PubMed

Nikolova, Ivanka; Galabov, Angel S; Petkova, Rumena; Chakarov, Stoyan; Atanasov, Boris

2011-01-01

Disoxaril inhibits enterovirus replication by binding to the hydrophobic pocket within the VP1 coat protein, thus stabilizing the virion and blocking its uncoating. Disoxaril-resistant (RES) mutants of the Coxsackievirus B1 (CVB1/RES) were derived from the wild disoxaril-sensitive (SOF) strain (CVB1/SOF) using a selection approach. A disoxaril-dependent (DEP) mutant (CVB1/DEP) was obtained following nine consecutive passages of the disoxaril-resistant mutant in the presence of disoxaril. Phenotypic characteristics of the disoxaril mutants were investigated. A timing-of-addition study of the CVB1/DEP replication demonstrated that in the absence of disoxaril the virus particle assembly stopped. VP1 RNA sequences of disoxaril mutants were compared with the existing Gen Bank CVB1 reference structure. The amino acid sequence of a large VP1 196-258 peptide (disoxaril-binding region) of CVB1/RES was significantly different from that of the CVB1/SOF. Crucially important changes in CVB1/RES were two point mutations, M213H and F237L, both in the ligand-binding pocket. The sequence analysis of the CVB1/DEP showed some reversion to CVB1/SOF. The amino acid sequences of the three VP1 proteins are presented.

Understanding the fate of the oxyallyl cation following Nazarov electrocyclization: sequential Wagner-Meerwein migrations and the synthesis of spirocyclic cyclopentenones

PubMed Central

Huang, Jie; Lebœuf, David; Frontier, Alison J.

2011-01-01

A general reaction sequence is described that involves Nazarov cyclization followed by two sequential Wagner Meerwein migrations, to afford spirocyclic compounds from divinyl ketones in the presence of one equivalent of copper(II) complexes. A detailed investigation of this sequence is described including a study of substrate scope and limitations. It was found that after 4π electrocyclization, two different pathways are available to the oxyallyl cation intermediate: elimination of a proton can give the usual Nazarov cycloadduct, or ring contraction can give an alternative tertiary carbocation. After ring contraction, either [1,2]-hydride or carbon migration can occur, depending upon the substitution pattern of the substrate, to furnish spirocyclic products. The rearrangement pathway is favored over the elimination pathway when catalyst loading was high and the copper(II) counterion is noncoordinating. Several ligands were found to be effective for the reaction. Thus, the reaction sequence can be controlled by judicious choice of reaction conditions to allow selective generation of richly functionalized spirocycles. The three steps of the sequence are stereospecific: electrocyclization followed by two [1,2]-suprafacial Wagner-Meerwein shifts: the ring contraction and then an hydride, alkenyl or aryl shift. The method allows stereospecific installation of adjacent stereocenters or adjacent quaternary centers arrayed around a cyclopentenone ring. PMID:21466152

A Novel Bioinformatics Strategy to Analyze Microbial Big Sequence Data for Efficient Knowledge Discovery: Batch-Learning Self-Organizing Map (BLSOM).

PubMed

Iwasaki, Yuki; Abe, Takashi; Wada, Kennosuke; Wada, Yoshiko; Ikemura, Toshimichi

2013-11-20

With the remarkable increase of genomic sequence data of microorganisms, novel tools are needed for comprehensive analyses of the big sequence data available. The self-organizing map (SOM) is an effective tool for clustering and visualizing high-dimensional data, such as oligonucleotide composition on one map. By modifying the conventional SOM, we developed batch-learning SOM (BLSOM), which allowed classification of sequence fragments (e.g., 1 kb) according to phylotypes, solely depending on oligonucleotide composition. Metagenomics studies of uncultivable microorganisms in clinical and environmental samples should allow extensive surveys of genes important in life sciences. BLSOM is most suitable for phylogenetic assignment of metagenomic sequences, because fragmental sequences can be clustered according to phylotypes, solely depending on oligonucleotide composition. We first constructed oligonucleotide BLSOMs for all available sequences from genomes of known species, and by mapping metagenomic sequences on these large-scale BLSOMs, we can predict phylotypes of individual metagenomic sequences, revealing a microbial community structure of uncultured microorganisms, including viruses. BLSOM has shown that influenza viruses isolated from humans and birds clearly differ in oligonucleotide composition. Based on this host-dependent oligonucleotide composition, we have proposed strategies for predicting directional changes of virus sequences and for surveilling potentially hazardous strains when introduced into humans from non-human sources.

A decade of pig genome sequencing: a window on pig domestication and evolution.

PubMed

Groenen, Martien A M

2016-03-29

Insight into how genomes change and adapt due to selection addresses key questions in evolutionary biology and in domestication of animals and plants by humans. In that regard, the pig and its close relatives found in Africa and Eurasia represent an excellent group of species that enables studies of the effect of both natural and human-mediated selection on the genome. The recent completion of the draft genome sequence of a domestic pig and the development of next-generation sequencing technology during the past decade have created unprecedented possibilities to address these questions in great detail. In this paper, I review recent whole-genome sequencing studies in the pig and closely-related species that provide insight into the demography, admixture and selection of these species and, in particular, how domestication and subsequent selection of Sus scrofa have shaped the genomes of these animals.

Sequence analysis of MHC class I α2 from sockeye salmon (Oncorhynchus nerka).

PubMed

McClelland, Erin K; Ming, Tobi J; Tabata, Amy; Miller, Kristina M

2011-09-01

Most studies assessing adaptive MHC diversity in salmon populations have focused on the classical class II DAB or DAA loci, as these have been most amenable to single PCR amplifications due to their relatively low level of sequence divergence. Herein, we report the characterization of the classical class I UBA α2 locus based on collections taken throughout the species range of sockeye salmon (Oncorhynchus nerka). Through use of multiple lineage-specific primer sets, denaturing gradient gel electrophoresis and sequencing, we identified thirty-four alleles from three highly divergent lineages. Sequence identity between lineages ranged from 30.0% to 56.8% but was relatively high within lineages. Allelic identity within the antigen recognition site (ARS) was greater than for the longer sequence. Global positive selection on UBA was seen at the sequence level (dN:dS = 1.012) with four codons under positive selection and 12 codons under negative selection. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

Generate Optimized Genetic Rhythm for Enzyme Expression in Non-native systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

2016-11-03

Most amino acids are represented by more than one codon, resulting in redundancy in the genetic code. Silent codon substitutions that do not alter the amino acid sequence still have an effect on protein expression. We have developed an algorithm, GoGREEN, to enhance the expression of foreign proteins in a host organism. GoGREEN selects codons according to frequency patterns seen in the gene of interest using the codon usage table from the host organism. GoGREEN is also designed to accommodate gaps in the sequence.This software takes for input (1) the aligned protein sequences for genes the user wishes to express,more » (2) the codon usage table for the host organism, (3) and the DNA sequence for the target protein found in the host organism. The program will select codons based on codon usage patterns for the target DNA sequence. The program will also select codons for “gaps” found in the aligned protein sequences using the codon usage table from the host organism.« less

Pseudouridines have context-dependent mutation and stop rates in high-throughput sequencing.

PubMed

Zhou, Katherine I; Clark, Wesley C; Pan, David W; Eckwahl, Matthew J; Dai, Qing; Pan, Tao

2018-05-11

The abundant RNA modification pseudouridine (Ψ) has been mapped transcriptome-wide by chemically modifying pseudouridines with carbodiimide and detecting the resulting reverse transcription stops in high-throughput sequencing. However, these methods have limited sensitivity and specificity, in part due to the use of reverse transcription stops. We sought to use mutations rather than just stops in sequencing data to identify pseudouridine sites. Here, we identify reverse transcription conditions that allow read-through of carbodiimide-modified pseudouridine (CMC-Ψ), and we show that pseudouridines in carbodiimide-treated human ribosomal RNA have context-dependent mutation and stop rates in high-throughput sequencing libraries prepared under these conditions. Furthermore, accounting for the context-dependence of mutation and stop rates can enhance the detection of pseudouridine sites. Similar approaches could contribute to the sequencing-based detection of many RNA modifications.

PFAAT version 2.0: a tool for editing, annotating, and analyzing multiple sequence alignments.

PubMed

Caffrey, Daniel R; Dana, Paul H; Mathur, Vidhya; Ocano, Marco; Hong, Eun-Jong; Wang, Yaoyu E; Somaroo, Shyamal; Caffrey, Brian E; Potluri, Shobha; Huang, Enoch S

2007-10-11

By virtue of their shared ancestry, homologous sequences are similar in their structure and function. Consequently, multiple sequence alignments are routinely used to identify trends that relate to function. This type of analysis is particularly productive when it is combined with structural and phylogenetic analysis. Here we describe the release of PFAAT version 2.0, a tool for editing, analyzing, and annotating multiple sequence alignments. Support for multiple annotations is a key component of this release as it provides a framework for most of the new functionalities. The sequence annotations are accessible from the alignment and tree, where they are typically used to label sequences or hyperlink them to related databases. Sequence annotations can be created manually or extracted automatically from UniProt entries. Once a multiple sequence alignment is populated with sequence annotations, sequences can be easily selected and sorted through a sophisticated search dialog. The selected sequences can be further analyzed using statistical methods that explicitly model relationships between the sequence annotations and residue properties. Residue annotations are accessible from the alignment viewer and are typically used to designate binding sites or properties for a particular residue. Residue annotations are also searchable, and allow one to quickly select alignment columns for further sequence analysis, e.g. computing percent identities. Other features include: novel algorithms to compute sequence conservation, mapping conservation scores to a 3D structure in Jmol, displaying secondary structure elements, and sorting sequences by residue composition. PFAAT provides a framework whereby end-users can specify knowledge for a protein family in the form of annotation. The annotations can be combined with sophisticated analysis to test hypothesis that relate to sequence, structure and function.

A Selective-Echo Method for Chemical-Shift Imaging of Two-Component Systems

NASA Astrophysics Data System (ADS)

Gerald, Rex E., II; Krasavin, Anatoly O.; Botto, Robert E.

A simple and effective method for selectively imaging either one of two chemical species in a two-component system is presented and demonstrated experimentally. The pulse sequence employed, selective- echo chemical- shift imaging (SECSI), is a hybrid (frequency-selective/ T1-contrast) technique that is executed in a short period of time, utilizes the full Boltzmann magnetization of each chemical species to form the corresponding image, and requires only hard pulses of quadrature phase. This approach provides a direct and unambiguous representation of the spatial distribution of the two chemical species. In addition, the performance characteristics and the advantages of the SECSI sequence are compared on a common basis to those of other pulse sequences.

An Evaluation of a Progressive High-Probability Instructional Sequence Combined with Low-Probability Demand Fading in the Treatment of Food Selectivity

ERIC Educational Resources Information Center

Penrod, Becky; Gardella, Laura; Fernand, Jonathan

2012-01-01

Few studies have examined the effects of the high-probability instructional sequence in the treatment of food selectivity, and results of these studies have been mixed (e.g., Dawson et al., 2003; Patel et al., 2007). The present study extended previous research on the high-probability instructional sequence by combining this procedure with…

Single nucleotide polymorphisms from Theobroma cacao expressed sequence tags associated with witches' broom disease in cacao.

PubMed

Lima, L S; Gramacho, K P; Carels, N; Novais, R; Gaiotto, F A; Lopes, U V; Gesteira, A S; Zaidan, H A; Cascardo, J C M; Pires, J L; Micheli, F

2009-07-14

In order to increase the efficiency of cacao tree resistance to witches' broom disease, which is caused by Moniliophthora perniciosa (Tricholomataceae), we looked for molecular markers that could help in the selection of resistant cacao genotypes. Among the different markers useful for developing marker-assisted selection, single nucleotide polymorphisms (SNPs) constitute the most common type of sequence difference between alleles and can be easily detected by in silico analysis from expressed sequence tag libraries. We report the first detection and analysis of SNPs from cacao-M. perniciosa interaction expressed sequence tags, using bioinformatics. Selection based on analysis of these SNPs should be useful for developing cacao varieties resistant to this devastating disease.

Mixed culture polyhydroxyalkanoates production from sugar molasses: the use of a 2-stage CSTR system for culture selection.

PubMed

Albuquerque, M G E; Concas, S; Bengtsson, S; Reis, M A M

2010-09-01

Polyhydroxyalkanoates (PHAs) are promising biodegradable polymers. The use of mixed microbial cultures (MMC) and low cost feedstocks have a positive impact on the cost-effectiveness of the process. It has typically been carried out in Sequencing Batch Reactors (SBR). In this study, a 2-stage CSTR system (under Feast and Famine conditions) was used to effectively select for PHA-storing organisms using fermented molasses as feedstock. The effect of influent substrate concentration (60-120 Cmmol VFA/L) and HRT ratio between the reactors (0.2-0.5h/h) on the system's selection efficiency was assessed. It was shown that Feast reactor residual substrate concentration impacted on the selective pressure for PHA storage (due to substrate-dependent kinetic limitation). Moreover, a residual substrate concentration coming from the Feast to the Famine reactor did not jeopardize the physiological adaptation required for enhanced PHA storage. The culture reached a maximum PHA content of 61%. This success opens new perspectives to the use of wastewater treatment infrastructure for PHA production, thus valorizing either excess sludge or wastewaters. Copyright 2010 Elsevier Ltd. All rights reserved.

An innovative approach for highly selective direct conversion of CO₂ into propanol using C₂H₄ and H₂.

PubMed

Ahlers, Stefan J; Bentrup, Ursula; Linke, David; Kondratenko, Evgenii V

2014-09-01

Multifunctional catalysts are developed for converting CO2 with C2H4 and H2 into propanol. Au nanoparticles (NP) supported on TiO2 are found to facilitate this reaction. The activity and selectivity strongly depend on NP size, which can be tuned by the method of Au deposition and by promoting with K. The promoter improves the selectivity to propanol. Under optimized reaction conditions (2 MPa, 473 K, and CO2/H2/C2H4=1:1:1), CO2 is continuously converted into propanol with a near-to-100% selectivity. Catalytic tests as well as mechanistic studies by in situ FTIR and temporal analysis of products with isotopic tracers allow the overall reaction scheme to be determined. Propanol is formed through a sequence of reactions starting with reverse water-gas shift to reduce CO2 to CO, which is further consumed in the hydroformylation of ethylene to propanal. The latter is finally hydrogenated to propanol, while propanol hydrogenation to propane is suppressed. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Improving the time efficiency of the Fourier synthesis method for slice selection in magnetic resonance imaging.

PubMed

Tahayori, B; Khaneja, N; Johnston, L A; Farrell, P M; Mareels, I M Y

2016-01-01

The design of slice selective pulses for magnetic resonance imaging can be cast as an optimal control problem. The Fourier synthesis method is an existing approach to solve these optimal control problems. In this method the gradient field as well as the excitation field are switched rapidly and their amplitudes are calculated based on a Fourier series expansion. Here, we provide a novel insight into the Fourier synthesis method via representing the Bloch equation in spherical coordinates. Based on the spherical Bloch equation, we propose an alternative sequence of pulses that can be used for slice selection which is more time efficient compared to the original method. Simulation results demonstrate that while the performance of both methods is approximately the same, the required time for the proposed sequence of pulses is half of the original sequence of pulses. Furthermore, the slice selectivity of both sequences of pulses changes with radio frequency field inhomogeneities in a similar way. We also introduce a measure, referred to as gradient complexity, to compare the performance of both sequences of pulses. This measure indicates that for a desired level of uniformity in the excited slice, the gradient complexity for the proposed sequence of pulses is less than the original sequence. Copyright © 2015 Associazione Italiana di Fisica Medica. Published by Elsevier Ltd. All rights reserved.

Theoretical studies on the selective mechanisms of GSK3β and CDK2 by molecular dynamics simulations and free energy calculations.

PubMed

Zhao, Sufang; Zhu, Jingyu; Xu, Lei; Jin, Jian

2017-06-01

Glycogen synthase kinase 3 (GSK3) is a serine/threonine protein kinase which is widely involved in cell signaling and controls a broad number of cellular functions. GSK3 contains α and β isoforms, and GSK3β has received more attention and becomes an attractive drug target for the treatment of several diseases. The binding pocket of cyclin-dependent kinase 2 (CDK2) shares high sequence identity to that of GSK3β, and therefore, the design of highly selective inhibitors toward GSK3β remains a big challenge. In this study, a computational strategy, which combines molecular docking, molecular dynamics simulations, free energy calculations, and umbrella sampling simulations, was employed to explore the binding mechanisms of two selective inhibitors to GSK3β and CDK2. The simulation results highlighted the key residues critical for GSK3β selectivity. It was observed that although GSK3β and CDK2 share the conserved ATP-binding pockets, some different residues have significant contributions to protein selectivity. This study provides valuable information for understanding the GSK3β-selective binding mechanisms and the rational design of selective GSK3β inhibitors. © 2016 John Wiley & Sons A/S.

Modeling the Embrace of a Mutator: APOBEC Selection of Nucleic Acid Ligands.

PubMed

Salter, Jason D; Smith, Harold C

2018-05-23

The 11-member APOBEC (apolipoprotein B mRNA editing catalytic polypeptide-like) family of zinc-dependent cytidine deaminases bind to RNA and single-stranded DNA (ssDNA) and, in specific contexts, modify select (deoxy)cytidines to (deoxy)uridines. In this review, we describe advances made through high-resolution co-crystal structures of APOBECs bound to mono- or oligonucleotides that reveal potential substrate-specific binding sites at the active site and non-sequence-specific nucleic acid binding sites distal to the active site. We also discuss the effect of APOBEC oligomerization on functionality. Future structural studies will need to address how ssDNA binding away from the active site may enhance catalysis and the mechanism by which RNA binding may modulate catalytic activity on ssDNA. Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.

An improved SELEX technique for selection of DNA aptamers binding to M-type 11 of Streptococcus pyogenes.

PubMed

Hamula, Camille L A; Peng, Hanyong; Wang, Zhixin; Tyrrell, Gregory J; Li, Xing-Fang; Le, X Chris

2016-03-15

Streptococcus pyogenes is a clinically important pathogen consisting of various serotypes determined by different M proteins expressed on the cell surface. The M type is therefore a useful marker to monitor the spread of invasive S. pyogenes in a population. Serotyping and nucleic acid amplification/sequencing methods for the identification of M types are laborious, inconsistent, and usually confined to reference laboratories. The primary objective of this work is to develop a technique that enables generation of aptamers binding to specific M-types of S. pyogenes. We describe here an in vitro technique that directly used live bacterial cells and the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) strategy. Live S. pyogenes cells were incubated with DNA libraries consisting of 40-nucleotides randomized sequences. Those sequences that bound to the cells were separated, amplified using polymerase chain reaction (PCR), purified using gel electrophoresis, and served as the input DNA pool for the next round of SELEX selection. A specially designed forward primer containing extended polyA20/5Sp9 facilitated gel electrophoresis purification of ssDNA after PCR amplification. A counter-selection step using non-target cells was introduced to improve selectivity. DNA libraries of different starting sequence diversity (10(16) and 10(14)) were compared. Aptamer pools from each round of selection were tested for their binding to the target and non-target cells using flow cytometry. Selected aptamer pools were then cloned and sequenced. Individual aptamer sequences were screened on the basis of their binding to the 10 M-types that were used as targets. Aptamer pools obtained from SELEX rounds 5-8 showed high affinity to the target S. pyogenes cells. Tests against non-target Streptococcus bovis, Streptococcus pneumoniae, and Enterococcus species demonstrated selectivity of these aptamers for binding to S. pyogenes. Several aptamer sequences were found to bind preferentially to the M11 M-type of S. pyogenes. Estimated binding dissociation constants (Kd) were in the low nanomolar range for the M11 specific sequences; for example, sequence E-CA20 had a Kd of 7±1 nM. These affinities are comparable to those of a monoclonal antibody. The improved bacterial cell-SELEX technique is successful in generating aptamers selective for S. pyogenes and some of its M-types. These aptamers are potentially useful for detecting S. pyogenes, achieving binding profiles of the various M-types, and developing new M-typing technologies for non-specialized laboratories or point-of-care testing. Copyright © 2015 Elsevier Inc. All rights reserved.

Contrasting patterns of selection between MHC I and II across populations of Humboldt and Magellanic penguins.

PubMed

Sallaberry-Pincheira, Nicole; González-Acuña, Daniel; Padilla, Pamela; Dantas, Gisele P M; Luna-Jorquera, Guillermo; Frere, Esteban; Valdés-Velásquez, Armando; Vianna, Juliana A

2016-10-01

The evolutionary and adaptive potential of populations or species facing an emerging infectious disease depends on their genetic diversity in genes, such as the major histocompatibility complex (MHC). In birds, MHC class I deals predominantly with intracellular infections (e.g., viruses) and MHC class II with extracellular infections (e.g., bacteria). Therefore, patterns of MHC I and II diversity may differ between species and across populations of species depending on the relative effect of local and global environmental selective pressures, genetic drift, and gene flow. We hypothesize that high gene flow among populations of Humboldt and Magellanic penguins limits local adaptation in MHC I and MHC II, and signatures of selection differ between markers, locations, and species. We evaluated the MHC I and II diversity using 454 next-generation sequencing of 100 Humboldt and 75 Magellanic penguins from seven different breeding colonies. Higher genetic diversity was observed in MHC I than MHC II for both species, explained by more than one MHC I loci identified. Large population sizes, high gene flow, and/or similar selection pressures maintain diversity but limit local adaptation in MHC I. A pattern of isolation by distance was observed for MHC II for Humboldt penguin suggesting local adaptation, mainly on the northernmost studied locality. Furthermore, trans-species alleles were found due to a recent speciation for the genus or convergent evolution. High MHC I and MHC II gene diversity described is extremely advantageous for the long-term survival of the species.

Pairwise Sequence Alignment Library

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeff Daily, PNNL

2015-05-20

Vector extensions, such as SSE, have been part of the x86 CPU since the 1990s, with applications in graphics, signal processing, and scientific applications. Although many algorithms and applications can naturally benefit from automatic vectorization techniques, there are still many that are difficult to vectorize due to their dependence on irregular data structures, dense branch operations, or data dependencies. Sequence alignment, one of the most widely used operations in bioinformatics workflows, has a computational footprint that features complex data dependencies. The trend of widening vector registers adversely affects the state-of-the-art sequence alignment algorithm based on striped data layouts. Therefore, amore » novel SIMD implementation of a parallel scan-based sequence alignment algorithm that can better exploit wider SIMD units was implemented as part of the Parallel Sequence Alignment Library (parasail). Parasail features: Reference implementations of all known vectorized sequence alignment approaches. Implementations of Smith Waterman (SW), semi-global (SG), and Needleman Wunsch (NW) sequence alignment algorithms. Implementations across all modern CPU instruction sets including AVX2 and KNC. Language interfaces for C/C++ and Python.« less

Contemporary NMR Studies of Protein Electrostatics.

PubMed

Hass, Mathias A S; Mulder, Frans A A

2015-01-01

Electrostatics play an important role in many aspects of protein chemistry. However, the accurate determination of side chain proton affinity in proteins by experiment and theory remains challenging. In recent years the field of nuclear magnetic resonance spectroscopy has advanced the way that protonation states are measured, allowing researchers to examine electrostatic interactions at an unprecedented level of detail and accuracy. Experiments are now in place that follow pH-dependent (13)C and (15)N chemical shifts as spatially close as possible to the sites of protonation, allowing all titratable amino acid side chains to be probed sequence specifically. The strong and telling response of carefully selected reporter nuclei allows individual titration events to be monitored. At the same time, improved frameworks allow researchers to model multiple coupled protonation equilibria and to identify the underlying pH-dependent contributions to the chemical shifts.

RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer

NASA Astrophysics Data System (ADS)

Wolfe, Andrew L.; Singh, Kamini; Zhong, Yi; Drewe, Philipp; Rajasekhar, Vinagolu K.; Sanghvi, Viraj R.; Mavrakis, Konstantinos J.; Jiang, Man; Roderick, Justine E.; van der Meulen, Joni; Schatz, Jonathan H.; Rodrigo, Christina M.; Zhao, Chunying; Rondou, Pieter; de Stanchina, Elisa; Teruya-Feldstein, Julie; Kelliher, Michelle A.; Speleman, Frank; Porco, John A.; Pelletier, Jerry; Rätsch, Gunnar; Wendel, Hans-Guido

2014-09-01

The translational control of oncoprotein expression is implicated in many cancers. Here we report an eIF4A RNA helicase-dependent mechanism of translational control that contributes to oncogenesis and underlies the anticancer effects of silvestrol and related compounds. For example, eIF4A promotes T-cell acute lymphoblastic leukaemia development in vivo and is required for leukaemia maintenance. Accordingly, inhibition of eIF4A with silvestrol has powerful therapeutic effects against murine and human leukaemic cells in vitro and in vivo. We use transcriptome-scale ribosome footprinting to identify the hallmarks of eIF4A-dependent transcripts. These include 5' untranslated region (UTR) sequences such as the 12-nucleotide guanine quartet (CGG)4 motif that can form RNA G-quadruplex structures. Notably, among the most eIF4A-dependent and silvestrol-sensitive transcripts are a number of oncogenes, superenhancer-associated transcription factors, and epigenetic regulators. Hence, the 5' UTRs of select cancer genes harbour a targetable requirement for the eIF4A RNA helicase.

Control of neuronal excitability by Group I metabotropic glutamate receptors.

PubMed

Correa, Ana Maria Bernal; Guimarães, Jennifer Diniz Soares; Dos Santos E Alhadas, Everton; Kushmerick, Christopher

2017-10-01

Metabotropic glutamate (mGlu) receptors couple through G proteins to regulate a large number of cell functions. Eight mGlu receptor isoforms have been cloned and classified into three Groups based on sequence, signal transduction mechanisms and pharmacology. This review will focus on Group I mGlu receptors, comprising the isoforms mGlu 1 and mGlu 5 . Activation of these receptors initiates both G protein-dependent and -independent signal transduction pathways. The G-protein-dependent pathway involves mainly Gα q , which can activate PLCβ, leading initially to the formation of IP 3 and diacylglycerol. IP 3 can release Ca 2+ from cellular stores resulting in activation of Ca 2+ -dependent ion channels. Intracellular Ca 2+ , together with diacylglycerol, activates PKC, which has many protein targets, including ion channels. Thus, activation of the G-protein-dependent pathway affects cellular excitability though several different effectors. In parallel, G protein-independent pathways lead to activation of non-selective cationic currents and metabotropic synaptic currents and potentials. Here, we provide a survey of the membrane transport proteins responsible for these electrical effects of Group I metabotropic glutamate receptors.

A systematic evaluation of three different cardiac T2-mapping sequences at 1.5 and 3T in healthy volunteers.

PubMed

Baeßler, Bettina; Schaarschmidt, Frank; Stehning, Christian; Schnackenburg, Bernhard; Maintz, David; Bunck, Alexander C

2015-11-01

Previous studies showed that myocardial T2 relaxation times measured by cardiac T2-mapping vary significantly depending on sequence and field strength. Therefore, a systematic comparison of different T2-mapping sequences and the establishment of dedicated T2 reference values is mandatory for diagnostic decision-making. Phantom experiments using gel probes with a range of different T1 and T2 times were performed on a clinical 1.5T and 3T scanner. In addition, 30 healthy volunteers were examined at 1.5 and 3T in immediate succession. In each examination, three different T2-mapping sequences were performed at three short-axis slices: Multi Echo Spin Echo (MESE), T2-prepared balanced SSFP (T2prep), and Gradient Spin Echo with and without fat saturation (GraSEFS/GraSE). Segmented T2-Maps were generated according to the AHA 16-segment model and statistical analysis was performed. Significant intra-individual differences between mean T2 times were observed for all sequences. In general, T2prep resulted in lowest and GraSE in highest T2 times. A significant variation with field strength was observed for mean T2 in phantom as well as in vivo, with higher T2 values at 1.5T compared to 3T, regardless of the sequence used. Segmental T2 values for each sequence at 1.5 and 3T are presented. Despite a careful selection of sequence parameters and volunteers, significant variations of the measured T2 values were observed between field strengths, MR sequences and myocardial segments. Therefore, we present segmental T2 values for each sequence at 1.5 and 3T with the inherent potential to serve as reference values for future studies. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Interactive toxicity of chlorpyrifos and parathion in neonatal rats: Role of esterases in exposure sequence-dependent toxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kacham, R.; Karanth, S.; Baireddy, P.

2006-01-15

We previously reported that sequence of exposure to chlorpyrifos and parathion in adult rats can markedly influence toxic outcome. In the present study, we evaluated the interactive toxicity of chlorpyrifos (8 mg/kg, po) and parathion (0.5 mg/kg, po) in neonatal (7 days old) rats. Rats were exposed to the insecticides either concurrently or sequentially (separated by 4 h) and sacrificed at 4, 8, and 24 h after the first exposure for biochemical measurements (cholinesterase activity in brain, plasma, and diaphragm and carboxylesterase activity in plasma and liver). The concurrently-exposed group showed more cumulative lethality (15/24) than either of the sequentialmore » dosing groups. With sequential dosing, rats treated initially with chlorpyrifos prior to parathion (C/P) exhibited higher lethality (7/23) compared to those treated with parathion before chlorpyrifos (P/C; 1/24). At 8 h after initial dosing, brain cholinesterase inhibition was significantly greater in the C/P group (59%) compared to the P/C group (28%). Diaphragm and plasma cholinesterase activity also followed a relatively similar pattern of inhibition. Carboxylesterase inhibition in plasma and liver was relatively similar among the treatment groups across time-points. Similar sequence-dependent differences in brain cholinesterase inhibition were also noted with lower binary exposures to chlorpyrifos (2 mg/kg) and parathion (0.35 mg/kg). In vitro and ex vivo studies compared relative oxon detoxification of carboxylesterases (calcium-insensitive) and A-esterases (calcium-sensitive) in liver homogenates from untreated and insecticide pretreated rats. Using tissues from untreated rats, carboxylesterases detoxified both chlorpyrifos oxon and paraoxon, while A-esterases only detoxified chlorpyrifos oxon. With parathion pretreatment, A-esterases still detoxified chlorpyrifos oxon while liver from chlorpyrifos pretreated rats had little apparent effect on paraoxon. We conclude that while neonatal rats are less capable than adults at detoxifying many organophosphorus insecticides including chlorpyrifos and parathion, toxicant-selective differences in detoxification play a role in sequence-dependent toxicity in both neonatal and adult rats with these two insecticides.« less

Sequence polymorphism in an insect RNA virus field population: A snapshot from a single point in space and time reveals stochastic differences among and within individual hosts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stenger, Drake C., E-mail: drake.stenger@ars.usda.

Population structure of Homalodisca coagulata Virus-1 (HoCV-1) among and within field-collected insects sampled from a single point in space and time was examined. Polymorphism in complete consensus sequences among single-insect isolates was dominated by synonymous substitutions. The mutant spectrum of the C2 helicase region within each single-insect isolate was unique and dominated by nonsynonymous singletons. Bootstrapping was used to correct the within-isolate nonsynonymous:synonymous arithmetic ratio (N:S) for RT-PCR error, yielding an N:S value ~one log-unit greater than that of consensus sequences. Probability of all possible single-base substitutions for the C2 region predicted N:S values within 95% confidence limits of themore » corrected within-isolate N:S when the only constraint imposed was viral polymerase error bias for transitions over transversions. These results indicate that bottlenecks coupled with strong negative/purifying selection drive consensus sequences toward neutral sequence space, and that most polymorphism within single-insect isolates is composed of newly-minted mutations sampled prior to selection. -- Highlights: •Sampling protocol minimized differential selection/history among isolates. •Polymorphism among consensus sequences dominated by negative/purifying selection. •Within-isolate N:S ratio corrected for RT-PCR error by bootstrapping. •Within-isolate mutant spectrum dominated by new mutations yet to undergo selection.« less

STAT1:DNA sequence-dependent binding modulation by phosphorylation, protein:protein interactions and small-molecule inhibition

PubMed Central

Bonham, Andrew J.; Wenta, Nikola; Osslund, Leah M.; Prussin, Aaron J.; Vinkemeier, Uwe; Reich, Norbert O.

2013-01-01

The DNA-binding specificity and affinity of the dimeric human transcription factor (TF) STAT1, were assessed by total internal reflectance fluorescence protein-binding microarrays (TIRF-PBM) to evaluate the effects of protein phosphorylation, higher-order polymerization and small-molecule inhibition. Active, phosphorylated STAT1 showed binding preferences consistent with prior characterization, whereas unphosphorylated STAT1 showed a weak-binding preference for one-half of the GAS consensus site, consistent with recent models of STAT1 structure and function in response to phosphorylation. This altered-binding preference was further tested by use of the inhibitor LLL3, which we show to disrupt STAT1 binding in a sequence-dependent fashion. To determine if this sequence-dependence is specific to STAT1 and not a general feature of human TF biology, the TF Myc/Max was analysed and tested with the inhibitor Mycro3. Myc/Max inhibition by Mycro3 is sequence independent, suggesting that the sequence-dependent inhibition of STAT1 may be specific to this system and a useful target for future inhibitor design. PMID:23180800

Identification of distal silencing elements in the murine interferon-A11 gene promoter.

PubMed

Roffet, P; Lopez, S; Navarro, S; Bandu, M T; Coulombel, C; Vignal, M; Doly, J; Vodjdani, G

1996-08-01

The murine interferon-A11 (Mu IFN-A11) gene is a member of the IFN-A multigenic family. In mouse L929 cells, the weak response of the gene's promoter to viral induction is due to a combination of both a point mutation in the virus responsive element (VRE) and the presence of negatively regulating sequences surrounding the VRE. In the distal part of the promoter, the negatively acting E1E2 sequence was delimited. This sequence displays an inhibitory effect in either orientation or position on the inducibility of a virus-responsive heterologous promoter. It selectively represses VRE-dependent transcription but is not able to reduce the transcriptional activity of a VRE-lacking promoter. In a transient transfection assay, an E1E2-containing DNA competitor was able to derepress the native Mu IFN-A11 promoter. Specific nuclear factors bind to this sequence; thus the binding of trans-regulators participates in the repression of the Mu IFN-A11 gene. The E1E2 sequence contains an IFN regulatory factor (IRF)-binding site. Recombinant IRF2 binds this sequence and anti-IRF2 antibodies supershift a major complex formed with nuclear extracts. The protein composing the complex is 50 kDa in size, indicating the presence of IRF2 or antigenically related proteins in the complex. The Mu IFN-A11 gene is the first example within the murine IFN-A family, in which a distal promoter element has been identified that can negatively modulate the transcriptional response to viral induction.

BEST: Improved Prediction of B-Cell Epitopes from Antigen Sequences

PubMed Central

Gao, Jianzhao; Faraggi, Eshel; Zhou, Yaoqi; Ruan, Jishou; Kurgan, Lukasz

2012-01-01

Accurate identification of immunogenic regions in a given antigen chain is a difficult and actively pursued problem. Although accurate predictors for T-cell epitopes are already in place, the prediction of the B-cell epitopes requires further research. We overview the available approaches for the prediction of B-cell epitopes and propose a novel and accurate sequence-based solution. Our BEST (B-cell Epitope prediction using Support vector machine Tool) method predicts epitopes from antigen sequences, in contrast to some method that predict only from short sequence fragments, using a new architecture based on averaging selected scores generated from sliding 20-mers by a Support Vector Machine (SVM). The SVM predictor utilizes a comprehensive and custom designed set of inputs generated by combining information derived from the chain, sequence conservation, similarity to known (training) epitopes, and predicted secondary structure and relative solvent accessibility. Empirical evaluation on benchmark datasets demonstrates that BEST outperforms several modern sequence-based B-cell epitope predictors including ABCPred, method by Chen et al. (2007), BCPred, COBEpro, BayesB, and CBTOPE, when considering the predictions from antigen chains and from the chain fragments. Our method obtains a cross-validated area under the receiver operating characteristic curve (AUC) for the fragment-based prediction at 0.81 and 0.85, depending on the dataset. The AUCs of BEST on the benchmark sets of full antigen chains equal 0.57 and 0.6, which is significantly and slightly better than the next best method we tested. We also present case studies to contrast the propensity profiles generated by BEST and several other methods. PMID:22761950

Deep sequencing of natural and experimental populations of Drosophila melanogaster reveals biases in the spectrum of new mutations.

PubMed

Assaf, Zoe June; Tilk, Susanne; Park, Jane; Siegal, Mark L; Petrov, Dmitri A

2017-12-01

Mutations provide the raw material of evolution, and thus our ability to study evolution depends fundamentally on having precise measurements of mutational rates and patterns. We generate a data set for this purpose using (1) de novo mutations from mutation accumulation experiments and (2) extremely rare polymorphisms from natural populations. The first, mutation accumulation (MA) lines are the product of maintaining flies in tiny populations for many generations, therefore rendering natural selection ineffective and allowing new mutations to accrue in the genome. The second, rare genetic variation from natural populations allows the study of mutation because extremely rare polymorphisms are relatively unaffected by the filter of natural selection. We use both methods in Drosophila melanogaster , first generating our own novel data set of sequenced MA lines and performing a meta-analysis of all published MA mutations (∼2000 events) and then identifying a high quality set of ∼70,000 extremely rare (≤0.1%) polymorphisms that are fully validated with resequencing. We use these data sets to precisely measure mutational rates and patterns. Highlights of our results include: a high rate of multinucleotide mutation events at both short (∼5 bp) and long (∼1 kb) genomic distances, showing that mutation drives GC content lower in already GC-poor regions, and using our precise context-dependent mutation rates to predict long-term evolutionary patterns at synonymous sites. We also show that de novo mutations from independent MA experiments display similar patterns of single nucleotide mutation and well match the patterns of mutation found in natural populations. © 2017 Assaf et al.; Published by Cold Spring Harbor Laboratory Press.

Identification of natural and artificial DNA substrates for the light-activated LOV-HTH transcription factor EL222

PubMed Central

Rivera-Cancel, Giomar; Motta-Mena, Laura B.; Gardner, Kevin H.

2012-01-01

Light-oxygen-voltage (LOV) domains serve as the photosensory modules for a wide range of plant and bacterial proteins, conferring blue light dependent regulation to effector activities as diverse as enzymes and DNA binding. LOV domains can also be engineered into a variety of exogenous targets, enabling similar regulation for new protein-based reagents. Common to these proteins is the ability for LOV domains to reversibly form a photochemical adduct between an internal flavin chromophore and the surrounding protein, using this to trigger conformational changes that affect output activity. Using the Erythrobacter litoralis protein EL222 model system which links LOV regulation to a helix-turn-helix (HTH) DNA binding domain, we demonstrated that the LOV domain binds and inhibits the HTH domain in the dark, releasing these interactions upon illumination [Nash et al. (2011) Proc. Natl. Acad. Sci. USA 108, 9449–9454]. Here we combine genomic and in vitro selection approaches to identify optimal DNA binding sites for EL222. Within the bacterial host, we observe binding several genomic sites using a 12 bp sequence consensus that is also found by in vitro selection methods. Sequence-specific alterations in the DNA consensus reduce EL222-binding affinity in a manner consistent with the expected binding mode: a protein dimer binding to two repeats. Finally, we demonstrate the light-dependent activation of transcription of two genes adjacent to an EL222 binding site. Taken together, these results shed light on the native function of EL222 and provide useful reagents for further basic and applications research of this versatile protein. PMID:23205774

Targeting helicase-dependent amplification products with an electrochemical genosensor for reliable and sensitive screening of genetically modified organisms.

PubMed

Moura-Melo, Suely; Miranda-Castro, Rebeca; de-Los-Santos-Álvarez, Noemí; Miranda-Ordieres, Arturo J; Dos Santos Junior, J Ribeiro; da Silva Fonseca, Rosana A; Lobo-Castañón, Maria Jesús

2015-08-18

Cultivation of genetically modified organisms (GMOs) and their use in food and feed is constantly expanding; thus, the question of informing consumers about their presence in food has proven of significant interest. The development of sensitive, rapid, robust, and reliable methods for the detection of GMOs is crucial for proper food labeling. In response, we have experimentally characterized the helicase-dependent isothermal amplification (HDA) and sequence-specific detection of a transgene from the Cauliflower Mosaic Virus 35S Promoter (CaMV35S), inserted into most transgenic plants. HDA is one of the simplest approaches for DNA amplification, emulating the bacterial replication machinery, and resembling PCR but under isothermal conditions. However, it usually suffers from a lack of selectivity, which is due to the accumulation of spurious amplification products. To improve the selectivity of HDA, which makes the detection of amplification products more reliable, we have developed an electrochemical platform targeting the central sequence of HDA copies of the transgene. A binary monolayer architecture is built onto a thin gold film where, upon the formation of perfect nucleic acid duplexes with the amplification products, these are enzyme-labeled and electrochemically transduced. The resulting combined system increases genosensor detectability up to 10(6)-fold, allowing Yes/No detection of GMOs with a limit of detection of ∼30 copies of the CaMV35S genomic DNA. A set of general utility rules in the design of genosensors for detection of HDA amplicons, which may assist in the development of point-of-care tests, is also included. The method provides a versatile tool for detecting nucleic acids with extremely low abundance not only for food safety control but also in the diagnostics and environmental control areas.

Effective assessment of egfr mutation status in bronchoalveolar lavage and pleural fluids by next-generation sequencing.

PubMed

Buttitta, Fiamma; Felicioni, Lara; Del Grammastro, Maela; Filice, Giampaolo; Di Lorito, Alessia; Malatesta, Sara; Viola, Patrizia; Centi, Irene; D'Antuono, Tommaso; Zappacosta, Roberta; Rosini, Sandra; Cuccurullo, Franco; Marchetti, Antonio

2013-02-01

The therapeutic choice for patients with lung adenocarcinoma depends on the presence of EGF receptor (EGFR) mutations. In many cases, only cytologic samples are available for molecular diagnosis. Bronchoalveolar lavage (BAL) and pleural fluid, which represent a considerable proportion of cytologic specimens, cannot always be used for molecular testing because of low rate of tumor cells. We tested the feasibility of EGFR mutation analysis on BAL and pleural fluid samples by next-generation sequencing (NGS), an innovative and extremely sensitive platform. The study was devised to extend the EGFR test to those patients who could not get it due to the paucity of biologic material. A series of 830 lung cytology specimens was used to select 48 samples (BAL and pleural fluid) from patients with EGFR mutations in resected tumors. These samples included 36 cases with 0.3% to 9% of neoplastic cells (series A) and 12 cases without evidence of tumor (series B). All samples were analyzed by Sanger sequencing and NGS on 454 Roche platform. A mean of 21,130 ± 2,370 sequences per sample were obtained by NGS. In series A, EGFR mutations were detected in 16% of cases by Sanger sequencing and in 81% of cases by NGS. Seventy-seven percent of cases found to be negative by Sanger sequencing showed mutations by NGS. In series B, all samples were negative for EGFR mutation by Sanger sequencing whereas 42% of them were positive by NGS. The very sensitive EGFR-NGS assay may open up to the possibility of specific treatments for patients otherwise doomed to re-biopsies or nontargeted therapies.

Natural Selection and Functional Potentials of Human Noncoding Elements Revealed by Analysis of Next Generation Sequencing Data

PubMed Central

Xu, Shuhua

2015-01-01

Noncoding DNA sequences (NCS) have attracted much attention recently due to their functional potentials. Here we attempted to reveal the functional roles of noncoding sequences from the point of view of natural selection that typically indicates the functional potentials of certain genomic elements. We analyzed nearly 37 million single nucleotide polymorphisms (SNPs) of Phase I data of the 1000 Genomes Project. We estimated a series of key parameters of population genetics and molecular evolution to characterize sequence variations of the noncoding genome within and between populations, and identified the natural selection footprints in NCS in worldwide human populations. Our results showed that purifying selection is prevalent and there is substantial constraint of variations in NCS, while positive selectionis more likely to be specific to some particular genomic regions and regional populations. Intriguingly, we observed larger fraction of non-conserved NCS variants with lower derived allele frequency in the genome, indicating possible functional gain of non-conserved NCS. Notably, NCS elements are enriched for potentially functional markers such as eQTLs, TF motif, and DNase I footprints in the genome. More interestingly, some NCS variants associated with diseases such as Alzheimer's disease, Type 1 diabetes, and immune-related bowel disorder (IBD) showed signatures of positive selection, although the majority of NCS variants, reported as risk alleles by genome-wide association studies, showed signatures of negative selection. Our analyses provided compelling evidence of natural selection forces on noncoding sequences in the human genome and advanced our understanding of their functional potentials that play important roles in disease etiology and human evolution. PMID:26053627

Signatures of positive selection in the cis-regulatory sequences of the human oxytocin receptor (OXTR) and arginine vasopressin receptor 1a (AVPR1A) genes.

PubMed

Schaschl, Helmut; Huber, Susanne; Schaefer, Katrin; Windhager, Sonja; Wallner, Bernard; Fieder, Martin

2015-05-13

The evolutionary highly conserved neurohypophyseal hormones oxytocin and arginine vasopressin play key roles in regulating social cognition and behaviours. The effects of these two peptides are meditated by their specific receptors, which are encoded by the oxytocin receptor (OXTR) and arginine vasopressin receptor 1a genes (AVPR1A), respectively. In several species, polymorphisms in these genes have been linked to various behavioural traits. Little, however, is known about whether positive selection acts on sequence variants in genes influencing variation in human behaviours. We identified, in both neuroreceptor genes, signatures of balancing selection in the cis-regulative acting sequences such as transcription factor binding and enhancer sequences, as well as in a transcriptional repressor sequence motif. Additionally, in the intron 3 of the OXTR gene, the SNP rs59190448 appears to be under positive directional selection. For rs59190448, only one phenotypical association is known so far, but it is in high LD' (>0.8) with loci of known association; i.e., variants associated with key pro-social behaviours and mental disorders in humans. Only for one SNP on the OXTR gene (rs59190448) was a sign of positive directional selection detected with all three methods of selection detection. For rs59190448, however, only one phenotypical association is known, but rs59190448 is in high LD' (>0.8), with variants associated with important pro-social behaviours and mental disorders in humans. We also detected various signatures of balancing selection on both neuroreceptor genes.

Genetic screens in human cells using the CRISPR-Cas9 system.

PubMed

Wang, Tim; Wei, Jenny J; Sabatini, David M; Lander, Eric S

2014-01-03

The bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system for genome editing has greatly expanded the toolbox for mammalian genetics, enabling the rapid generation of isogenic cell lines and mice with modified alleles. Here, we describe a pooled, loss-of-function genetic screening approach suitable for both positive and negative selection that uses a genome-scale lentiviral single-guide RNA (sgRNA) library. sgRNA expression cassettes were stably integrated into the genome, which enabled a complex mutant pool to be tracked by massively parallel sequencing. We used a library containing 73,000 sgRNAs to generate knockout collections and performed screens in two human cell lines. A screen for resistance to the nucleotide analog 6-thioguanine identified all expected members of the DNA mismatch repair pathway, whereas another for the DNA topoisomerase II (TOP2A) poison etoposide identified TOP2A, as expected, and also cyclin-dependent kinase 6, CDK6. A negative selection screen for essential genes identified numerous gene sets corresponding to fundamental processes. Last, we show that sgRNA efficiency is associated with specific sequence motifs, enabling the prediction of more effective sgRNAs. Collectively, these results establish Cas9/sgRNA screens as a powerful tool for systematic genetic analysis in mammalian cells.

Culture-dependent and culture-independent diversity of Actinobacteria associated with the marine sponge Hymeniacidon perleve from the South China Sea.

PubMed

Sun, Wei; Dai, Shikun; Jiang, Shumei; Wang, Guanghua; Liu, Guohui; Wu, Houbo; Li, Xiang

2010-06-01

In this report, the diversity of Actinobacteria associated with the marine sponge Hymeniacidon perleve collected from a remote island of the South China Sea was investigated employing classical cultivation and characterization, 16S rDNA library construction, 16S rDNA-restriction fragment length polymorphism (rDNA-RFLP) and phylogenetic analysis. A total of 184 strains were isolated using seven different media and 24 isolates were selected according to their morphological characteristics for phylogenetic analysis on the basis of their 16S rRNA gene sequences. Results showed that the 24 isolates were assigned to six genera including Salinispora, Gordonia, Mycobacterium, Nocardia, Rhodococcus and Streptomyces. This is the first report that Salinispora is present in a marine sponge from the South China Sea. Subsequently, 26 rDNA clones were selected from 191 clones in an Actinobacteria-specific 16S rDNA library of the H. perleve sample, using the RFLP technique for sequencing and phylogenetic analysis. In total, 26 phylotypes were clustered in eight known genera of Actinobacteria including Mycobacterium, Amycolatopsis, Arthrobacter, Brevibacterium, Microlunatus, Nocardioides, Pseudonocardia and Streptomyces. This study contributes to our understanding of actinobacterial diversity in the marine sponge H. perleve from the South China Sea.

Uncovering the design rules for peptide synthesis of metal nanoparticles.

PubMed

Tan, Yen Nee; Lee, Jim Yang; Wang, Daniel I C

2010-04-28

Peptides are multifunctional reagents (reducing and capping agents) that can be used for the synthesis of biocompatible metal nanoparticles under relatively mild conditions. However, the progress in peptide synthesis of metal nanoparticles has been slow due to the lack of peptide design rules. It is difficult to establish sequence-reactivity relationships from peptides isolated from biological sources (e.g., biomineralizing organisms) or selected by combinatorial display libraries because of their widely varying compositions and structures. The abundance of random and inactive amino acid sequences in the peptides also increases the difficulty in knowledge extraction. In this study, a "bottom-up" approach was used to formulate a set of rudimentary rules for the size- and shape-controlled peptide synthesis of gold nanoparticles from the properties of the 20 natural alpha-amino acids for AuCl(4)(-) reduction and binding to Au(0). It was discovered that the reduction capability of a peptide depends on the presence of certain reducing amino acid residues, whose activity may be regulated by neighboring residues with different Au(0) binding strengths. Another finding is the effect of peptide net charge on the nucleation and growth of the Au nanoparticles. On the basis of these understandings, several multifunctional peptides were designed to synthesize gold nanoparticles in different morphologies (nanospheres and nanoplates) and with sizes tunable by the strategic placement of selected amino acid residues in the peptide sequence. The methodology presented here and the findings are useful for establishing the scientific basis for the rational design of peptides for the synthesis of metal nanostructures.

Signatures of selection among sex-determining alleles of the honey bee.

PubMed

Hasselmann, Martin; Beye, Martin

2004-04-06

Patterns of DNA polymorphisms are a primary tool for dissecting signatures of selection; however, the underlying selective forces are poorly understood for most genes. A classical example of diversifying selection is the complementary sex-determining locus that is found in the very large insect order Hymenoptera (bees, wasps, ants, and sawflies). The gene responsible for sex determination, the complementary sex determiner (csd), has been most recently identified in the honey bee. Females are heterozygous at this locus. Males result when there is only one functional allele present, as a result of either homozygosity (fertilized eggs) or, more commonly, hemizygosity (unfertilized eggs). The homozygotes, diploid males, do not reproduce and have zero fitness, which implies positive selection in favor of rare alleles. Large differences in csd cDNA sequences within and between four populations were found that fall into two major groups, types I and II. Type I consists of several allelic lineages that were maintained over an extended period, an indication of balancing selection. Diversifying selection has operated on several confined parts of the protein, as shown by an excess of nonsynonymous differences. Elevated sequence differences indicate another selected part near a repeat region. These findings have general implications about the understanding of both the function of the multiallelic mechanism and the adaptive processes on the level of nucleotide sequences. Moreover, the first csd sequence data are a notable basis for the avoidance of diploid males in bee selection programs by allele-assisted breeding.

Fossil rhabdoviral sequences integrated into arthropod genomes: ontogeny, evolution, and potential functionality.

PubMed

Fort, Philippe; Albertini, Aurélie; Van-Hua, Aurélie; Berthomieu, Arnaud; Roche, Stéphane; Delsuc, Frédéric; Pasteur, Nicole; Capy, Pierre; Gaudin, Yves; Weill, Mylène

2012-01-01

Retroelements represent a considerable fraction of many eukaryotic genomes and are considered major drives for adaptive genetic innovations. Recent discoveries showed that despite not normally using DNA intermediates like retroviruses do, Mononegaviruses (i.e., viruses with nonsegmented, negative-sense RNA genomes) can integrate gene fragments into the genomes of their hosts. This was shown for Bornaviridae and Filoviridae, the sequences of which have been found integrated into the germ line cells of many vertebrate hosts. Here, we show that Rhabdoviridae sequences, the major Mononegavirales family, have integrated only into the genomes of arthropod species. We identified 185 integrated rhabdoviral elements (IREs) coding for nucleoproteins, glycoproteins, or RNA-dependent RNA polymerases; they were mostly found in the genomes of the mosquito Aedes aegypti and the blacklegged tick Ixodes scapularis. Phylogenetic analyses showed that most IREs in A. aegypti derived from multiple independent integration events. Since RNA viruses are submitted to much higher substitution rates as compared with their hosts, IREs thus represent fossil traces of the diversity of extinct Rhabdoviruses. Furthermore, analyses of orthologous IREs in A. aegypti field mosquitoes sampled worldwide identified an integrated polymerase IRE fragment that appeared under purifying selection within several million years, which supports a functional role in the host's biology. These results show that A. aegypti was subjected to repeated Rhabdovirus infectious episodes during its evolution history, which led to the accumulation of many integrated sequences. They also suggest that like retroviruses, integrated rhabdoviral sequences may participate actively in the evolution of their hosts.

Analysing grouping of nucleotides in DNA sequences using lumped processes constructed from Markov chains.

PubMed

Guédon, Yann; d'Aubenton-Carafa, Yves; Thermes, Claude

2006-03-01

The most commonly used models for analysing local dependencies in DNA sequences are (high-order) Markov chains. Incorporating knowledge relative to the possible grouping of the nucleotides enables to define dedicated sub-classes of Markov chains. The problem of formulating lumpability hypotheses for a Markov chain is therefore addressed. In the classical approach to lumpability, this problem can be formulated as the determination of an appropriate state space (smaller than the original state space) such that the lumped chain defined on this state space retains the Markov property. We propose a different perspective on lumpability where the state space is fixed and the partitioning of this state space is represented by a one-to-many probabilistic function within a two-level stochastic process. Three nested classes of lumped processes can be defined in this way as sub-classes of first-order Markov chains. These lumped processes enable parsimonious reparameterizations of Markov chains that help to reveal relevant partitions of the state space. Characterizations of the lumped processes on the original transition probability matrix are derived. Different model selection methods relying either on hypothesis testing or on penalized log-likelihood criteria are presented as well as extensions to lumped processes constructed from high-order Markov chains. The relevance of the proposed approach to lumpability is illustrated by the analysis of DNA sequences. In particular, the use of lumped processes enables to highlight differences between intronic sequences and gene untranslated region sequences.

Identification of a novel plant amalgavirus (Amalgavirus, Amalgaviridae) genome sequence in Cistus incanus.

PubMed

Goh, C J; Park, D; Lee, J S; Sebastiani, F; Hahn, Y

2018-01-01

Amalgaviridae is a family of double-stranded, monosegmented RNA viruses that are associated with plants, fungi, microsporidians, and animals. A sequence contig derived from the transcriptome of a eudicot, Cistus incanus (the family Cistaceae; commonly known as hoary rockrose), was identified as the genome sequence of a novel plant RNA virus and named Cistus incanus RNA virus 1 (CiRV1). Sequence comparison and phylogenetic analysis indicated that CiRV1 is a novel species of the genus Amalgavirus in the family Amalgaviridae. The CiRV1 genome contig has two overlapping open reading frames (ORFs). ORF1 encodes a putative replication factory matrix-like protein, while ORF2 encodes a RNA-dependent RNA polymerase (RdRp) domain. An ORF1+2 fusion protein, which functions in viral RNA replication, is produced by a +1 programmed ribosomal frameshifting (PRF) mechanism. A +1 PRF motif UUU_CGU, which matches the conserved amalgavirus +1 PRF consensus sequence UUU_CGN, was found at the boundary of CiRV1 ORF1 and ORF2. Comparison of 25 amalgavirus ORF1+2 fusion proteins revealed that only three different positions within a 13-amino acid segment were recurrently used at the boundary, possibly being selected so as not to interfere with correct folding and function of the fusion protein. CiRV1 is the first virus found to be associated with the Cistus species and may be useful for studying amalgaviruses.

Development of a novel biosensing system based on the structural change of a polymerized guanine-quadruplex DNA nanostructure.

PubMed

Morita, Yo; Yoshida, Wataru; Savory, Nasa; Han, Sung Woong; Tera, Masayuki; Nagasawa, Kazuo; Nakamura, Chikashi; Sode, Koji; Ikebukuro, Kazunori

2011-08-15

By inserting an adenosine aptamer into an aptamer that forms a G-quadruplex, we developed an adaptor molecule, named the Gq-switch, which links an electrode with flavin adenine dinucleotide-dependent glucose dehydrogenase (FADGDH) that is capable of transferring electron to a electrode directly. First, we selected an FADGDH-binding aptamer and identified that its sequence is composed of two blocks of consecutive six guanine bases and it forms a polymerized G-quadruplex structure. Then, we inserted a sequence of an adenosine aptamer between the two blocks of consecutive guanine bases, and we found it also bound to adenosine. Then we named it as Gq-switch. In the absence of adenosine, the Gq-switch-FADGDH complex forms a 30-nm high bulb-shaped structure that changes in the presence of adenosine to give an 8-nm high wire-shaped structure. This structural change brings the FADGDH sufficiently close to the electrode for electron transfer to occur, and the adenosine can be detected from the current produced by the FADGDH. Adenosine was successfully detected with a concentration dependency using the Gq-switch-FADGDH complex immobilized Au electrode by measuring response current to the addition of glucose. Copyright © 2011 Elsevier B.V. All rights reserved.

Comprehensive thermodynamic analysis of 3′ double-nucleotide overhangs neighboring Watson–Crick terminal base pairs

PubMed Central

O'Toole, Amanda S.; Miller, Stacy; Haines, Nathan; Zink, M. Coleen; Serra, Martin J.

2006-01-01

Thermodynamic parameters are reported for duplex formation of 48 self-complementary RNA duplexes containing Watson–Crick terminal base pairs (GC, AU and UA) with all 16 possible 3′ double-nucleotide overhangs; mimicking the structures of short interfering RNAs (siRNA) and microRNAs (miRNA). Based on nearest-neighbor analysis, the addition of a second dangling nucleotide to a single 3′ dangling nucleotide increases stability of duplex formation up to 0.8 kcal/mol in a sequence dependent manner. Results from this study in conjunction with data from a previous study [A. S. O'Toole, S. Miller and M. J. Serra (2005) RNA, 11, 512.] allows for the development of a refined nearest-neighbor model to predict the influence of 3′ double-nucleotide overhangs on the stability of duplex formation. The model improves the prediction of free energy and melting temperature when tested against five oligomers with various core duplex sequences. Phylogenetic analysis of naturally occurring miRNAs was performed to support our results. Selection of the effector miR strand of the mature miRNA duplex appears to be dependent upon the identity of the 3′ double-nucleotide overhang. Thermodynamic parameters for 3′ single terminal overhangs adjacent to a UA pair are also presented. PMID:16820533

The Effects of CBI Lesson Sequence Type and Field Dependence on Learning from Computer-Based Cooperative Instruction in Web

ERIC Educational Resources Information Center

Ipek, Ismail

2010-01-01

The purpose of this study was to investigate the effects of CBI lesson sequence type and cognitive style of field dependence on learning from Computer-Based Cooperative Instruction (CBCI) in WEB on the dependent measures, achievement, reading comprehension and reading rate. Eighty-seven college undergraduate students were randomly assigned to…

Accounting for rate-dependent category boundary shifts in speech perception.

PubMed

Bosker, Hans Rutger

2017-01-01

The perception of temporal contrasts in speech is known to be influenced by the speech rate in the surrounding context. This rate-dependent perception is suggested to involve general auditory processes because it is also elicited by nonspeech contexts, such as pure tone sequences. Two general auditory mechanisms have been proposed to underlie rate-dependent perception: durational contrast and neural entrainment. This study compares the predictions of these two accounts of rate-dependent speech perception by means of four experiments, in which participants heard tone sequences followed by Dutch target words ambiguous between /ɑs/ "ash" and /a:s/ "bait". Tone sequences varied in the duration of tones (short vs. long) and in the presentation rate of the tones (fast vs. slow). Results show that the duration of preceding tones did not influence target perception in any of the experiments, thus challenging durational contrast as explanatory mechanism behind rate-dependent perception. Instead, the presentation rate consistently elicited a category boundary shift, with faster presentation rates inducing more /a:s/ responses, but only if the tone sequence was isochronous. Therefore, this study proposes an alternative, neurobiologically plausible account of rate-dependent perception involving neural entrainment of endogenous oscillations to the rate of a rhythmic stimulus.

Transcriptome analysis of sika deer in China.

PubMed

Jia, Bo-Yin; Ba, Heng-Xing; Wang, Gui-Wu; Yang, Ying; Cui, Xue-Zhe; Peng, Ying-Hua; Zheng, Jun-Jun; Xing, Xiu-Mei; Yang, Fu-He

2016-10-01

Sika deer is of great commercial value because their antlers are used in tonics and alternative medicine and their meat is healthy and delicious. The goal of this study was to generate transcript sequences from sika deer for functional genomic analyses and to identify the transcripts that demonstrate tissue-specific, age-dependent differential expression patterns. These sequences could enhance our understanding of the molecular mechanisms underlying sika deer growth and development. In the present study, we performed de novo transcriptome assembly and profiling analysis across ten tissue types and four developmental stages (juvenile, adolescent, adult, and aged) of sika deer, using Illumina paired-end tag (PET) sequencing technology. A total of 1,752,253 contigs with an average length of 799 bp were generated, from which 1,348,618 unigenes with an average length of 590 bp were defined. Approximately 33.2 % of these (447,931 unigenes) were then annotated in public protein databases. Many sika deer tissue-specific, age-dependent unigenes were identified. The testes have the largest number of tissue-enriched unigenes, and some of them were prone to develop new functions for other tissues. Additionally, our transcriptome revealed that the juvenile-adolescent transition was the most complex and important stage of the sika deer life cycle. The present work represents the first multiple tissue transcriptome analysis of sika deer across four developmental stages. The generated data not only provide a functional genomics resource for future biological research on sika deer but also guide the selection and manipulation of genes controlling growth and development.

Simulation of Crack Propagation in Engine Rotating Components under Variable Amplitude Loading

NASA Technical Reports Server (NTRS)

Bonacuse, P. J.; Ghosn, L. J.; Telesman, J.; Calomino, A. M.; Kantzos, P.

1998-01-01

The crack propagation life of tested specimens has been repeatedly shown to strongly depend on the loading history. Overloads and extended stress holds at temperature can either retard or accelerate the crack growth rate. Therefore, to accurately predict the crack propagation life of an actual component, it is essential to approximate the true loading history. In military rotorcraft engine applications, the loading profile (stress amplitudes, temperature, and number of excursions) can vary significantly depending on the type of mission flown. To accurately assess the durability of a fleet of engines, the crack propagation life distribution of a specific component should account for the variability in the missions performed (proportion of missions flown and sequence). In this report, analytical and experimental studies are described that calibrate/validate the crack propagation prediction capability ]or a disk alloy under variable amplitude loading. A crack closure based model was adopted to analytically predict the load interaction effects. Furthermore, a methodology has been developed to realistically simulate the actual mission mix loading on a fleet of engines over their lifetime. A sequence of missions is randomly selected and the number of repeats of each mission in the sequence is determined assuming a Poisson distributed random variable with a given mean occurrence rate. Multiple realizations of random mission histories are generated in this manner and are used to produce stress, temperature, and time points for fracture mechanics calculations. The result is a cumulative distribution of crack propagation lives for a given, life limiting, component location. This information can be used to determine a safe retirement life or inspection interval for the given location.

Using machine learning for sequence-level automated MRI protocol selection in neuroradiology.

PubMed

Brown, Andrew D; Marotta, Thomas R

2018-05-01

Incorrect imaging protocol selection can lead to important clinical findings being missed, contributing to both wasted health care resources and patient harm. We present a machine learning method for analyzing the unstructured text of clinical indications and patient demographics from magnetic resonance imaging (MRI) orders to automatically protocol MRI procedures at the sequence level. We compared 3 machine learning models - support vector machine, gradient boosting machine, and random forest - to a baseline model that predicted the most common protocol for all observations in our test set. The gradient boosting machine model significantly outperformed the baseline and demonstrated the best performance of the 3 models in terms of accuracy (95%), precision (86%), recall (80%), and Hamming loss (0.0487). This demonstrates the feasibility of automating sequence selection by applying machine learning to MRI orders. Automated sequence selection has important safety, quality, and financial implications and may facilitate improvements in the quality and safety of medical imaging service delivery.

The Evolution of Vp1 Gene in Enterovirus C Species Sub-Group That Contains Types CVA-21, CVA-24, EV-C95, EV-C96 and EV-C99

PubMed Central

Smura, Teemu; Blomqvist, Soile; Vuorinen, Tytti; Ivanova, Olga; Samoilovich, Elena; Al-Hello, Haider; Savolainen-Kopra, Carita; Hovi, Tapani; Roivainen, Merja

2014-01-01

Genus Enterovirus (Family Picornaviridae,) consists of twelve species divided into genetically diverse types by their capsid protein VP1 coding sequences. Each enterovirus type can further be divided into intra-typic sub-clusters (genotypes). The aim of this study was to elucidate what leads to the emergence of novel enterovirus clades (types and genotypes). An evolutionary analysis was conducted for a sub-group of Enterovirus C species that contains types Coxsackievirus A21 (CVA-21), CVA-24, Enterovirus C95 (EV-C95), EV-C96 and EV-C99. VP1 gene datasets were collected and analysed to infer the phylogeny, rate of evolution, nucleotide and amino acid substitution patterns and signs of selection. In VP1 coding gene, high intra-typic sequence diversities and robust grouping into distinct genotypes within each type were detected. Within each type the majority of nucleotide substitutions were synonymous and the non-synonymous substitutions tended to cluster in distinct highly polymorphic sites. Signs of positive selection were detected in some of these highly polymorphic sites, while strong negative selection was indicated in most of the codons. Despite robust clustering to intra-typic genotypes, only few genotype-specific ‘signature’ amino acids were detected. In contrast, when different enterovirus types were compared, there was a clear tendency towards fixation of type-specific ‘signature’ amino acids. The results suggest that permanent fixation of type-specific amino acids is a hallmark associated with evolution of different enterovirus types, whereas neutral evolution and/or (frequency-dependent) positive selection in few highly polymorphic amino acid sites are the dominant forms of evolution when strains within an enterovirus type are compared. PMID:24695547

The evolution of Vp1 gene in enterovirus C species sub-group that contains types CVA-21, CVA-24, EV-C95, EV-C96 and EV-C99.

PubMed

Smura, Teemu; Blomqvist, Soile; Vuorinen, Tytti; Ivanova, Olga; Samoilovich, Elena; Al-Hello, Haider; Savolainen-Kopra, Carita; Hovi, Tapani; Roivainen, Merja

2014-01-01

Genus Enterovirus (Family Picornaviridae,) consists of twelve species divided into genetically diverse types by their capsid protein VP1 coding sequences. Each enterovirus type can further be divided into intra-typic sub-clusters (genotypes). The aim of this study was to elucidate what leads to the emergence of novel enterovirus clades (types and genotypes). An evolutionary analysis was conducted for a sub-group of Enterovirus C species that contains types Coxsackievirus A21 (CVA-21), CVA-24, Enterovirus C95 (EV-C95), EV-C96 and EV-C99. VP1 gene datasets were collected and analysed to infer the phylogeny, rate of evolution, nucleotide and amino acid substitution patterns and signs of selection. In VP1 coding gene, high intra-typic sequence diversities and robust grouping into distinct genotypes within each type were detected. Within each type the majority of nucleotide substitutions were synonymous and the non-synonymous substitutions tended to cluster in distinct highly polymorphic sites. Signs of positive selection were detected in some of these highly polymorphic sites, while strong negative selection was indicated in most of the codons. Despite robust clustering to intra-typic genotypes, only few genotype-specific 'signature' amino acids were detected. In contrast, when different enterovirus types were compared, there was a clear tendency towards fixation of type-specific 'signature' amino acids. The results suggest that permanent fixation of type-specific amino acids is a hallmark associated with evolution of different enterovirus types, whereas neutral evolution and/or (frequency-dependent) positive selection in few highly polymorphic amino acid sites are the dominant forms of evolution when strains within an enterovirus type are compared.

Selection and characterization of Argentine isolates of Trichoderma harzianum for effective biocontrol of Septoria leaf blotch of wheat.

PubMed

Stocco, Marina C; Mónaco, Cecilia I; Abramoff, Cecilia; Lampugnani, Gladys; Salerno, Graciela; Kripelz, Natalia; Cordo, Cristina A; Consolo, Verónica F

2016-03-01

Species of the genus Trichoderma are economically important as biocontrol agents, serving as a potential alternative to chemical control. The applicability of Trichoderma isolates to different ecozones will depend on the behavior of the strains selected from each zone. The present study was undertaken to isolate biocontrol populations of Trichoderma spp. from the Argentine wheat regions and to select and characterize the best strains of Trichoderma harzianum by means of molecular techniques. A total of 84 out of the 240 strains of Trichoderma were able to reduce the disease severity of the leaf blotch of wheat. Thirty-seven strains were selected for the reduction equal to or greater than 50% of the severity, compared with the control. The percentage values of reduction of the pycnidial coverage ranged between 45 and 80%. The same last strains were confirmed as T. harzianum by polymerase chain reaction amplification of internal transcribed spacers, followed by sequencing. Inter-simple sequence repeat was used to examine the genetic variability among isolates. This resulted in a total of 132 bands. Further numerical analysis revealed 19 haplotypes, grouped in three clusters (I, II, III). Shared strains, with different geographical origins and isolated in different years, were observed within each cluster. The origin of the isolates and the genetic group were partially related. All isolates from Paraná were in cluster I, all isolates from Lobería were in cluster II, and all isolates from Pergamino and Santa Fe were in cluster III. Our results suggest that the 37 native strains of T. harzianum are important in biocontrol programs and could be advantageous for the preparation of biopesticides adapted to the agroecological conditions of wheat culture.

Molecular principle of the cyclin-dependent kinase selectivity of 4-(thiazol-5-yl)-2-(phenylamino) pyrimidine-5-carbonitrile derivatives revealed by molecular modeling studies.

PubMed

Kong, Xiaotian; Sun, Huiyong; Pan, Peichen; Tian, Sheng; Li, Dan; Li, Youyong; Hou, Tingjun

2016-01-21

Due to the high sequence identity of the binding pockets of cyclin-dependent kinases (CDKs), designing highly selective inhibitors towards a specific CDK member remains a big challenge. 4-(thiazol-5-yl)-2-(phenylamino) pyrimidine derivatives are effective inhibitors of CDKs, among which the most promising inhibitor 12u demonstrates high binding affinity to CDK9 and attenuated binding affinity to other homologous kinases, such as CDK2. In this study, in order to rationalize the principle of the binding preference towards CDK9 over CDK2 and to explore crucial information that may aid the design of selective CDK9 inhibitors, MM/GBSA calculations based on conventional molecular dynamics (MD) simulations and enhanced sampling simulations (umbrella sampling and steered MD simulations) were carried out on two representative derivatives (12u and 4). The calculation results show that the binding specificity of 12u to CDK9 is primarily controlled by conformational change of the G-loop and variation of the van der Waals interactions. Furthermore, the enhanced sampling simulations revealed the different reaction coordinates and transient interactions of inhibitors 12u and 4 as they dissociate from the binding pockets of CDK9 and CDK2. The physical principles obtained from this study may facilitate the discovery and rational design of novel and specific inhibitors of CDK9.

Inducible Alkylation of DNA by a Quinone Methide-Peptide Nucleic Acid Conjugate†

PubMed Central

Liu, Yang; Rokita, Steven E.

2012-01-01

The reversibility of alkylation by a quinone methide intermediate (QM) avoids the irreversible consumption that plagues most reagents based on covalent chemistry and allows for site specific reaction that is controlled by the thermodynamics rather than kinetics of target association. This characteristic was originally examined with an oligonucleotide QM conjugate but broad application depends on alternative derivatives that are compatible with a cellular environment. Now, a peptide nucleic acid (PNA) derivative has been constructed and shown to exhibit an equivalent ability to delivery the reactive QM in a controlled manner. This new conjugate demonstrates high selectivity for a complementary sequence of DNA even when challenged with an alternative sequence containing a single T/T mismatch. Alkylation of non-complementary sequences is only possible when a template strand is present to co-localize the conjugate and its target. For efficient alkylation in this example, a single-stranded region of the target is required adjacent to the QM conjugate. Most importantly, the intrastrand self adducts formed between the PNA and its attached QM remained active and reversible over more than eight days in aqueous solution prior to reaction with a chosen target added subsequently. PMID:22243337

Sequence variants in the bovine silent information regulator 6, their linkage and their associations with body measurements and carcass quality traits in Qinchuan cattle.

PubMed

Gui, Linsheng; Jiang, Bijie; Zhang, Yaran; Zan, Linsen

2015-03-15

Silent information regulator 6 (SIRT6) belongs to the family of class III nicotinamide adenine dinucleotide (NAD)-dependent deacetylase and plays an essential role in DNA repair and metabolism. This study was conducted to detect potential polymorphisms of the bovine SIRT6 gene and explore their relationships with body measurement and carcass quality in Qinchuan cattle. Four sequence variants (SVs) were identified in intron 6, exon 7, exon 9, and 3' UTR, via sequencing technology conducted in 468 individual Qinchuan cattle. Eleven different haplotypes were identified, of which two major haplotypes had a frequency of 45.7% (-CACT-) and 14.8% (-CGTC-). Three SVs (SV2, SV3 and SV4) were significantly associated with some of the body measurements and carcass quality traits (P<0.05 or P<0.01), and the H2H7 (CC-GA-TT-TC) diplotype had better performance than other combinations. Our results suggest that some polymorphisms in SIRT6 are associated with production traits and may be used as candidates for marker-assisted selection (MAS) and management in beef cattle breeding programs. Copyright © 2015 Elsevier B.V. All rights reserved.

Simulation of gene evolution under directional mutational pressure

NASA Astrophysics Data System (ADS)

Dudkiewicz, Małgorzata; Mackiewicz, Paweł; Kowalczuk, Maria; Mackiewicz, Dorota; Nowicka, Aleksandra; Polak, Natalia; Smolarczyk, Kamila; Banaszak, Joanna; R. Dudek, Mirosław; Cebrat, Stanisław

2004-05-01

The two main mechanisms generating the genetic diversity, mutation and recombination, have random character but they are biased which has an effect on the generation of asymmetry in the bacterial chromosome structure and in the protein coding sequences. Thus, like in a case of two chiral molecules-the two possible orientations of a gene in relation to the topology of a chromosome are not equivalent. Assuming that the sequence of a gene may oscillate only between certain limits of its structural composition means that the gene could be forced out of these limits by the directional mutation pressure, in the course of evolution. The probability of the event depends on the time the gene stays under the same mutation pressure. Inversion of the gene changes the directional mutational pressure to the reciprocal one and hence it changes the distance of the gene to its lower and upper bound of the structural tolerance. Using Monte Carlo methods we were able to simulate the evolution of genes under experimentally found mutational pressure, assuming simple mechanisms of selection. We found that the mutation and recombination should work in accordance to lower their negative effects on the function of the products of coding sequences.

Adaptive molecular evolution of the two-pore channel 1 gene TPC1 in the karst-adapted genus Primulina (Gesneriaceae)

PubMed Central

Tao, Junjie; Feng, Chao; Ai, Bin; Kang, Ming

2016-01-01

Background and Aims Limestone karst areas possess high floral diversity and endemism. The genus Primulina, which contributes to the unique calcicole flora, has high species richness and exhibit specific soil-based habitat associations that are mainly distributed on calcareous karst soils. The adaptive molecular evolutionary mechanism of the genus to karst calcium-rich environments is still not well understood. The Ca2+-permeable channel TPC1 was used in this study to test whether its gene is involved in the local adaptation of Primulina to karst high-calcium soil environments. Methods Specific amplification and sequencing primers were designed and used to amplify the full-length coding sequences of TPC1 from cDNA of 76 Primulina species. The sequence alignment without recombination and the corresponding reconstructed phylogeny tree were used in molecular evolutionary analyses at the nucleic acid level and amino acid level, respectively. Finally, the identified sites under positive selection were labelled on the predicted secondary structure of TPC1. Key Results Seventy-six full-length coding sequences of Primulina TPC1 were obtained. The length of the sequences varied between 2220 and 2286 bp and the insertion/deletion was located at the 5′ end of the sequences. No signal of substitution saturation was detected in the sequences, while significant recombination breakpoints were detected. The molecular evolutionary analyses showed that TPC1 was dominated by purifying selection and the selective pressures were not significantly different among species lineages. However, significant signals of positive selection were detected at both TPC1 codon level and amino acid level, and five sites under positive selective pressure were identified by at least three different methods. Conclusions The Ca2+-permeable channel TPC1 may be involved in the local adaptation of Primulina to karst Ca2+-rich environments. Different species lineages suffered similar selective pressure associated with calcium in karst environments, and episodic diversifying selection at a few sites may play a major role in the molecular evolution of Primulina TPC1. PMID:27582362

Genomic prediction using preselected DNA variants from a GWAS with whole-genome sequence data in Holstein-Friesian cattle.

PubMed

Veerkamp, Roel F; Bouwman, Aniek C; Schrooten, Chris; Calus, Mario P L

2016-12-01

Whole-genome sequence data is expected to capture genetic variation more completely than common genotyping panels. Our objective was to compare the proportion of variance explained and the accuracy of genomic prediction by using imputed sequence data or preselected SNPs from a genome-wide association study (GWAS) with imputed whole-genome sequence data. Phenotypes were available for 5503 Holstein-Friesian bulls. Genotypes were imputed up to whole-genome sequence (13,789,029 segregating DNA variants) by using run 4 of the 1000 bull genomes project. The program GCTA was used to perform GWAS for protein yield (PY), somatic cell score (SCS) and interval from first to last insemination (IFL). From the GWAS, subsets of variants were selected and genomic relationship matrices (GRM) were used to estimate the variance explained in 2087 validation animals and to evaluate the genomic prediction ability. Finally, two GRM were fitted together in several models to evaluate the effect of selected variants that were in competition with all the other variants. The GRM based on full sequence data explained only marginally more genetic variation than that based on common SNP panels: for PY, SCS and IFL, genomic heritability improved from 0.81 to 0.83, 0.83 to 0.87 and 0.69 to 0.72, respectively. Sequence data also helped to identify more variants linked to quantitative trait loci and resulted in clearer GWAS peaks across the genome. The proportion of total variance explained by the selected variants combined in a GRM was considerably smaller than that explained by all variants (less than 0.31 for all traits). When selected variants were used, accuracy of genomic predictions decreased and bias increased. Although 35 to 42 variants were detected that together explained 13 to 19% of the total variance (18 to 23% of the genetic variance) when fitted alone, there was no advantage in using dense sequence information for genomic prediction in the Holstein data used in our study. Detection and selection of variants within a single breed are difficult due to long-range linkage disequilibrium. Stringent selection of variants resulted in more biased genomic predictions, although this might be due to the training population being the same dataset from which the selected variants were identified.

Inability of Prevotella bryantii to Form a Functional Shine-Dalgarno Interaction Reflects Unique Evolution of Ribosome Binding Sites in Bacteroidetes

PubMed Central

Accetto, Tomaž; Avguštin, Gorazd

2011-01-01

The Shine-Dalgarno (SD) sequence is a key element directing the translation to initiate at the authentic start codons and also enabling translation initiation to proceed in 5′ untranslated mRNA regions (5′-UTRs) containing moderately strong secondary structures. Bioinformatic analysis of almost forty genomes from the major bacterial phylum Bacteroidetes revealed, however, a general absence of SD sequence, drop in GC content and consequently reduced tendency to form secondary structures in 5′-UTRs. The experiments using the Prevotella bryantii TC1-1 expression system were in agreement with these findings: neither addition nor omission of SD sequence in the unstructured 5′-UTR affected the level of the reporter protein, non-specific nuclease NucB. Further, NucB level in P. bryantii TC1-1, contrary to hMGFP level in Escherichia coli, was five times lower when SD sequence formed part of the secondary structure with a folding energy -5,2 kcal/mol. Also, the extended SD sequences did not affect protein levels as in E. coli. It seems therefore that a functional SD interaction does not take place during the translation initiation in P. bryanttii TC1-1 and possibly other members of phylum Bacteroidetes although the anti SD sequence is present in 16S rRNA genes of their genomes. We thus propose that in the absence of the SD sequence interaction, the selection of genuine start codons in Bacteroidetes is accomplished by binding of ribosomal protein S1 to unstructured 5′-UTR as opposed to coding region which is inaccessible due to mRNA secondary structure. Additionally, we found that sequence logos of region preceding the start codons may be used as taxonomical markers. Depending on whether complete sequence logo or only part of it, such as information content and base proportion at specific positions, is used, bacterial genera or families and in some cases even bacterial phyla can be distinguished. PMID:21857964

SENCA: A Multilayered Codon Model to Study the Origins and Dynamics of Codon Usage

PubMed Central

Pouyet, Fanny; Bailly-Bechet, Marc; Mouchiroud, Dominique; Guéguen, Laurent

2016-01-01

Gene sequences are the target of evolution operating at different levels, including the nucleotide, codon, and amino acid levels. Disentangling the impact of those different levels on gene sequences requires developing a probabilistic model with three layers. Here we present SENCA (site evolution of nucleotides, codons, and amino acids), a codon substitution model that separately describes 1) nucleotide processes which apply on all sites of a sequence such as the mutational bias, 2) preferences between synonymous codons, and 3) preferences among amino acids. We argue that most synonymous substitutions are not neutral and that SENCA provides more accurate estimates of selection compared with more classical codon sequence models. We study the forces that drive the genomic content evolution, intraspecifically in the core genome of 21 prokaryotes and interspecifically for five Enterobacteria. We retrieve the existence of a universal mutational bias toward AT, and that taking into account selection on synonymous codon usage has consequences on the measurement of selection on nonsynonymous substitutions. We also confirm that codon usage bias is mostly driven by selection on preferred codons. We propose new summary statistics to measure the relative importance of the different evolutionary processes acting on sequences. PMID:27401173

Evolutionary characterization of hemagglutinin gene of H9N2 influenza viruses isolated from Asia.

PubMed

Shahsavandi, Shahla; Salmanian, Ali-Hatef; Ghorashi, Seyed Ali; Masoudi, Shahin; Ebrahimi, Mohammad Majid

2012-08-01

The full length hemagglutinin (HA) genes of 287 H9N2 AI strains isolated from chickens in Asia during the period 1994-2009 were genetically analyzed. Phylogenetic analysis showed that G1-like viruses circulated in the Middle East and Indian sub-continent countries, whereas other sublineages existed in Far East countries. It also revealed G1-like viruses with an average 96.7% identity clustered into two subgroups largely based on their time of isolation. The Ka/Ks ratio was calculated 0.34 for subgroup 1 and 0.57 for subgroup 2 indicates purifying/stabilizing selection, but despite this there is evidence of localized positive selection when comparing the subgroups 1 and 2 protein sequences. Five sites in HA H9N2 viruses had a posterior probability >0.5 using the Bayesian method, indicating these sites were under positive selection. These sites were found to be associated with the globular head region of HA. To identify sites under positive selection; amino acid substitution classified depends on their radicalism and neutrality. The results indicate that, although most positions in HAs were under purifying selection and can be eliminated, a few positions located in the antigenic regions and receptor binding sites were subject to positive selection. Copyright © 2011 Elsevier Ltd. All rights reserved.

Context-Dependent Learning in People With Parkinson's Disease.

PubMed

Lee, Ya-Yun; Winstein, Carolee J; Gordon, James; Petzinger, Giselle M; Zelinski, Elizabeth M; Fisher, Beth E

2016-01-01

Context-dependent learning is a phenomenon in which people demonstrate superior performance in the context in which they originally learned a skill but perform less well in a novel context. This study investigated context-dependent learning in people with Parkinson's disease (PD) and age-matched nondisabled adults. All participants practiced 3 finger sequences, each embedded within a unique context (colors and locations on a computer screen). One day after practice, the participants were tested either under the sequence-context associations remained the same as during practice, or the sequence-context associations were changed (SWITCH). Compared with nondisabled adults, people with PD demonstrated significantly greater decrement in performance (especially movement time) under the SWITCH condition, suggesting that individuals with PD are more context dependent than nondisabled adults.

Epigenomic Views of Innate Lymphoid Cells.

PubMed

Sciumè, Giuseppe; Shih, Han-Yu; Mikami, Yohei; O'Shea, John J

2017-01-01

The discovery of innate lymphoid cells (ILCs) with selective production of cytokines typically attributed to subsets of T helper cells forces immunologists to reassess the mechanisms by which selective effector functions arise. The parallelism between ILCs and T cells extends beyond these two cell types and comprises other innate-like T lymphocytes. Beyond the recognition of specialized effector functionalities in diverse lymphocytes, features typical of T cells, such as plasticity and memory, are also relevant for innate lymphocytes. Herein, we review what we have learned in terms of the molecular mechanisms underlying these shared functions, focusing on insights provided by next generation sequencing technologies. We review data on the role of lineage-defining- and signal-dependent transcription factors (TFs). ILC regulomes emerge developmentally whereas the much of the open chromatin regions of T cells are generated acutely, in an activation-dependent manner. And yet, these regions of open chromatin in T cells and ILCs have remarkable overlaps, suggesting that though accessibility is acquired by distinct modes, the end result is that convergent signaling pathways may be involved. Although much is left to be learned, substantial progress has been made in understanding how TFs and epigenomic status contribute to ILC biology in terms of differentiation, specification, and plasticity.

Epigenomic Views of Innate Lymphoid Cells

PubMed Central

Sciumè, Giuseppe; Shih, Han-Yu; Mikami, Yohei; O’Shea, John J.

2017-01-01

The discovery of innate lymphoid cells (ILCs) with selective production of cytokines typically attributed to subsets of T helper cells forces immunologists to reassess the mechanisms by which selective effector functions arise. The parallelism between ILCs and T cells extends beyond these two cell types and comprises other innate-like T lymphocytes. Beyond the recognition of specialized effector functionalities in diverse lymphocytes, features typical of T cells, such as plasticity and memory, are also relevant for innate lymphocytes. Herein, we review what we have learned in terms of the molecular mechanisms underlying these shared functions, focusing on insights provided by next generation sequencing technologies. We review data on the role of lineage-defining- and signal-dependent transcription factors (TFs). ILC regulomes emerge developmentally whereas the much of the open chromatin regions of T cells are generated acutely, in an activation-dependent manner. And yet, these regions of open chromatin in T cells and ILCs have remarkable overlaps, suggesting that though accessibility is acquired by distinct modes, the end result is that convergent signaling pathways may be involved. Although much is left to be learned, substantial progress has been made in understanding how TFs and epigenomic status contribute to ILC biology in terms of differentiation, specification, and plasticity. PMID:29250060

Bi-functional, substrate mimicking RNA inhibits MSK1-mediated cAMP-response element-binding protein phosphorylation and reveals magnesium ion-dependent conformational changes of the kinase.

PubMed

Hamm, Jorg; Alessi, Dario R; Biondi, Ricardo M

2002-11-29

The design of specific inhibitors for protein kinases is an important step toward elucidation of intracellular signal transduction pathways and to guide drug discovery programs. We devised a model approach to generate specific, competitive kinase inhibitors by isolating substrate mimics containing two independent binding sites with an anti-idiotype strategy from combinatorial RNA libraries. As a general test for the ability to generate highly specific kinase inhibitors, we selected the transcription factor cAMP-response element-binding protein (CREB) that is phosphorylated on the same serine residue by the protein kinase MSK1 as well as by RSK1. The sequences and structures of these kinases are very similar, about 60% of their amino acids are identical. Nevertheless, we can demonstrate that the selected RNA inhibitors inhibit specifically CREB phosphorylation by MSK1 but do not affect CREB phosphorylation by RSK1. The inhibitors interact preferentially with the inactive form of MSK1. Furthermore, we demonstrate that RNA ligands can be conformation-specific probes, and this feature allowed us to describe magnesium ion-dependent conformational changes of MSK1 upon activation.

DNA capture elements for rapid detection and identification of biological agents

NASA Astrophysics Data System (ADS)

Kiel, Johnathan L.; Parker, Jill E.; Holwitt, Eric A.; Vivekananda, Jeeva

2004-08-01

DNA capture elements (DCEs; aptamers) are artificial DNA sequences, from a random pool of sequences, selected for their specific binding to potential biological warfare agents. These sequences were selected by an affinity method using filters to which the target agent was attached and the DNA isolated and amplified by polymerase chain reaction (PCR) in an iterative, increasingly stringent, process. Reporter molecules were attached to the finished sequences. To date, we have made DCEs to Bacillus anthracis spores, Shiga toxin, Venezuelan Equine Encephalitis (VEE) virus, and Francisella tularensis. These DCEs have demonstrated specificity and sensitivity equal to or better than antibody.

Positive Selection Underlies Faster-Z Evolution of Gene Expression in Birds.

PubMed

Dean, Rebecca; Harrison, Peter W; Wright, Alison E; Zimmer, Fabian; Mank, Judith E

2015-10-01

The elevated rate of evolution for genes on sex chromosomes compared with autosomes (Fast-X or Fast-Z evolution) can result either from positive selection in the heterogametic sex or from nonadaptive consequences of reduced relative effective population size. Recent work in birds suggests that Fast-Z of coding sequence is primarily due to relaxed purifying selection resulting from reduced relative effective population size. However, gene sequence and gene expression are often subject to distinct evolutionary pressures; therefore, we tested for Fast-Z in gene expression using next-generation RNA-sequencing data from multiple avian species. Similar to studies of Fast-Z in coding sequence, we recover clear signatures of Fast-Z in gene expression; however, in contrast to coding sequence, our data indicate that Fast-Z in expression is due to positive selection acting primarily in females. In the soma, where gene expression is highly correlated between the sexes, we detected Fast-Z in both sexes, although at a higher rate in females, suggesting that many positively selected expression changes in females are also expressed in males. In the gonad, where intersexual correlations in expression are much lower, we detected Fast-Z for female gene expression, but crucially, not males. This suggests that a large amount of expression variation is sex-specific in its effects within the gonad. Taken together, our results indicate that Fast-Z evolution of gene expression is the product of positive selection acting on recessive beneficial alleles in the heterogametic sex. More broadly, our analysis suggests that the adaptive potential of Z chromosome gene expression may be much greater than that of gene sequence, results which have important implications for the role of sex chromosomes in speciation and sexual selection. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Dynamics of actin evolution in dinoflagellates.

PubMed

Kim, Sunju; Bachvaroff, Tsvetan R; Handy, Sara M; Delwiche, Charles F

2011-04-01

Dinoflagellates have unique nuclei and intriguing genome characteristics with very high DNA content making complete genome sequencing difficult. In dinoflagellates, many genes are found in multicopy gene families, but the processes involved in the establishment and maintenance of these gene families are poorly understood. Understanding the dynamics of gene family evolution in dinoflagellates requires comparisons at different evolutionary scales. Studies of closely related species provide fine-scale information relative to species divergence, whereas comparisons of more distantly related species provides broad context. We selected the actin gene family as a highly expressed conserved gene previously studied in dinoflagellates. Of the 142 sequences determined in this study, 103 were from the two closely related species, Dinophysis acuminata and D. caudata, including full length and partial cDNA sequences as well as partial genomic amplicons. For these two Dinophysis species, at least three types of sequences could be identified. Most copies (79%) were relatively similar and in nucleotide trees, the sequences formed two bushy clades corresponding to the two species. In comparisons within species, only eight to ten nucleotide differences were found between these copies. The two remaining types formed clades containing sequences from both species. One type included the most similar sequences in between-species comparisons with as few as 12 nucleotide differences between species. The second type included the most divergent sequences in comparisons between and within species with up to 93 nucleotide differences between sequences. In all the sequences, most variation occurred in synonymous sites or the 5' UnTranslated Region (UTR), although there was still limited amino acid variation between most sequences. Several potential pseudogenes were found (approximately 10% of all sequences depending on species) with incomplete open reading frames due to frameshifts or early stop codons. Overall, variation in the actin gene family fits best with the "birth and death" model of evolution based on recent duplications, pseudogenes, and incomplete lineage sorting. Divergence between species was similar to variation within species, so that actin may be too conserved to be useful for phylogenetic estimation of closely related species.

Rare missense variants in CHRNB3 and CHRNA3 are associated with risk of alcohol and cocaine dependence.

PubMed

Haller, Gabe; Kapoor, Manav; Budde, John; Xuei, Xiaoling; Edenberg, Howard; Nurnberger, John; Kramer, John; Brooks, Andy; Tischfield, Jay; Almasy, Laura; Agrawal, Arpana; Bucholz, Kathleen; Rice, John; Saccone, Nancy; Bierut, Laura; Goate, Alison

2014-02-01

Previous findings have demonstrated that variants in nicotinic receptor genes are associated with nicotine, alcohol and cocaine dependence. Because of the substantial comorbidity, it has often been unclear whether a variant is associated with multiple substances or whether the association is actually with a single substance. To investigate the possible contribution of rare variants to the development of substance dependencies other than nicotine dependence, specifically alcohol and cocaine dependence, we undertook pooled sequencing of the coding regions and flanking sequence of CHRNA5, CHRNA3, CHRNB4, CHRNA6 and CHRNB3 in 287 African American and 1028 European American individuals from the Collaborative Study of the Genetics of Alcoholism (COGA). All members of families for whom any individual was sequenced (2504 African Americans and 7318 European Americans) were then genotyped for all variants identified by sequencing. For each gene, we then tested for association using FamSKAT. For European Americans, we find increased DSM-IV cocaine dependence symptoms (FamSKAT P = 2 × 10(-4)) and increased DSM-IV alcohol dependence symptoms (FamSKAT P = 5 × 10(-4)) among carriers of missense variants in CHRNB3. Additionally, one variant (rs149775276; H329Y) shows association with both cocaine dependence symptoms (P = 7.4 × 10(-5), β = 2.04) and alcohol dependence symptoms (P = 2.6 × 10(-4), β = 2.04). For African Americans, we find decreased cocaine dependence symptoms among carriers of missense variants in CHRNA3 (FamSKAT P = 0.005). Replication in an independent sample supports the role of rare variants in CHRNB3 and alcohol dependence (P = 0.006). These are the first results to implicate rare variants in CHRNB3 or CHRNA3 in risk for alcohol dependence or cocaine dependence.

The nearly neutral and selection theories of molecular evolution under the fisher geometrical framework: substitution rate, population size, and complexity.

PubMed

Razeto-Barry, Pablo; Díaz, Javier; Vásquez, Rodrigo A

2012-06-01

The general theories of molecular evolution depend on relatively arbitrary assumptions about the relative distribution and rate of advantageous, deleterious, neutral, and nearly neutral mutations. The Fisher geometrical model (FGM) has been used to make distributions of mutations biologically interpretable. We explored an FGM-based molecular model to represent molecular evolutionary processes typically studied by nearly neutral and selection models, but in which distributions and relative rates of mutations with different selection coefficients are a consequence of biologically interpretable parameters, such as the average size of the phenotypic effect of mutations and the number of traits (complexity) of organisms. A variant of the FGM-based model that we called the static regime (SR) represents evolution as a nearly neutral process in which substitution rates are determined by a dynamic substitution process in which the population's phenotype remains around a suboptimum equilibrium fitness produced by a balance between slightly deleterious and slightly advantageous compensatory substitutions. As in previous nearly neutral models, the SR predicts a negative relationship between molecular evolutionary rate and population size; however, SR does not have the unrealistic properties of previous nearly neutral models such as the narrow window of selection strengths in which they work. In addition, the SR suggests that compensatory mutations cannot explain the high rate of fixations driven by positive selection currently found in DNA sequences, contrary to what has been previously suggested. We also developed a generalization of SR in which the optimum phenotype can change stochastically due to environmental or physiological shifts, which we called the variable regime (VR). VR models evolution as an interplay between adaptive processes and nearly neutral steady-state processes. When strong environmental fluctuations are incorporated, the process becomes a selection model in which evolutionary rate does not depend on population size, but is critically dependent on the complexity of organisms and mutation size. For SR as well as VR we found that key parameters of molecular evolution are linked by biological factors, and we showed that they cannot be fixed independently by arbitrary criteria, as has usually been assumed in previous molecular evolutionary models.

The Nearly Neutral and Selection Theories of Molecular Evolution Under the Fisher Geometrical Framework: Substitution Rate, Population Size, and Complexity

PubMed Central

Razeto-Barry, Pablo; Díaz, Javier; Vásquez, Rodrigo A.

2012-01-01

The general theories of molecular evolution depend on relatively arbitrary assumptions about the relative distribution and rate of advantageous, deleterious, neutral, and nearly neutral mutations. The Fisher geometrical model (FGM) has been used to make distributions of mutations biologically interpretable. We explored an FGM-based molecular model to represent molecular evolutionary processes typically studied by nearly neutral and selection models, but in which distributions and relative rates of mutations with different selection coefficients are a consequence of biologically interpretable parameters, such as the average size of the phenotypic effect of mutations and the number of traits (complexity) of organisms. A variant of the FGM-based model that we called the static regime (SR) represents evolution as a nearly neutral process in which substitution rates are determined by a dynamic substitution process in which the population’s phenotype remains around a suboptimum equilibrium fitness produced by a balance between slightly deleterious and slightly advantageous compensatory substitutions. As in previous nearly neutral models, the SR predicts a negative relationship between molecular evolutionary rate and population size; however, SR does not have the unrealistic properties of previous nearly neutral models such as the narrow window of selection strengths in which they work. In addition, the SR suggests that compensatory mutations cannot explain the high rate of fixations driven by positive selection currently found in DNA sequences, contrary to what has been previously suggested. We also developed a generalization of SR in which the optimum phenotype can change stochastically due to environmental or physiological shifts, which we called the variable regime (VR). VR models evolution as an interplay between adaptive processes and nearly neutral steady-state processes. When strong environmental fluctuations are incorporated, the process becomes a selection model in which evolutionary rate does not depend on population size, but is critically dependent on the complexity of organisms and mutation size. For SR as well as VR we found that key parameters of molecular evolution are linked by biological factors, and we showed that they cannot be fixed independently by arbitrary criteria, as has usually been assumed in previous molecular evolutionary models. PMID:22426879

Combining phage display with de novo protein sequencing for reverse engineering of monoclonal antibodies.

PubMed

Rickert, Keith W; Grinberg, Luba; Woods, Robert M; Wilson, Susan; Bowen, Michael A; Baca, Manuel

2016-01-01

The enormous diversity created by gene recombination and somatic hypermutation makes de novo protein sequencing of monoclonal antibodies a uniquely challenging problem. Modern mass spectrometry-based sequencing will rarely, if ever, provide a single unambiguous sequence for the variable domains. A more likely outcome is computation of an ensemble of highly similar sequences that can satisfy the experimental data. This outcome can result in the need for empirical testing of many candidate sequences, sometimes iteratively, to identity one which can replicate the activity of the parental antibody. Here we describe an improved approach to antibody protein sequencing by using phage display technology to generate a combinatorial library of sequences that satisfy the mass spectrometry data, and selecting for functional candidates that bind antigen. This approach was used to reverse engineer 2 commercially-obtained monoclonal antibodies against murine CD137. Proteomic data enabled us to assign the majority of the variable domain sequences, with the exception of 3-5% of the sequence located within or adjacent to complementarity-determining regions. To efficiently resolve the sequence in these regions, small phage-displayed libraries were generated and subjected to antigen binding selection. Following enrichment of antigen-binding clones, 2 clones were selected for each antibody and recombinantly expressed as antigen-binding fragments (Fabs). In both cases, the reverse-engineered Fabs exhibited identical antigen binding affinity, within error, as Fabs produced from the commercial IgGs. This combination of proteomic and protein engineering techniques provides a useful approach to simplifying the technically challenging process of reverse engineering monoclonal antibodies from protein material.

Combining phage display with de novo protein sequencing for reverse engineering of monoclonal antibodies

PubMed Central

Rickert, Keith W.; Grinberg, Luba; Woods, Robert M.; Wilson, Susan; Bowen, Michael A.; Baca, Manuel

2016-01-01

ABSTRACT The enormous diversity created by gene recombination and somatic hypermutation makes de novo protein sequencing of monoclonal antibodies a uniquely challenging problem. Modern mass spectrometry-based sequencing will rarely, if ever, provide a single unambiguous sequence for the variable domains. A more likely outcome is computation of an ensemble of highly similar sequences that can satisfy the experimental data. This outcome can result in the need for empirical testing of many candidate sequences, sometimes iteratively, to identity one which can replicate the activity of the parental antibody. Here we describe an improved approach to antibody protein sequencing by using phage display technology to generate a combinatorial library of sequences that satisfy the mass spectrometry data, and selecting for functional candidates that bind antigen. This approach was used to reverse engineer 2 commercially-obtained monoclonal antibodies against murine CD137. Proteomic data enabled us to assign the majority of the variable domain sequences, with the exception of 3–5% of the sequence located within or adjacent to complementarity-determining regions. To efficiently resolve the sequence in these regions, small phage-displayed libraries were generated and subjected to antigen binding selection. Following enrichment of antigen-binding clones, 2 clones were selected for each antibody and recombinantly expressed as antigen-binding fragments (Fabs). In both cases, the reverse-engineered Fabs exhibited identical antigen binding affinity, within error, as Fabs produced from the commercial IgGs. This combination of proteomic and protein engineering techniques provides a useful approach to simplifying the technically challenging process of reverse engineering monoclonal antibodies from protein material. PMID:26852694

Streptococcal phosphoenolpyruvate-sugar phosphotransferase system: amino acid sequence and site of ATP-dependent phosphorylation of HPr

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deutscher, J.; Pevec, B.; Beyreuther, K.

1986-10-21

The amino acid sequence of histidine-containing protein (HPr) from Streptococcus faecalis has been determined by direct Edman degradation of intact HPr and by amino acid sequence analysis of tryptic peptides, V8 proteolyptic peptides, thermolytic peptides, and cyanogen bromide cleavage products. HPr from S. faecalis was found to contain 89 amino acid residues, corresponding to a molecular weight of 9438. The amino acid sequence of HPr from S. faecalis shows extended homology to the primary structure of HPr proteins from other bacteria. Besides the phosphoenolpyruvate-dependent phosphorylation of a histidyl residue in HPr, catalyzed by enzyme I of the bacterial phosphotransferase system,more » HPr was also found to be phosphorylated at a seryl residue in an ATP-dependent protein kinase catalyzed reaction. The site of ATP-dependent phosphorylation in HPr of S faecalis has now been determined. (/sup 32/P)P-Ser-HPr was digested with three different proteases, and in each case, a single labeled peptide was isolated. Following digestion with subtilisin, they obtained a peptide with the sequence -(P)Ser-Ile-Met-. Using chymotrypsin, they isolated a peptide with the sequence -Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-Gly-Val-Met-. The longest labeled peptide was obtained with V8 staphylococcal protease. According to amino acid analysis, this peptide contained 36 out of the 89 amino acid residues of HPr. The following sequence of 12 amino acid residues of the V8 peptide was determined: -Tyr-Lys-Gly-Lys-Ser-Val-Asn-Leu-Lys-(P)Ser-Ile-Met-. Thus, the site of ATP-dependent phosphorylation was determined to be Ser-46 within the primary structure of HPr.« less

Stability of Solutions to Classes of Traveling Salesman Problems.

PubMed

Niendorf, Moritz; Kabamba, Pierre T; Girard, Anouck R

2016-04-01

By performing stability analysis on an optimal tour for problems belonging to classes of the traveling salesman problem (TSP), this paper derives margins of optimality for a solution with respect to disturbances in the problem data. Specifically, we consider the asymmetric sequence-dependent TSP, where the sequence dependence is driven by the dynamics of a stack. This is a generalization of the symmetric non sequence-dependent version of the TSP. Furthermore, we also consider the symmetric sequence-dependent variant and the asymmetric non sequence-dependent variant. Amongst others these problems have applications in logistics and unmanned aircraft mission planning. Changing external conditions such as traffic or weather may alter task costs, which can render an initially optimal itinerary suboptimal. Instead of optimizing the itinerary every time task costs change, stability criteria allow for fast evaluation of whether itineraries remain optimal. This paper develops a method to compute stability regions for the best tour in a set of tours for the symmetric TSP and extends the results to the asymmetric problem as well as their sequence-dependent counterparts. As the TSP is NP-hard, heuristic methods are frequently used to solve it. The presented approach is also applicable to analyze stability regions for a tour obtained through application of the k -opt heuristic with respect to the k -neighborhood. A dimensionless criticality metric for edges is proposed, such that a high criticality of an edge indicates that the optimal tour is more susceptible to cost changes in that edge. Multiple examples demonstrate the application of the developed stability computation method as well as the edge criticality measure that facilitates an intuitive assessment of instances of the TSP.

Identification of (R)-selective ω-aminotransferases by exploring evolutionary sequence space.

PubMed

Kim, Eun-Mi; Park, Joon Ho; Kim, Byung-Gee; Seo, Joo-Hyun

2018-03-01

Several (R)-selective ω-aminotransferases (R-ωATs) have been reported. The existence of additional R-ωATs having different sequence characteristics from previous ones is highly expected. In addition, it is generally accepted that R-ωATs are variants of aminotransferase group III. Based on these backgrounds, sequences in RefSeq database were scored using family profiles of branched-chain amino acid aminotransferase (BCAT) and d-alanine aminotransferase (DAT) to predict and identify putative R-ωATs. Sequences with two profile analysis scores were plotted on two-dimensional score space. Candidates with relatively similar scores in both BCAT and DAT profiles (i.e., profile analysis score using BCAT profile was similar to profile analysis score using DAT profile) were selected. Experimental results for selected candidates showed that putative R-ωATs from Saccharopolyspora erythraea (R-ωAT_Sery), Bacillus cellulosilyticus (R-ωAT_Bcel), and Bacillus thuringiensis (R-ωAT_Bthu) had R-ωAT activity. Additional experiments revealed that R-ωAT_Sery also possessed DAT activity while R-ωAT_Bcel and R-ωAT_Bthu had BCAT activity. Selecting putative R-ωATs from regions with similar profile analysis scores identified potential R-ωATs. Therefore, R-ωATs could be efficiently identified by using simple family profile analysis and exploring evolutionary sequence space. Copyright © 2017 Elsevier Inc. All rights reserved.

Selection and Screening of DNA Aptamers for Inorganic Nanomaterials.

PubMed

Zhou, Yibo; Huang, Zhicheng; Yang, Ronghua; Liu, Juewen

2018-02-21

Searching for DNA sequences that can strongly and selectively bind to inorganic surfaces is a long-standing topic in bionanotechnology, analytical chemistry and biointerface research. This can be achieved either by aptamer selection starting with a very large library of ≈10 14 random DNA sequences, or by careful screening of a much smaller library (usually from a few to a few hundred) with rationally designed sequences. Unlike typical molecular targets, inorganic surfaces often have quite strong DNA adsorption affinities due to polyvalent binding and even chemical interactions. This leads to a very high background binding making aptamer selection difficult. Screening, on the other hand, can be designed to compare relative binding affinities of different DNA sequences and could be more appropriate for inorganic surfaces. The resulting sequences have been used for DNA-directed assembly, sorting of carbon nanotubes, and DNA-controlled growth of inorganic nanomaterials. It was recently discovered that poly-cytosine (C) DNA can strongly bind to a diverse range of nanomaterials including nanocarbons (graphene oxide and carbon nanotubes), various metal oxides and transition-metal dichalcogenides. In this Concept article, we articulate the need for screening and potential artifacts associated with traditional aptamer selection methods for inorganic surfaces. Representative examples of application are discussed, and a few future research opportunities are proposed towards the end of this article. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of the species Malassezia pachydermatis and re-evaluation of its lipid dependence using a synthetic agar medium

PubMed Central

Puig, Laura; Castellá, Gemma

2017-01-01

The genus Malassezia includes lipophilic yeasts, which are part of the skin microbiota of various mammals and birds. Unlike the rest of Malassezia species, M. pachydermatis is described as non-lipid-dependent, as it is able to grow on Sabouraud glucose agar (SGA) without lipid supplementation. In this study we have examined the phenotypic variability within M. pachydermatis and confirmed its lipid-dependent nature using a synthetic agar medium. We used a selection of representative non-lipid-dependent strains from different animal species and three atypical lipid-dependent strains of this species, which were not able to grow after multiple passages on SGA. More than 400 lipid-dependent Malassezia isolates from animals were studied in order to detect the three lipid-dependent strains of M. pachydermatis. The identity of the atypical strains was confirmed by DNA sequencing. On the other hand, we have modified the Tween diffusion test, which is widely used in the characterization of these yeasts, by using a synthetic agar-based medium instead of SGA. This modification has proved to be useful for differentiation of M. pachydermatis strains, providing reproducible results and a straightforward interpretation. The finding of these peculiar lipid-dependent strains exemplifies the large variability within the species M. pachydermatis, which involves rare atypical strains with particular growth requirements. PMID:28586389

Characterization of the species Malassezia pachydermatis and re-evaluation of its lipid dependence using a synthetic agar medium.

PubMed

Puig, Laura; Bragulat, M Rosa; Castellá, Gemma; Cabañes, F Javier

2017-01-01

The genus Malassezia includes lipophilic yeasts, which are part of the skin microbiota of various mammals and birds. Unlike the rest of Malassezia species, M. pachydermatis is described as non-lipid-dependent, as it is able to grow on Sabouraud glucose agar (SGA) without lipid supplementation. In this study we have examined the phenotypic variability within M. pachydermatis and confirmed its lipid-dependent nature using a synthetic agar medium. We used a selection of representative non-lipid-dependent strains from different animal species and three atypical lipid-dependent strains of this species, which were not able to grow after multiple passages on SGA. More than 400 lipid-dependent Malassezia isolates from animals were studied in order to detect the three lipid-dependent strains of M. pachydermatis. The identity of the atypical strains was confirmed by DNA sequencing. On the other hand, we have modified the Tween diffusion test, which is widely used in the characterization of these yeasts, by using a synthetic agar-based medium instead of SGA. This modification has proved to be useful for differentiation of M. pachydermatis strains, providing reproducible results and a straightforward interpretation. The finding of these peculiar lipid-dependent strains exemplifies the large variability within the species M. pachydermatis, which involves rare atypical strains with particular growth requirements.

Recent horizontal transfer of mellifera subfamily mariner transposons into insect lineages representing four different orders shows that selection acts only during horizontal transfer.

PubMed

Lampe, David J; Witherspoon, David J; Soto-Adames, Felipe N; Robertson, Hugh M

2003-04-01

We report the isolation and sequencing of genomic copies of mariner transposons involved in recent horizontal transfers into the genomes of the European earwig, Forficula auricularia; the European honey bee, Apis mellifera; the Mediterranean fruit fly, Ceratitis capitata; and a blister beetle, Epicauta funebris, insects from four different orders. These elements are in the mellifera subfamily and are the second documented example of full-length mariner elements involved in this kind of phenomenon. We applied maximum likelihood methods to the coding sequences and determined that the copies in each genome were evolving neutrally, whereas reconstructed ancestral coding sequences appeared to be under selection, which strengthens our previous hypothesis that the primary selective constraint on mariner sequence evolution is the act of horizontal transfer between genomes.

Genomic analysis and temperature-dependent transcriptome profiles of the rhizosphere originating strain Pseudomonas aeruginosa M18

PubMed Central

2011-01-01

Background Our previously published reports have described an effective biocontrol agent named Pseudomonas sp. M18 as its 16S rDNA sequence and several regulator genes share homologous sequences with those of P. aeruginosa, but there are several unusual phenotypic features. This study aims to explore its strain specific genomic features and gene expression patterns at different temperatures. Results The complete M18 genome is composed of a single chromosome of 6,327,754 base pairs containing 5684 open reading frames. Seven genomic islands, including two novel prophages and five specific non-phage islands were identified besides the conserved P. aeruginosa core genome. Each prophage contains a putative chitinase coding gene, and the prophage II contains a capB gene encoding a putative cold stress protein. The non-phage genomic islands contain genes responsible for pyoluteorin biosynthesis, environmental substance degradation and type I and III restriction-modification systems. Compared with other P. aeruginosa strains, the fewest number (3) of insertion sequences and the most number (3) of clustered regularly interspaced short palindromic repeats in M18 genome may contribute to the relative genome stability. Although the M18 genome is most closely related to that of P. aeruginosa strain LESB58, the strain M18 is more susceptible to several antimicrobial agents and easier to be erased in a mouse acute lung infection model than the strain LESB58. The whole M18 transcriptomic analysis indicated that 10.6% of the expressed genes are temperature-dependent, with 22 genes up-regulated at 28°C in three non-phage genomic islands and one prophage but none at 37°C. Conclusions The P. aeruginosa strain M18 has evolved its specific genomic structures and temperature dependent expression patterns to meet the requirement of its fitness and competitiveness under selective pressures imposed on the strain in rhizosphere niche. PMID:21884571

Taxonomic structure and stability of the bacterial community in belgian sourdough ecosystems as assessed by culture and population fingerprinting.

PubMed

Scheirlinck, Ilse; Van der Meulen, Roel; Van Schoor, Ann; Vancanneyt, Marc; De Vuyst, Luc; Vandamme, Peter; Huys, Geert

2008-04-01

A total of 39 traditional sourdoughs were sampled at 11 bakeries located throughout Belgium which were visited twice with a 1-year interval. The taxonomic structure and stability of the bacterial communities occurring in these traditional sourdoughs were assessed using both culture-dependent and culture-independent methods. A total of 1,194 potential lactic acid bacterium (LAB) isolates were tentatively grouped and identified by repetitive element sequence-based PCR, followed by sequence-based identification using 16S rRNA and pheS genes from a selection of genotypically unique LAB isolates. In parallel, all samples were analyzed by denaturing gradient gel electrophoresis (DGGE) of V3-16S rRNA gene amplicons. In addition, extensive metabolite target analysis of more than 100 different compounds was performed. Both culturing and DGGE analysis showed that the species Lactobacillus sanfranciscensis, Lactobacillus paralimentarius, Lactobacillus plantarum, and Lactobacillus pontis dominated the LAB population of Belgian type I sourdoughs. In addition, DGGE band sequence analysis demonstrated the presence of Acetobacter sp. and a member of the Erwinia/Enterobacter/Pantoea group in some samples. Overall, the culture-dependent and culture-independent approaches each exhibited intrinsic limitations in assessing bacterial LAB diversity in Belgian sourdoughs. Irrespective of the LAB biodiversity, a large majority of the sugar and amino acid metabolites were detected in all sourdough samples. Principal component-based analysis of biodiversity and metabolic data revealed only little variation among the two samples of the sourdoughs produced at the same bakery. The rare cases of instability observed could generally be linked with variations in technological parameters or differences in detection capacity between culture-dependent and culture-independent approaches. Within a sampling interval of 1 year, this study reinforces previous observations that the bakery environment rather than the type or batch of flour largely determines the development of a stable LAB population in sourdoughs.

Taxonomic Structure and Stability of the Bacterial Community in Belgian Sourdough Ecosystems as Assessed by Culture and Population Fingerprinting▿ †

PubMed Central

Scheirlinck, Ilse; Van der Meulen, Roel; Van Schoor, Ann; Vancanneyt, Marc; De Vuyst, Luc; Vandamme, Peter; Huys, Geert

2008-01-01

A total of 39 traditional sourdoughs were sampled at 11 bakeries located throughout Belgium which were visited twice with a 1-year interval. The taxonomic structure and stability of the bacterial communities occurring in these traditional sourdoughs were assessed using both culture-dependent and culture-independent methods. A total of 1,194 potential lactic acid bacterium (LAB) isolates were tentatively grouped and identified by repetitive element sequence-based PCR, followed by sequence-based identification using 16S rRNA and pheS genes from a selection of genotypically unique LAB isolates. In parallel, all samples were analyzed by denaturing gradient gel electrophoresis (DGGE) of V3-16S rRNA gene amplicons. In addition, extensive metabolite target analysis of more than 100 different compounds was performed. Both culturing and DGGE analysis showed that the species Lactobacillus sanfranciscensis, Lactobacillus paralimentarius, Lactobacillus plantarum, and Lactobacillus pontis dominated the LAB population of Belgian type I sourdoughs. In addition, DGGE band sequence analysis demonstrated the presence of Acetobacter sp. and a member of the Erwinia/Enterobacter/Pantoea group in some samples. Overall, the culture-dependent and culture-independent approaches each exhibited intrinsic limitations in assessing bacterial LAB diversity in Belgian sourdoughs. Irrespective of the LAB biodiversity, a large majority of the sugar and amino acid metabolites were detected in all sourdough samples. Principal component-based analysis of biodiversity and metabolic data revealed only little variation among the two samples of the sourdoughs produced at the same bakery. The rare cases of instability observed could generally be linked with variations in technological parameters or differences in detection capacity between culture-dependent and culture-independent approaches. Within a sampling interval of 1 year, this study reinforces previous observations that the bakery environment rather than the type or batch of flour largely determines the development of a stable LAB population in sourdoughs. PMID:18310426

Searching for evidence of selection in avian DNA barcodes.

PubMed

Kerr, Kevin C R

2011-11-01

The barcode of life project has assembled a tremendous number of mitochondrial cytochrome c oxidase I (COI) sequences. Although these sequences were gathered to develop a DNA-based system for species identification, it has been suggested that further biological inferences may also be derived from this wealth of data. Recurrent selective sweeps have been invoked as an evolutionary mechanism to explain limited intraspecific COI diversity, particularly in birds, but this hypothesis has not been formally tested. In this study, I collated COI sequences from previous barcoding studies on birds and tested them for evidence of selection. Using this expanded data set, I re-examined the relationships between intraspecific diversity and interspecific divergence and sampling effort, respectively. I employed the McDonald-Kreitman test to test for neutrality in sequence evolution between closely related pairs of species. Because amino acid sequences were generally constrained between closely related pairs, I also included broader intra-order comparisons to quantify patterns of protein variation in avian COI sequences. Lastly, using 22 published whole mitochondrial genomes, I compared the evolutionary rate of COI against the other 12 protein-coding mitochondrial genes to assess intragenomic variability. I found no conclusive evidence of selective sweeps. Most evidence pointed to an overall trend of strong purifying selection and functional constraint. The COI protein did vary across the class Aves, but to a very limited extent. COI was the least variable gene in the mitochondrial genome, suggesting that other genes might be more informative for probing factors constraining mitochondrial variation within species. © 2011 Blackwell Publishing Ltd.

Different evolutionary patterns of SNPs between domains and unassigned regions in human protein-coding sequences.

PubMed

Pang, Erli; Wu, Xiaomei; Lin, Kui

2016-06-01

Protein evolution plays an important role in the evolution of each genome. Because of their functional nature, in general, most of their parts or sites are differently constrained selectively, particularly by purifying selection. Most previous studies on protein evolution considered individual proteins in their entirety or compared protein-coding sequences with non-coding sequences. Less attention has been paid to the evolution of different parts within each protein of a given genome. To this end, based on PfamA annotation of all human proteins, each protein sequence can be split into two parts: domains or unassigned regions. Using this rationale, single nucleotide polymorphisms (SNPs) in protein-coding sequences from the 1000 Genomes Project were mapped according to two classifications: SNPs occurring within protein domains and those within unassigned regions. With these classifications, we found: the density of synonymous SNPs within domains is significantly greater than that of synonymous SNPs within unassigned regions; however, the density of non-synonymous SNPs shows the opposite pattern. We also found there are signatures of purifying selection on both the domain and unassigned regions. Furthermore, the selective strength on domains is significantly greater than that on unassigned regions. In addition, among all of the human protein sequences, there are 117 PfamA domains in which no SNPs are found. Our results highlight an important aspect of protein domains and may contribute to our understanding of protein evolution.

Continuous- and discrete-time stimulus sequences for high stimulus rate paradigm in evoked potential studies.

PubMed

Wang, Tao; Huang, Jiang-hua; Lin, Lin; Zhan, Chang'an A

2013-01-01

To obtain reliable transient auditory evoked potentials (AEPs) from EEGs recorded using high stimulus rate (HSR) paradigm, it is critical to design the stimulus sequences of appropriate frequency properties. Traditionally, the individual stimulus events in a stimulus sequence occur only at discrete time points dependent on the sampling frequency of the recording system and the duration of stimulus sequence. This dependency likely causes the implementation of suboptimal stimulus sequences, sacrificing the reliability of resulting AEPs. In this paper, we explicate the use of continuous-time stimulus sequence for HSR paradigm, which is independent of the discrete electroencephalogram (EEG) recording system. We employ simulation studies to examine the applicability of the continuous-time stimulus sequences and the impacts of sampling frequency on AEPs in traditional studies using discrete-time design. Results from these studies show that the continuous-time sequences can offer better frequency properties and improve the reliability of recovered AEPs. Furthermore, we find that the errors in the recovered AEPs depend critically on the sampling frequencies of experimental systems, and their relationship can be fitted using a reciprocal function. As such, our study contributes to the literature by demonstrating the applicability and advantages of continuous-time stimulus sequences for HSR paradigm and by revealing the relationship between the reliability of AEPs and sampling frequencies of the experimental systems when discrete-time stimulus sequences are used in traditional manner for the HSR paradigm.

(abstract) Synthesis of Speaker Facial Movements to Match Selected Speech Sequences

NASA Technical Reports Server (NTRS)

Scott, Kenneth C.

1994-01-01

We are developing a system for synthesizing image sequences the simulate the facial motion of a speaker. To perform this synthesis, we are pursuing two major areas of effort. We are developing the necessary computer graphics technology to synthesize a realistic image sequence of a person speaking selected speech sequences. Next, we are developing a model that expresses the relation between spoken phonemes and face/mouth shape. A subject is video taped speaking an arbitrary text that contains expression of the full list of desired database phonemes. The subject is video taped from the front speaking normally, recording both audio and video detail simultaneously. Using the audio track, we identify the specific video frames on the tape relating to each spoken phoneme. From this range we digitize the video frame which represents the extreme of mouth motion/shape. Thus, we construct a database of images of face/mouth shape related to spoken phonemes. A selected audio speech sequence is recorded which is the basis for synthesizing a matching video sequence; the speaker need not be the same as used for constructing the database. The audio sequence is analyzed to determine the spoken phoneme sequence and the relative timing of the enunciation of those phonemes. Synthesizing an image sequence corresponding to the spoken phoneme sequence is accomplished using a graphics technique known as morphing. Image sequence keyframes necessary for this processing are based on the spoken phoneme sequence and timing. We have been successful in synthesizing the facial motion of a native English speaker for a small set of arbitrary speech segments. Our future work will focus on advancement of the face shape/phoneme model and independent control of facial features.

Error Analysis of Deep Sequencing of Phage Libraries: Peptides Censored in Sequencing

PubMed Central

Matochko, Wadim L.; Derda, Ratmir

2013-01-01

Next-generation sequencing techniques empower selection of ligands from phage-display libraries because they can detect low abundant clones and quantify changes in the copy numbers of clones without excessive selection rounds. Identification of errors in deep sequencing data is the most critical step in this process because these techniques have error rates >1%. Mechanisms that yield errors in Illumina and other techniques have been proposed, but no reports to date describe error analysis in phage libraries. Our paper focuses on error analysis of 7-mer peptide libraries sequenced by Illumina method. Low theoretical complexity of this phage library, as compared to complexity of long genetic reads and genomes, allowed us to describe this library using convenient linear vector and operator framework. We describe a phage library as N × 1 frequency vector n = ||ni||, where ni is the copy number of the ith sequence and N is the theoretical diversity, that is, the total number of all possible sequences. Any manipulation to the library is an operator acting on n. Selection, amplification, or sequencing could be described as a product of a N × N matrix and a stochastic sampling operator (S a). The latter is a random diagonal matrix that describes sampling of a library. In this paper, we focus on the properties of S a and use them to define the sequencing operator (S e q). Sequencing without any bias and errors is S e q = S a IN, where IN is a N × N unity matrix. Any bias in sequencing changes IN to a nonunity matrix. We identified a diagonal censorship matrix (C E N), which describes elimination or statistically significant downsampling, of specific reads during the sequencing process. PMID:24416071

Highly Stable Double-Stranded DNA Containing Sequential Silver(I)-Mediated 7-Deazaadenine/Thymine Watson-Crick Base Pairs.

PubMed

Santamaría-Díaz, Noelia; Méndez-Arriaga, José M; Salas, Juan M; Galindo, Miguel A

2016-05-17

The oligonucleotide d(TX)9 , which consists of an octadecamer sequence with alternating non-canonical 7-deazaadenine (X) and canonical thymine (T) as the nucleobases, was synthesized and shown to hybridize into double-stranded DNA through the formation of hydrogen-bonded Watson-Crick base pairs. dsDNA with metal-mediated base pairs was then obtained by selectively replacing W-C hydrogen bonds by coordination bonds to central silver(I) ions. The oligonucleotide I adopts a duplex structure in the absence of Ag(+) ions, and its stability is significantly enhanced in the presence of Ag(+) ions while its double-helix structure is retained. Temperature-dependent UV spectroscopy, circular dichroism spectroscopy, and ESI mass spectrometry were used to confirm the selective formation of the silver(I)-mediated base pairs. This strategy could become useful for preparing stable metallo-DNA-based nanostructures. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Belov, Mikhail E.; Anderson, Gordon A.; Smith, Richard D.

Data-dependent selective external ion ejection with improved resolution is demonstrated with a 3.5 tesla FTICR instrument employing DREAMS (Dynamic Range Enhancement Applied to Mass Spectrometry) technology. To correct for the fringing rf-field aberrations each rod of the selection quadrupole has been segmented into three sections, so that ion excitation and ejection was performed by applying auxiliary rf-only waveforms in the region of the middle segments. Two different modes of external ion trapping and ejection were studied with the mixtures of model peptides and a tryptic digest of bovine serum albumin. A mass resolution of about 100 has been attained formore » rf-only dipolar ejection in a quadrupole operating at a Mathieu parameter q of{approx} 0.45. LC-ESI-DREAMS-FTICR analysis of a 0.1 mg/mL solution of bovine serum albumin digest resulted in detection of 82 unique tryptic peptides with mass measurement errors lower than 5 ppm, providing 100% sequence coverage of the protein.« less

DOE Office of Scientific and Technical Information (OSTI.GOV)

Belov, Mikhail E.; Anderson, Gordon A.; Smith, Richard D.

Data-dependent selective external ion ejection with improved resolution is demonstrated with a 3.5 tesla FTICR instrument employing DREAMS (Dynamic Range Enhancement Applied to Mass Spectrometry) technology. To correct for the fringing rf-field aberrations each rod of the selection quadrupole has been segmented into three sections, so that ion excitation and ejection was performed by applying auxiliary rf-only waveforms in the region of the middle segments. Two different modes of external ion trapping and ejection were studied with the mixtures of model peptides and a tryptic digest of bovine serum albumin. A mass resolution of about 100 had been attained formore » rf-only dipolar ejection in a quadrupole operating at a Mathieu parameter q of ~0.45. LC-ESI-DREAMS-FTICR analysis of a 0.1 mg/mL solution of bovine serum albumin digest resulted in detection of 82 unique tryptic peptides with mass measurement errors lower than 5 ppm, providing 100 % sequence coverage of the protein.« less

Homing endonuclease genes: the rise and fall and rise again of a selfish element.

PubMed

Burt, Austin; Koufopanou, Vassiliki

2004-12-01

Homing endonuclease genes (HEGs) are selfish genetic elements that spread by first cleaving chromosomes that do not contain them and then getting copied across to the broken chromosome as a byproduct of the repair process. The success of this strategy will depend on the opportunities for homing--in other words, the frequency with which HEG(+) and HEG(-) chromosomes come into contact--which varies widely among host taxa. HEGs are also unusual in that the selection pressure for endonuclease function disappears if they become fixed in a population, which makes them susceptible to degeneration and imposes a need for regular horizontal transmission between species. HEGs will be selected to reduce the harm done to the host organism, and this is expected to influence the evolution of their sequence specificity and maturase functions. HEGs may also be domesticated by their hosts, and are currently being put to human uses.

Optimal experimental designs for fMRI when the model matrix is uncertain.

PubMed

Kao, Ming-Hung; Zhou, Lin

2017-07-15

This study concerns optimal designs for functional magnetic resonance imaging (fMRI) experiments when the model matrix of the statistical model depends on both the selected stimulus sequence (fMRI design), and the subject's uncertain feedback (e.g. answer) to each mental stimulus (e.g. question) presented to her/him. While practically important, this design issue is challenging. This mainly is because that the information matrix cannot be fully determined at the design stage, making it difficult to evaluate the quality of the selected designs. To tackle this challenging issue, we propose an easy-to-use optimality criterion for evaluating the quality of designs, and an efficient approach for obtaining designs optimizing this criterion. Compared with a previously proposed method, our approach requires a much less computing time to achieve designs with high statistical efficiencies. Copyright © 2017 Elsevier Inc. All rights reserved.

Attention capture without awareness in a non-spatial selection task.

PubMed

Oriet, Chris; Pandey, Mamata; Kawahara, Jun-Ichiro

2017-02-01

Distractors presented prior to a critical target in a rapid sequence of visually-presented items induce a lag-dependent deficit in target identification, particularly when the distractor shares a task-relevant feature of the target. Presumably, such capture of central attention is important for bringing a target into awareness. The results of the present investigation suggest that greater capture of attention by a distractor is not accompanied by greater awareness of it. Moreover, awareness tends to be limited to superficial characteristics of the target such as colour. The findings are interpreted within the context of a model that assumes sudden increases in arousal trigger selection of information for consolidation in working memory. In this conceptualization, prolonged analysis of distractor items sharing task-relevant features leads to larger target identification deficits (i.e., greater capture) but no increase in awareness. Copyright © 2016 Elsevier Inc. All rights reserved.

Sexual Selection of Protamine 1 in Mammals.

PubMed

Lüke, Lena; Tourmente, Maximiliano; Roldan, Eduardo R S

2016-01-01

Protamines have a crucial role in male fertility. They are involved in sperm chromatin packaging and influence the shape of the sperm head and, hence, are important for sperm performance. Protamine structure is basic with numerous arginine-rich DNA-binding domains. Postcopulatory sexual selection is thought to play an important role in protamine sequence evolution and expression. Here, we analyze patterns of evolution and sexual selection (in the form of sperm competition) acting on protamine 1 gene sequence in 237 mammalian species. We assessed common patterns as well as differences between the major mammalian subclasses (Eutheria, Metatheria) and clades. We found that a high arginine content in protamine 1 associates with a lower sperm head width, which may have an impact on sperm swimming velocity. Increase in arginine content in protamine 1 across mammals appears to take place in a way consistent with sexual selection. In metatherians, increase in sequence length correlates with sexual selection. Differences in selective pressures on sequences and codon sites were observed between mammalian clades. Our study revealed a complex evolutionary pattern of protamine 1, with different selective constraints, and effects of sexual selection, between mammalian groups. In contrast, the effect of arginine content on head shape, and the possible involvement of sperm competition, was identified across all mammals. © The Author 2015. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

ARYANA: Aligning Reads by Yet Another Approach

PubMed Central

2014-01-01

Motivation Although there are many different algorithms and software tools for aligning sequencing reads, fast gapped sequence search is far from solved. Strong interest in fast alignment is best reflected in the $106 prize for the Innocentive competition on aligning a collection of reads to a given database of reference genomes. In addition, de novo assembly of next-generation sequencing long reads requires fast overlap-layout-concensus algorithms which depend on fast and accurate alignment. Contribution We introduce ARYANA, a fast gapped read aligner, developed on the base of BWA indexing infrastructure with a completely new alignment engine that makes it significantly faster than three other aligners: Bowtie2, BWA and SeqAlto, with comparable generality and accuracy. Instead of the time-consuming backtracking procedures for handling mismatches, ARYANA comes with the seed-and-extend algorithmic framework and a significantly improved efficiency by integrating novel algorithmic techniques including dynamic seed selection, bidirectional seed extension, reset-free hash tables, and gap-filling dynamic programming. As the read length increases ARYANA's superiority in terms of speed and alignment rate becomes more evident. This is in perfect harmony with the read length trend as the sequencing technologies evolve. The algorithmic platform of ARYANA makes it easy to develop mission-specific aligners for other applications using ARYANA engine. Availability ARYANA with complete source code can be obtained from http://github.com/aryana-aligner PMID:25252881

CloVR-Comparative: automated, cloud-enabled comparative microbial genome sequence analysis pipeline.

PubMed

Agrawal, Sonia; Arze, Cesar; Adkins, Ricky S; Crabtree, Jonathan; Riley, David; Vangala, Mahesh; Galens, Kevin; Fraser, Claire M; Tettelin, Hervé; White, Owen; Angiuoli, Samuel V; Mahurkar, Anup; Fricke, W Florian

2017-04-27

The benefit of increasing genomic sequence data to the scientific community depends on easy-to-use, scalable bioinformatics support. CloVR-Comparative combines commonly used bioinformatics tools into an intuitive, automated, and cloud-enabled analysis pipeline for comparative microbial genomics. CloVR-Comparative runs on annotated complete or draft genome sequences that are uploaded by the user or selected via a taxonomic tree-based user interface and downloaded from NCBI. CloVR-Comparative runs reference-free multiple whole-genome alignments to determine unique, shared and core coding sequences (CDSs) and single nucleotide polymorphisms (SNPs). Output includes short summary reports and detailed text-based results files, graphical visualizations (phylogenetic trees, circular figures), and a database file linked to the Sybil comparative genome browser. Data up- and download, pipeline configuration and monitoring, and access to Sybil are managed through CloVR-Comparative web interface. CloVR-Comparative and Sybil are distributed as part of the CloVR virtual appliance, which runs on local computers or the Amazon EC2 cloud. Representative datasets (e.g. 40 draft and complete Escherichia coli genomes) are processed in <36 h on a local desktop or at a cost of <$20 on EC2. CloVR-Comparative allows anybody with Internet access to run comparative genomics projects, while eliminating the need for on-site computational resources and expertise.

MRI image plane nonuniformity in evaluation of ferrous sulphate dosimeter gel (FeGel) by means of T1-relaxation time.

PubMed

Magnusson, P; Bäck, S A; Olsson, L E

1999-11-01

MR image nonuniformity can vary significantly with the spin-echo pulse sequence repetition time. When MR images with different nonuniformity shapes are used in a T1-calculation the resulting T1-image becomes nonuniform. As shown in this work the uniformity TR-dependence of the spin-echo pulse sequence is a critical property for T1 measurements in general and for ferrous sulfate dosimeter gel (FeGel) applications in particular. The purpose was to study the characteristics of the MR image plane nonuniformity in FeGel evaluation. This included studies of the possibility of decreasing nonuniformities by selecting uniformity optimized repetition times, studies of the transmitted and received RF-fields and studies of the effectiveness of the correction methods background subtraction and quotient correction. A pronounced MR image nonuniformity variation with repetition and T1 relaxation time was observed, and was found to originate from nonuniform RF-transmission in combination with the inherent differences in T1 relaxation for different repetition times. The T1 calculation itself, the uniformity optimized repetition times, nor none of the correction methods studied could sufficiently correct the nonuniformities observed in the T1 images. The nonuniformities were found to vary considerably less with inversion time for the inversion-recovery pulse sequence, than with repetition time for the spin-echo pulse sequence, resulting in considerably lower T1 image nonuniformity levels.

Salt bridges: geometrically specific, designable interactions.

PubMed

Donald, Jason E; Kulp, Daniel W; DeGrado, William F

2011-03-01

Salt bridges occur frequently in proteins, providing conformational specificity and contributing to molecular recognition and catalysis. We present a comprehensive analysis of these interactions in protein structures by surveying a large database of protein structures. Salt bridges between Asp or Glu and His, Arg, or Lys display extremely well-defined geometric preferences. Several previously observed preferences are confirmed, and others that were previously unrecognized are discovered. Salt bridges are explored for their preferences for different separations in sequence and in space, geometric preferences within proteins and at protein-protein interfaces, co-operativity in networked salt bridges, inclusion within metal-binding sites, preference for acidic electrons, apparent conformational side chain entropy reduction on formation, and degree of burial. Salt bridges occur far more frequently between residues at close than distant sequence separations, but, at close distances, there remain strong preferences for salt bridges at specific separations. Specific types of complex salt bridges, involving three or more members, are also discovered. As we observe a strong relationship between the propensity to form a salt bridge and the placement of salt-bridging residues in protein sequences, we discuss the role that salt bridges might play in kinetically influencing protein folding and thermodynamically stabilizing the native conformation. We also develop a quantitative method to select appropriate crystal structure resolution and B-factor cutoffs. Detailed knowledge of these geometric and sequence dependences should aid de novo design and prediction algorithms. Copyright © 2010 Wiley-Liss, Inc.

A flavivirus protein M-derived peptide directly permeabilizes mitochondrial membranes, triggers cell death and reduces human tumor growth in nude mice.

PubMed

Brabant, Magali; Baux, Ludwig; Casimir, Richard; Briand, Jean Paul; Chaloin, Olivier; Porceddu, Mathieu; Buron, Nelly; Chauvier, David; Lassalle, Myriam; Lecoeur, Hervé; Langonné, Alain; Dupont, Sylvie; Déas, Olivier; Brenner, Catherine; Rebouillat, Dominique; Muller, Sylviane; Borgne-Sanchez, Annie; Jacotot, Etienne

2009-10-01

Dengue viruses belong to the Flavivirus family and are responsible for hemorrhagic fever in Human. Dengue virus infection triggers apoptosis especially through the expression of the small membrane (M) protein. Using isolated mitochondria, we found that synthetic peptides containing the C-terminus part of the M ectodomain caused apoptosis-related mitochondrial membrane permeabilization (MMP) events. These events include matrix swelling and the dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)). Protein M Flavivirus sequence alignments and helical wheel projections reveal a conserved distribution of charged residues. Moreover, when combined to the cell penetrating HIV-1 Tat peptide transduction domain (Tat-PTD), this sequence triggers a caspase-dependent cell death associated with DeltaPsi(m) loss and cytochrome c release. Mutational approaches coupled to functional screening on isolated mitochondria resulted in the selection of a protein M derived sequence containing nine residues with potent MMP-inducing properties on isolated mitochondria. A chimeric peptide composed of a Tat-PTD linked to the 9-mer entity triggers MMP and cell death. Finally, local administration of this chimeric peptide induces growth inhibition of xenograft prostate PC3 tumors in immuno-compromised mice, and significantly enhances animal survival. Together, these findings support the notion of using viral genomes as valuable sources to discover mitochondria-targeted sequences that may lead to the development of new anticancer compounds.

ARYANA: Aligning Reads by Yet Another Approach.

PubMed

Gholami, Milad; Arbabi, Aryan; Sharifi-Zarchi, Ali; Chitsaz, Hamidreza; Sadeghi, Mehdi

2014-01-01

Although there are many different algorithms and software tools for aligning sequencing reads, fast gapped sequence search is far from solved. Strong interest in fast alignment is best reflected in the $10(6) prize for the Innocentive competition on aligning a collection of reads to a given database of reference genomes. In addition, de novo assembly of next-generation sequencing long reads requires fast overlap-layout-concensus algorithms which depend on fast and accurate alignment. We introduce ARYANA, a fast gapped read aligner, developed on the base of BWA indexing infrastructure with a completely new alignment engine that makes it significantly faster than three other aligners: Bowtie2, BWA and SeqAlto, with comparable generality and accuracy. Instead of the time-consuming backtracking procedures for handling mismatches, ARYANA comes with the seed-and-extend algorithmic framework and a significantly improved efficiency by integrating novel algorithmic techniques including dynamic seed selection, bidirectional seed extension, reset-free hash tables, and gap-filling dynamic programming. As the read length increases ARYANA's superiority in terms of speed and alignment rate becomes more evident. This is in perfect harmony with the read length trend as the sequencing technologies evolve. The algorithmic platform of ARYANA makes it easy to develop mission-specific aligners for other applications using ARYANA engine. ARYANA with complete source code can be obtained from http://github.com/aryana-aligner.

Metagenomic survey of bacterial diversity in the atmosphere of Mexico City using different sampling methods.

PubMed

Serrano-Silva, N; Calderón-Ezquerro, M C

2018-04-01

The identification of airborne bacteria has traditionally been performed by retrieval in culture media, but the bacterial diversity in the air is underestimated using this method because many bacteria are not readily cultured. Advances in DNA sequencing technology have produced a broad knowledge of genomics and metagenomics, which can greatly improve our ability to identify and study the diversity of airborne bacteria. However, researchers are facing several challenges, particularly the efficient retrieval of low-density microorganisms from the air and the lack of standardized protocols for sample collection and processing. In this study, we tested three methods for sampling bioaerosols - a Durham-type spore trap (Durham), a seven-day recording volumetric spore trap (HST), and a high-throughput 'Jet' spore and particle sampler (Jet) - and recovered metagenomic DNA for 16S rDNA sequencing. Samples were simultaneously collected with the three devices during one week, and the sequencing libraries were analyzed. A simple and efficient method for collecting bioaerosols and extracting good quality DNA for high-throughput sequencing was standardized. The Durham sampler collected preferentially Cyanobacteria, the HST Actinobacteria, Proteobacteria and Firmicutes, and the Jet mainly Proteobacteria and Firmicutes. The HST sampler collected the largest amount of airborne bacterial diversity. More experiments are necessary to select the right sampler, depending on study objectives, which may require monitoring and collecting specific airborne bacteria. Copyright © 2017 Elsevier Ltd. All rights reserved.

AlexSys: a knowledge-based expert system for multiple sequence alignment construction and analysis

PubMed Central

Aniba, Mohamed Radhouene; Poch, Olivier; Marchler-Bauer, Aron; Thompson, Julie Dawn

2010-01-01

Multiple sequence alignment (MSA) is a cornerstone of modern molecular biology and represents a unique means of investigating the patterns of conservation and diversity in complex biological systems. Many different algorithms have been developed to construct MSAs, but previous studies have shown that no single aligner consistently outperforms the rest. This has led to the development of a number of ‘meta-methods’ that systematically run several aligners and merge the output into one single solution. Although these methods generally produce more accurate alignments, they are inefficient because all the aligners need to be run first and the choice of the best solution is made a posteriori. Here, we describe the development of a new expert system, AlexSys, for the multiple alignment of protein sequences. AlexSys incorporates an intelligent inference engine to automatically select an appropriate aligner a priori, depending only on the nature of the input sequences. The inference engine was trained on a large set of reference multiple alignments, using a novel machine learning approach. Applying AlexSys to a test set of 178 alignments, we show that the expert system represents a good compromise between alignment quality and running time, making it suitable for high throughput projects. AlexSys is freely available from http://alnitak.u-strasbg.fr/∼aniba/alexsys. PMID:20530533

Genomic resources and their influence on the detection of the signal of positive selection in genome scans.

PubMed

Manel, S; Perrier, C; Pratlong, M; Abi-Rached, L; Paganini, J; Pontarotti, P; Aurelle, D

2016-01-01

Genome scans represent powerful approaches to investigate the action of natural selection on the genetic variation of natural populations and to better understand local adaptation. This is very useful, for example, in the field of conservation biology and evolutionary biology. Thanks to Next Generation Sequencing, genomic resources are growing exponentially, improving genome scan analyses in non-model species. Thousands of SNPs called using Reduced Representation Sequencing are increasingly used in genome scans. Besides, genome sequences are also becoming increasingly available, allowing better processing of short-read data, offering physical localization of variants, and improving haplotype reconstruction and data imputation. Ultimately, genome sequences are also becoming the raw material for selection inferences. Here, we discuss how the increasing availability of such genomic resources, notably genome sequences, influences the detection of signals of selection. Mainly, increasing data density and having the information of physical linkage data expand genome scans by (i) improving the overall quality of the data, (ii) helping the reconstruction of demographic history for the population studied to decrease false-positive rates and (iii) improving the statistical power of methods to detect the signal of selection. Of particular importance, the availability of a high-quality reference genome can improve the detection of the signal of selection by (i) allowing matching the potential candidate loci to linked coding regions under selection, (ii) rapidly moving the investigation to the gene and function and (iii) ensuring that the highly variable regions of the genomes that include functional genes are also investigated. For all those reasons, using reference genomes in genome scan analyses is highly recommended. © 2015 John Wiley & Sons Ltd.

Book Catalogs; Selected References.

ERIC Educational Resources Information Center

Brandhorst, Wesley T.

The 116 citations on book catalogs are divided into the following two main sections: (1) Selected References, in alphabetic sequence by personal or institutional author and (2) Anonymous Entries, in alphabetic sequence by title. One hundred and seven of the citations cover the years 1960 through March 1969. There are five scattered citations in…

Selection of a platinum-binding sequence in a loop of a four-helix bundle protein.

PubMed

Yagi, Sota; Akanuma, Satoshi; Kaji, Asumi; Niiro, Hiroya; Akiyama, Hayato; Uchida, Tatsuya; Yamagishi, Akihiko

2018-02-01

Protein-metal hybrids are functional materials with various industrial applications. For example, a redox enzyme immobilized on a platinum electrode is a key component of some biofuel cells and biosensors. To create these hybrid materials, protein molecules are bound to metal surfaces. Here, we report the selection of a novel platinum-binding sequence in a loop of a four-helix bundle protein, the Lac repressor four-helix protein (LARFH), an artificial protein in which four identical α-helices are connected via three identical loops. We created a genetic library in which the Ser-Gly-Gln-Gly-Gly-Ser sequence within the first inter-helical loop of LARFH was semi-randomly mutated. The library was then subjected to selection for platinum-binding affinity by using the T7 phage display method. The majority of the selected variants contained the Tyr-Lys-Arg-Gly-Tyr-Lys (YKRGYK) sequence in their randomized segment. We characterized the platinum-binding properties of mutant LARFH by using quartz crystal microbalance analysis. Mutant LARFH seemed to interact with platinum through its loop containing the YKRGYK sequence, as judged by the estimated exclusive area occupied by a single molecule. Furthermore, a 10-residue peptide containing the YKRGYK sequence bound to platinum with reasonably high affinity and basic side chains in the peptide were crucial in mediating this interaction. In conclusion, we have identified an amino acid sequence, YKRGYK, in the loop of a helix-loop-helix motif that shows high platinum-binding affinity. This sequence could be grafted into loops of other polypeptides as an approach to immobilize proteins on platinum electrodes for use as biosensors among other applications. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Measuring the amplification of attention

PubMed Central

Blaser, Erik; Sperling, George; Lu, Zhong-Lin

1999-01-01

An ambiguous motion paradigm, in which the direction of apparent motion is determined by salience (i.e., the extent to which an area is perceived as figure versus ground), is used to assay the amplification of color by attention to color. In the red–green colored gratings used in these experiments, without attention instructions, salience depends on the chromaticity difference between colored stripes embedded in the motion sequence and the yellow background. Selective attention to red (or to green) alters the perceived direction of motion and is found to be equivalent to increasing the physical redness (or greenness) by 25–117%, depending on the observer and color. Whereas attention to a color drastically alters the salience of that color, it leaves color appearance unchanged. A computational model, which embodies separate, parallel pathways for object perception and for salience, accounts for 99% of the variance of the experimental data. PMID:10500237

Measuring the amplification of attention.

PubMed

Blaser, E; Sperling, G; Lu, Z L

1999-09-28

An ambiguous motion paradigm, in which the direction of apparent motion is determined by salience (i.e., the extent to which an area is perceived as figure versus ground), is used to assay the amplification of color by attention to color. In the red-green colored gratings used in these experiments, without attention instructions, salience depends on the chromaticity difference between colored stripes embedded in the motion sequence and the yellow background. Selective attention to red (or to green) alters the perceived direction of motion and is found to be equivalent to increasing the physical redness (or greenness) by 25-117%, depending on the observer and color. Whereas attention to a color drastically alters the salience of that color, it leaves color appearance unchanged. A computational model, which embodies separate, parallel pathways for object perception and for salience, accounts for 99% of the variance of the experimental data.

Selective chemical labeling reveals the genome-wide distribution of 5-hydroxymethylcytosine.

PubMed

Song, Chun-Xiao; Szulwach, Keith E; Fu, Ye; Dai, Qing; Yi, Chengqi; Li, Xuekun; Li, Yujing; Chen, Chih-Hsin; Zhang, Wen; Jian, Xing; Wang, Jing; Zhang, Li; Looney, Timothy J; Zhang, Baichen; Godley, Lucy A; Hicks, Leslie M; Lahn, Bruce T; Jin, Peng; He, Chuan

2011-01-01

In contrast to 5-methylcytosine (5-mC), which has been studied extensively, little is known about 5-hydroxymethylcytosine (5-hmC), a recently identified epigenetic modification present in substantial amounts in certain mammalian cell types. Here we present a method for determining the genome-wide distribution of 5-hmC. We use the T4 bacteriophage β-glucosyltransferase to transfer an engineered glucose moiety containing an azide group onto the hydroxyl group of 5-hmC. The azide group can be chemically modified with biotin for detection, affinity enrichment and sequencing of 5-hmC-containing DNA fragments in mammalian genomes. Using this method, we demonstrate that 5-hmC is present in human cell lines beyond those previously recognized. We also find a gene expression level-dependent enrichment of intragenic 5-hmC in mouse cerebellum and an age-dependent acquisition of this modification in specific gene bodies linked to neurodegenerative disorders.

Footprinting reveals that nogalamycin and actinomycin shuffle between DNA binding sites.

PubMed Central

Fox, K R; Waring, M J

1986-01-01

The hypothesis that sequence-selective DNA-binding antibiotics locate their preferred binding sites by a process involving migration from nonspecific sites has been tested by footprinting with DNAase I. Footprinting patterns on the tyrT DNA fragment produced by nogalamycin and actinomycin change with time after mixing the antibiotic with the DNA. Sites of protection as well as enhanced cleavage are seen to develop in a fashion which is both temperature and concentration-dependent. At certain sites cutting is transiently enhanced, then blocked. Limited evidence for slow reaction with echinomycin and mithramycin is presented, but the kinetics of footprinting with daunomycin and distamycin appear instantaneous. The feasibility of adducing direct evidence for shuffling by footprinting seems to be governed by slow dissociation of the antibiotic-DNA complex. It may also be dependent upon the mode of binding, be it intercalative or non-intercalative in character. Images PMID:2421246

Mechanisms of viral mutation.

PubMed

Sanjuán, Rafael; Domingo-Calap, Pilar

2016-12-01

The remarkable capacity of some viruses to adapt to new hosts and environments is highly dependent on their ability to generate de novo diversity in a short period of time. Rates of spontaneous mutation vary amply among viruses. RNA viruses mutate faster than DNA viruses, single-stranded viruses mutate faster than double-strand virus, and genome size appears to correlate negatively with mutation rate. Viral mutation rates are modulated at different levels, including polymerase fidelity, sequence context, template secondary structure, cellular microenvironment, replication mechanisms, proofreading, and access to post-replicative repair. Additionally, massive numbers of mutations can be introduced by some virus-encoded diversity-generating elements, as well as by host-encoded cytidine/adenine deaminases. Our current knowledge of viral mutation rates indicates that viral genetic diversity is determined by multiple virus- and host-dependent processes, and that viral mutation rates can evolve in response to specific selective pressures.

Oligo Design: a computer program for development of probes for oligonucleotide microarrays.

PubMed

Herold, Keith E; Rasooly, Avraham

2003-12-01

Oligonucleotide microarrays have demonstrated potential for the analysis of gene expression, genotyping, and mutational analysis. Our work focuses primarily on the detection and identification of bacteria based on known short sequences of DNA. Oligo Design, the software described here, automates several design aspects that enable the improved selection of oligonucleotides for use with microarrays for these applications. Two major features of the program are: (i) a tiling algorithm for the design of short overlapping temperature-matched oligonucleotides of variable length, which are useful for the analysis of single nucleotide polymorphisms and (ii) a set of tools for the analysis of multiple alignments of gene families and related short DNA sequences, which allow for the identification of conserved DNA sequences for PCR primer selection and variable DNA sequences for the selection of unique probes for identification. Note that the program does not address the full genome perspective but, instead, is focused on the genetic analysis of short segments of DNA. The program is Internet-enabled and includes a built-in browser and the automated ability to download sequences from GenBank by specifying the GI number. The program also includes several utilities, including audio recital of a DNA sequence (useful for verifying sequences against a written document), a random sequence generator that provides insight into the relationship between melting temperature and GC content, and a PCR calculator.

A novel de novo mutation in ATP1A3 and childhood-onset schizophrenia

PubMed Central

Smedemark-Margulies, Niklas; Brownstein, Catherine A.; Vargas, Sigella; Tembulkar, Sahil K.; Towne, Meghan C.; Shi, Jiahai; Gonzalez-Cuevas, Elisa; Liu, Kevin X.; Bilguvar, Kaya; Kleiman, Robin J.; Han, Min-Joon; Torres, Alcy; Berry, Gerard T.; Yu, Timothy W.; Beggs, Alan H.; Agrawal, Pankaj B.; Gonzalez-Heydrich, Joseph

2016-01-01

We describe a child with onset of command auditory hallucinations and behavioral regression at 6 yr of age in the context of longer standing selective mutism, aggression, and mild motor delays. His genetic evaluation included chromosomal microarray analysis and whole-exome sequencing. Sequencing revealed a previously unreported heterozygous de novo mutation c.385G>A in ATP1A3, predicted to result in a p.V129M amino acid change. This gene codes for a neuron-specific isoform of the catalytic α-subunit of the ATP-dependent transmembrane sodium–potassium pump. Heterozygous mutations in this gene have been reported as causing both sporadic and inherited forms of alternating hemiplegia of childhood and rapid-onset dystonia parkinsonism. We discuss the literature on phenotypes associated with known variants in ATP1A3, examine past functional studies of the role of ATP1A3 in neuronal function, and describe a novel clinical presentation associated with mutation of this gene. PMID:27626066

Subrandom methods for multidimensional nonuniform sampling.

PubMed

Worley, Bradley

2016-08-01

Methods of nonuniform sampling that utilize pseudorandom number sequences to select points from a weighted Nyquist grid are commonplace in biomolecular NMR studies, due to the beneficial incoherence introduced by pseudorandom sampling. However, these methods require the specification of a non-arbitrary seed number in order to initialize a pseudorandom number generator. Because the performance of pseudorandom sampling schedules can substantially vary based on seed number, this can complicate the task of routine data collection. Approaches such as jittered sampling and stochastic gap sampling are effective at reducing random seed dependence of nonuniform sampling schedules, but still require the specification of a seed number. This work formalizes the use of subrandom number sequences in nonuniform sampling as a means of seed-independent sampling, and compares the performance of three subrandom methods to their pseudorandom counterparts using commonly applied schedule performance metrics. Reconstruction results using experimental datasets are also provided to validate claims made using these performance metrics. Copyright © 2016 Elsevier Inc. All rights reserved.

Rational Design of a Transferrin-Binding Peptide Sequence Tailored to Targeted Nanoparticle Internalization.

PubMed

Santi, Melissa; Maccari, Giuseppe; Mereghetti, Paolo; Voliani, Valerio; Rocchiccioli, Silvia; Ucciferri, Nadia; Luin, Stefano; Signore, Giovanni

2017-02-15

The transferrin receptor (TfR) is a promising target in cancer therapy owing to its overexpression in most solid tumors and on the blood-brain barrier. Nanostructures chemically derivatized with transferrin are employed in TfR targeting but often lose their functionality upon injection in the bloodstream. As an alternative strategy, we rationally designed a peptide coating able to bind transferrin on suitable pockets not involved in binding to TfR or iron by using an iterative multiscale-modeling approach coupled with quantitative structure-activity and relationship (QSAR) analysis and evolutionary algorithms. We tested that selected sequences have low aspecific protein adsorption and high binding energy toward transferrin, and one of them is efficiently internalized in cells with a transferrin-dependent pathway. Furthermore, it promotes transferrin-mediated endocytosis of gold nanoparticles by modifying their protein corona and promoting oriented adsorption of transferrin. This strategy leads to highly effective nanostructures, potentially useful in diagnostic and therapeutic applications, which exploit (and do not suffer) the protein solvation for achieving a better targeting.

Self-assembling peptide nanofiber hydrogels in tissue engineering and regenerative medicine: Progress, design guidelines, and applications.

PubMed

Koutsopoulos, Sotirios

2016-04-01

Until the mid-1980s, mainly biologists were conducting peptide research. This changed with discoveries that opened new paths of research involving the use of peptides in bioengineering, biotechnology, biomedicine, nanotechnology, and bioelectronics. Peptide engineering and rational design of novel peptide sequences with unique and tailor-made properties further expanded the field. The discovery of short self-assembling peptides, which upon association form well-defined supramolecular architectures, created new and exciting areas of research. Depending on the amino acid sequence, the pH, and the type of the electrolyte in the medium, peptide self-assembly leads to the formation of nanofibers, which are further organized to form a hydrogel. In this review, the application of ionic complementary peptides which self-assemble to form nanofiber hydrogels for tissue engineering and regenerative medicine will be discussed through a selective presentation of the most important work performed during the last 25 years. © 2016 Wiley Periodicals, Inc.

Online tracking of outdoor lighting variations for augmented reality with moving cameras.

PubMed

Liu, Yanli; Granier, Xavier

2012-04-01

In augmented reality, one of key tasks to achieve a convincing visual appearance consistency between virtual objects and video scenes is to have a coherent illumination along the whole sequence. As outdoor illumination is largely dependent on the weather, the lighting condition may change from frame to frame. In this paper, we propose a full image-based approach for online tracking of outdoor illumination variations from videos captured with moving cameras. Our key idea is to estimate the relative intensities of sunlight and skylight via a sparse set of planar feature-points extracted from each frame. To address the inevitable feature misalignments, a set of constraints are introduced to select the most reliable ones. Exploiting the spatial and temporal coherence of illumination, the relative intensities of sunlight and skylight are finally estimated by using an optimization process. We validate our technique on a set of real-life videos and show that the results with our estimations are visually coherent along the video sequences.

Convergent evolution and mimicry of protein linear motifs in host-pathogen interactions.

PubMed

Chemes, Lucía Beatriz; de Prat-Gay, Gonzalo; Sánchez, Ignacio Enrique

2015-06-01

Pathogen linear motif mimics are highly evolvable elements that facilitate rewiring of host protein interaction networks. Host linear motifs and pathogen mimics differ in sequence, leading to thermodynamic and structural differences in the resulting protein-protein interactions. Moreover, the functional output of a mimic depends on the motif and domain repertoire of the pathogen protein. Regulatory evolution mediated by linear motifs can be understood by measuring evolutionary rates, quantifying positive and negative selection and performing phylogenetic reconstructions of linear motif natural history. Convergent evolution of linear motif mimics is widespread among unrelated proteins from viral, prokaryotic and eukaryotic pathogens and can also take place within individual protein phylogenies. Statistics, biochemistry and laboratory models of infection link pathogen linear motifs to phenotypic traits such as tropism, virulence and oncogenicity. In vitro evolution experiments and analysis of natural sequences suggest that changes in linear motif composition underlie pathogen adaptation to a changing environment. Copyright © 2015 Elsevier Ltd. All rights reserved.

Single Machine Scheduling and Due Date Assignment with Past-Sequence-Dependent Setup Time and Position-Dependent Processing Time

PubMed Central

Zhao, Chuan-Li; Hsu, Hua-Feng

2014-01-01

This paper considers single machine scheduling and due date assignment with setup time. The setup time is proportional to the length of the already processed jobs; that is, the setup time is past-sequence-dependent (p-s-d). It is assumed that a job's processing time depends on its position in a sequence. The objective functions include total earliness, the weighted number of tardy jobs, and the cost of due date assignment. We analyze these problems with two different due date assignment methods. We first consider the model with job-dependent position effects. For each case, by converting the problem to a series of assignment problems, we proved that the problems can be solved in O(n 4) time. For the model with job-independent position effects, we proved that the problems can be solved in O(n 3) time by providing a dynamic programming algorithm. PMID:25258727

Single machine scheduling and due date assignment with past-sequence-dependent setup time and position-dependent processing time.

PubMed

Zhao, Chuan-Li; Hsu, Chou-Jung; Hsu, Hua-Feng

2014-01-01

This paper considers single machine scheduling and due date assignment with setup time. The setup time is proportional to the length of the already processed jobs; that is, the setup time is past-sequence-dependent (p-s-d). It is assumed that a job's processing time depends on its position in a sequence. The objective functions include total earliness, the weighted number of tardy jobs, and the cost of due date assignment. We analyze these problems with two different due date assignment methods. We first consider the model with job-dependent position effects. For each case, by converting the problem to a series of assignment problems, we proved that the problems can be solved in O(n(4)) time. For the model with job-independent position effects, we proved that the problems can be solved in O(n(3)) time by providing a dynamic programming algorithm.

Understanding protein evolution: from protein physics to Darwinian selection.

PubMed

Zeldovich, Konstantin B; Shakhnovich, Eugene I

2008-01-01

Efforts in whole-genome sequencing and structural proteomics start to provide a global view of the protein universe, the set of existing protein structures and sequences. However, approaches based on the selection of individual sequences have not been entirely successful at the quantitative description of the distribution of structures and sequences in the protein universe because evolutionary pressure acts on the entire organism, rather than on a particular molecule. In parallel to this line of study, studies in population genetics and phenomenological molecular evolution established a mathematical framework to describe the changes in genome sequences in populations of organisms over time. Here, we review both microscopic (physics-based) and macroscopic (organism-level) models of protein-sequence evolution and demonstrate that bridging the two scales provides the most complete description of the protein universe starting from clearly defined, testable, and physiologically relevant assumptions.

GuiTope: an application for mapping random-sequence peptides to protein sequences.

PubMed

Halperin, Rebecca F; Stafford, Phillip; Emery, Jack S; Navalkar, Krupa Arun; Johnston, Stephen Albert

2012-01-03

Random-sequence peptide libraries are a commonly used tool to identify novel ligands for binding antibodies, other proteins, and small molecules. It is often of interest to compare the selected peptide sequences to the natural protein binding partners to infer the exact binding site or the importance of particular residues. The ability to search a set of sequences for similarity to a set of peptides may sometimes enable the prediction of an antibody epitope or a novel binding partner. We have developed a software application designed specifically for this task. GuiTope provides a graphical user interface for aligning peptide sequences to protein sequences. All alignment parameters are accessible to the user including the ability to specify the amino acid frequency in the peptide library; these frequencies often differ significantly from those assumed by popular alignment programs. It also includes a novel feature to align di-peptide inversions, which we have found improves the accuracy of antibody epitope prediction from peptide microarray data and shows utility in analyzing phage display datasets. Finally, GuiTope can randomly select peptides from a given library to estimate a null distribution of scores and calculate statistical significance. GuiTope provides a convenient method for comparing selected peptide sequences to protein sequences, including flexible alignment parameters, novel alignment features, ability to search a database, and statistical significance of results. The software is available as an executable (for PC) at http://www.immunosignature.com/software and ongoing updates and source code will be available at sourceforge.net.

Simultaneous improvement of grain yield and protein content in durum wheat by different phenotypic indices and genomic selection.

PubMed

Rapp, M; Lein, V; Lacoudre, F; Lafferty, J; Müller, E; Vida, G; Bozhanova, V; Ibraliu, A; Thorwarth, P; Piepho, H P; Leiser, W L; Würschum, T; Longin, C F H

2018-06-01

Simultaneous improvement of protein content and grain yield by index selection is possible but its efficiency largely depends on the weighting of the single traits. The genetic architecture of these indices is similar to that of the primary traits. Grain yield and protein content are of major importance in durum wheat breeding, but their negative correlation has hampered their simultaneous improvement. To account for this in wheat breeding, the grain protein deviation (GPD) and the protein yield were proposed as targets for selection. The aim of this work was to investigate the potential of different indices to simultaneously improve grain yield and protein content in durum wheat and to evaluate their genetic architecture towards genomics-assisted breeding. To this end, we investigated two different durum wheat panels comprising 159 and 189 genotypes, which were tested in multiple field locations across Europe and genotyped by a genotyping-by-sequencing approach. The phenotypic analyses revealed significant genetic variances for all traits and heritabilities of the phenotypic indices that were in a similar range as those of grain yield and protein content. The GPD showed a high and positive correlation with protein content, whereas protein yield was highly and positively correlated with grain yield. Thus, selecting for a high GPD would mainly increase the protein content whereas a selection based on protein yield would mainly improve grain yield, but a combination of both indices allows to balance this selection. The genome-wide association mapping revealed a complex genetic architecture for all traits with most QTL having small effects and being detected only in one germplasm set, thus limiting the potential of marker-assisted selection for trait improvement. By contrast, genome-wide prediction appeared promising but its performance strongly depends on the relatedness between training and prediction sets.

Exome Sequencing Analysis Reveals Variants in Primary Immunodeficiency Genes in Patients With Very Early Onset Inflammatory Bowel Disease

PubMed Central

Kelsen, Judith R.; Dawany, Noor; Moran, Christopher J.; Petersen, Britt-Sabina; Sarmady, Mahdi; Sasson, Ariella; Pauly-Hubbard, Helen; Martinez, Alejandro; Maurer, Kelly; Soong, Joanne; Rappaport, Eric; Franke, Andre; Keller, Andreas; Winter, Harland S.; Mamula, Petar; Piccoli, David; Artis, David; Sonnenberg, Gregory F.; Daly, Mark; Sullivan, Kathleen E.; Baldassano, Robert N.; Devoto, Marcella

2016-01-01

Background & Aims Very early onset inflammatory bowel disease (VEO-IBD), IBD diagnosed ≤5 y of age, frequently presents with a different and more severe phenotype than older-onset IBD. We investigated whether patients with VEO-IBD carry rare or novel variants in genes associated with immunodeficiencies that might contribute to disease development. Methods Patients with VEO-IBD and parents (when available) were recruited from the Children's Hospital of Philadelphia from March 2013 through July 2014. We analyzed DNA from 125 patients with VEO-IBD (ages 3 weeks to 4 y) and 19 parents, 4 of whom also had IBD. Exome capture was performed by Agilent SureSelect V4, and sequencing was performed using the Illumina HiSeq platform. Alignment to human genome GRCh37 was achieved followed by post-processing and variant calling. Following functional annotation, candidate variants were analyzed for change in protein function, minor allele frequency <0.1%, and scaled combined annotation dependent depletion scores ≤10. We focused on genes associated with primary immunodeficiencies and related pathways. An additional 210 exome samples from patients with pediatric IBD (n=45) or adult-onset Crohn's disease (n=20) and healthy individuals (controls, n=145) were obtained from the University of Kiel, Germany and used as control groups. Results Four-hundred genes and regions associated with primary immunodeficiency, covering approximately 6500 coding exons totaling > 1 Mbp of coding sequence, were selected from the whole exome data. Our analysis revealed novel and rare variants within these genes that could contribute to the development of VEO-IBD, including rare heterozygous missense variants in IL10RA and previously unidentified variants in MSH5 and CD19. Conclusions In an exome sequence analysis of patients with VEO-IBD and their parents, we identified variants in genes that regulate B- and T-cell functions and could contribute to pathogenesis. Our analysis could lead to the identification of previously unidentified IBD-associated variants. PMID:26193622

Adenosine-to-Inosine Editing of MicroRNA-487b Alters Target Gene Selection After Ischemia and Promotes Neovascularization.

PubMed

van der Kwast, Reginald V C T; van Ingen, Eva; Parma, Laura; Peters, Hendrika A B; Quax, Paul H A; Nossent, A Yaël

2018-02-02

Adenosine-to-inosine editing of microRNAs has the potential to cause a shift in target site selection. 2'-O-ribose-methylation of adenosine residues, however, has been shown to inhibit adenosine-to-inosine editing. To investigate whether angiomiR miR487b is subject to adenosine-to-inosine editing or 2'-O-ribose-methylation during neovascularization. Complementary DNA was prepared from C57BL/6-mice subjected to hindlimb ischemia. Using Sanger sequencing and endonuclease digestion, we identified and validated adenosine-to-inosine editing of the miR487b seed sequence. In the gastrocnemius muscle, pri-miR487b editing increased from 6.7±0.4% before to 11.7±1.6% ( P =0.02) 1 day after ischemia. Edited pri-miR487b is processed into a novel microRNA, edited miR487b, which is also upregulated after ischemia. We confirmed editing of miR487b in multiple human primary vascular cell types. Short interfering RNA-mediated knockdown demonstrated that editing is adenosine deaminase acting on RNA 1 and 2 dependent. Using reverse-transcription at low dNTP concentrations followed by quantitative-PCR, we found that the same adenosine residue is methylated in mice and human primary cells. In the murine gastrocnemius, the estimated methylation fraction increased from 32.8±14% before to 53.6±12% 1 day after ischemia. Short interfering RNA knockdown confirmed that methylation is fibrillarin dependent. Although we could not confirm that methylation directly inhibits editing, we do show that adenosine deaminase acting on RNA 1 and 2 and fibrillarin negatively influence each other's expression. Using multiple luciferase reporter gene assays, we could demonstrate that editing results in a complete switch of target site selection. In human primary cells, we confirmed the shift in miR487b targeting after editing, resulting in a edited miR487b targetome that is enriched for multiple proangiogenic pathways. Furthermore, overexpression of edited miR487b, but not wild-type miR487b, stimulates angiogenesis in both in vitro and ex vivo assays. MiR487b is edited in the seed sequence in mice and humans, resulting in a novel, proangiogenic microRNA with a unique targetome. The rate of miR487b editing, as well as 2'-O-ribose-methylation, is increased in murine muscle tissue during postischemic neovascularization. Our findings suggest miR487b editing plays an intricate role in postischemic neovascularization. © 2017 American Heart Association, Inc.

Geno2pheno[HCV] – A Web-based Interpretation System to Support Hepatitis C Treatment Decisions in the Era of Direct-Acting Antiviral Agents

PubMed Central

Kalaghatgi, Prabhav; Sikorski, Anna Maria; Knops, Elena; Rupp, Daniel; Sierra, Saleta; Heger, Eva; Neumann-Fraune, Maria; Beggel, Bastian; Walker, Andreas; Timm, Jörg; Walter, Hauke; Obermeier, Martin; Kaiser, Rolf; Bartenschlager, Ralf; Lengauer, Thomas

2016-01-01

The face of hepatitis C virus (HCV) therapy is changing dramatically. Direct-acting antiviral agents (DAAs) specifically targeting HCV proteins have been developed and entered clinical practice in 2011. However, despite high sustained viral response (SVR) rates of more than 90%, a fraction of patients do not eliminate the virus and in these cases treatment failure has been associated with the selection of drug resistance mutations (RAMs). RAMs may be prevalent prior to the start of treatment, or can be selected under therapy, and furthermore they can persist after cessation of treatment. Additionally, certain DAAs have been approved only for distinct HCV genotypes and may even have subtype specificity. Thus, sequence analysis before start of therapy is instrumental for managing DAA-based treatment strategies. We have created the interpretation system geno2pheno[HCV] (g2p[HCV]) to analyse HCV sequence data with respect to viral subtype and to predict drug resistance. Extensive reviewing and weighting of literature related to HCV drug resistance was performed to create a comprehensive list of drug resistance rules for inhibitors of the HCV protease in non-structural protein 3 (NS3-protease: Boceprevir, Paritaprevir, Simeprevir, Asunaprevir, Grazoprevir and Telaprevir), the NS5A replicase factor (Daclatasvir, Ledipasvir, Elbasvir and Ombitasvir), and the NS5B RNA-dependent RNA polymerase (Dasabuvir and Sofosbuvir). Upon submission of up to eight sequences, g2p[HCV] aligns the input sequences, identifies the genomic region(s), predicts the HCV geno- and subtypes, and generates for each DAA a drug resistance prediction report. g2p[HCV] offers easy-to-use and fast subtype and resistance analysis of HCV sequences, is continuously updated and freely accessible under http://hcv.geno2pheno.org/index.php. The system was partially validated with respect to the NS3-protease inhibitors Boceprevir, Telaprevir and Simeprevir by using data generated with recombinant, phenotypic cell culture assays obtained from patients’ virus variants. PMID:27196673

Design of a tobacco exon array with application to investigate the differential cadmium accumulation property in two tobacco varieties

PubMed Central

2012-01-01

Background For decades the tobacco plant has served as a model organism in plant biology to answer fundamental biological questions in the areas of plant development, physiology, and genetics. Due to the lack of sufficient coverage of genomic sequences, however, none of the expressed sequence tag (EST)-based chips developed to date cover gene expression from the whole genome. The availability of Tobacco Genome Initiative (TGI) sequences provides a useful resource to build a whole genome exon array, even if the assembled sequences are highly fragmented. Here, the design of a Tobacco Exon Array is reported and an application to improve the understanding of genes regulated by cadmium (Cd) in tobacco is described. Results From the analysis and annotation of the 1,271,256 Nicotiana tabacum fasta and quality files from methyl filtered genomic survey sequences (GSS) obtained from the TGI and ~56,000 ESTs available in public databases, an exon array with 272,342 probesets was designed (four probes per exon) and tested on two selected tobacco varieties. Two tobacco varieties out of 45 accumulating low and high cadmium in leaf were identified based on the GGE biplot analysis, which is analysis of the genotype main effect (G) plus analysis of the genotype by environment interaction (GE) of eight field trials (four fields over two years) showing reproducibility across the trials. The selected varieties were grown under greenhouse conditions in two different soils and subjected to exon array analyses using root and leaf tissues to understand the genetic make-up of the Cd accumulation. Conclusions An Affymetrix Exon Array was developed to cover a large (~90%) proportion of the tobacco gene space. The Tobacco Exon Array will be available for research use through Affymetrix array catalogue. As a proof of the exon array usability, we have demonstrated that the Tobacco Exon Array is a valuable tool for studying Cd accumulation in tobacco leaves. Data from field and greenhouse experiments supported by gene expression studies strongly suggested that the difference in leaf Cd accumulation between the two specific tobacco cultivars is dependent solely on genetic factors and genetic variability rather than on the environment. PMID:23190529

Genotypic and Phenotypic Characterization of “Streptococcus milleri” Group Isolates from a Veterans Administration Hospital Population

PubMed Central

Clarridge, Jill E.; Osting, Cheryl; Jalali, Mehri; Osborne, Janet; Waddington, Michael

1999-01-01

Because identification of the species within the “Streptococcus milleri” group is difficult for the clinical laboratory as the species share overlapping phenotypic characteristics, we wished to confirm biochemical identification with identification by 16S rRNA gene sequence analysis. Ninety-four clinical isolates previously identified as the “Streptococcus milleri” group were reclassified as S. anginosus, S. constellatus, or S. intermedius with the API 20 Strep system (bioMerieux Vikek, Hazelton, Mo.) and the Fluo-card (Key Scientific, Round Rock, Tex.). In addition, we determined the Lancefield group, hemolysis, colony size, colony texture, repetitive extragenic palindromic PCR (rep-PCR) pattern, and cellular fatty acid (CFA) profile (MIDI, Newark, Del.). 16S rRNA gene sequence analysis with 40 selected representative strains showed three distinct groups, with S. constellatus and S. intermedius found to be more closely related to each other than to S. anginosus, and further distinguished a biochemically distinct group of urogenital isolates within the S. anginosus group of isolates. Except for strains unreactive with the Fluo-card (8%), all S. anginosus and S. intermedius strains identified by sequencing were similarly identified by biochemical testing. However, 23% of the selected S. constellatus isolates identified by sequencing (9% of all S. constellatus isolates) would have been identified as S. anginosus or S. intermedius by biochemical tests. Although most S. anginosus strains formed one unique cluster by CFA analysis and most S. constellatus strains showed similar rep-PCR patterns, neither method was sufficiently dependable for identification. Whereas Lancefield group or lactose fermentation did not correspond to sequence or biochemical type, S. constellatus was most likely to be beta-hemolytic and S. intermedius was most likely to have a dry colony type. The most frequent isolate in our population was S. constellatus, followed by S. anginosus. There was an association of S. anginosus with a gastrointestinal or urogenital source, and there was an association of S. constellatus and S. intermedius with both the respiratory tract and upper-body abscesses. PMID:10523574

Alu sequence involvement in transcriptional insulation of the keratin 18 gene in transgenic mice.

PubMed Central

Thorey, I S; Ceceña, G; Reynolds, W; Oshima, R G

1993-01-01

The human keratin 18 (K18) gene is expressed in a variety of adult simple epithelial tissues, including liver, intestine, lung, and kidney, but is not normally found in skin, muscle, heart, spleen, or most of the brain. Transgenic animals derived from the cloned K18 gene express the transgene in appropriate tissues at levels directly proportional to the copy number and independently of the sites of integration. We have investigated in transgenic mice the dependence of K18 gene expression on the distal 5' and 3' flanking sequences and upon the RNA polymerase III promoter of an Alu repetitive DNA transcription unit immediately upstream of the K18 promoter. Integration site-independent expression of tandemly duplicated K18 transgenes requires the presence of either an 825-bp fragment of the 5' flanking sequence or the 3.5-kb 3' flanking sequence. Mutation of the RNA polymerase III promoter of the Alu element within the 825-bp fragment abolishes copy number-dependent expression in kidney but does not abolish integration site-independent expression when assayed in the absence of the 3' flanking sequence of the K18 gene. The characteristics of integration site-independent expression and copy number-dependent expression are separable. In addition, the formation of the chromatin state of the K18 gene, which likely restricts the tissue-specific expression of this gene, is not dependent upon the distal flanking sequences of the 10-kb K18 gene but rather may depend on internal regulatory regions of the gene. Images PMID:7692231

Pseudorandom number generation using chaotic true orbits of the Bernoulli map

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saito, Asaki, E-mail: saito@fun.ac.jp; Yamaguchi, Akihiro

We devise a pseudorandom number generator that exactly computes chaotic true orbits of the Bernoulli map on quadratic algebraic integers. Moreover, we describe a way to select the initial points (seeds) for generating multiple pseudorandom binary sequences. This selection method distributes the initial points almost uniformly (equidistantly) in the unit interval, and latter parts of the generated sequences are guaranteed not to coincide. We also demonstrate through statistical testing that the generated sequences possess good randomness properties.

Use of Sequence-Independent, Single-Primer-Amplification (SISPA) for rapid detection, identification, and characterization of avian RNA viruses

USDA-ARS?s Scientific Manuscript database

Current technologies with next generation sequencing have revolutionized metagenomics analysis of clinical samples. To achieve the non-selective amplification and recovery of low abundance genetic sequences, a simplified Sequence-Independent, Single-Primer Amplification (SISPA) technique in combinat...

Effect of ionic strength and cationic DNA affinity binders on the DNA sequence selective alkylation of guanine N7-positions by nitrogen mustards

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hartley, J.A.; Forrow, S.M.; Souhami, R.L.

Large variations in alkylation intensities exist among guanines in a DNA sequence following treatment with chemotherapeutic alkylating agents such as nitrogen mustards, and the substituent attached to the reactive group can impose a distinct sequence preference for reaction. In order to understand further the structural and electrostatic factors which determine the sequence selectivity of alkylation reactions, the effect of increase ionic strength, the intercalator ethidium bromide, AT-specific minor groove binders distamycin A and netropsin, and the polyamine spermine on guanine N7-alkylation by L-phenylalanine mustard (L-Pam), uracil mustard (UM), and quinacrine mustard (QM) was investigated with a modification of the guanine-specificmore » chemical cleavage technique for DNA sequencing. The result differed with both the nitrogen mustard and the cationic agent used. The effect, which resulted in both enhancement and suppression of alkylation sites, was most striking in the case of netropsin and distamycin A, which differed from each other. DNA footprinting indicated that selective binding to AT sequences in the minor groove of DNA can have long-range effects on the alkylation pattern of DNA in the major groove.« less

Comparison of sequence of trunk and arm motions between short and long official distance groups in javelin throwing.

PubMed

Liu, Hui; Leigh, Steve; Yu, Bing

2014-03-01

The purpose of this study was to determine the effects of sequences of the trunk and arm angular motions on the performance of javelin throwing. In this study, 32 male and 30 female elite javelin throwers participated and were separated into a short official distance group or a long official distance group in each gender. Three-dimensional coordinates of 21 body landmarks and 3 marks on the javelin in the best trial were collected for each subject. Joint center linear velocities and selected trunk and arm segment and joint angles and angular velocities were calculated. The times of the initiations of the selected segment and joint angular motions and maximum angular velocities were determined. The sequences of the initiations of the selected segment and joint angular motions and maximum angular velocities were compared between short and long official distance groups and between genders. The results demonstrated that short and long official distance groups employed similar sequences of the trunk and arm motions. Male and female javelin throwers employed different sequences of the trunk and arm motions. The sequences of the trunk and arm motions were different from those of the maximal joint center linear velocities.

Optimization process planning using hybrid genetic algorithm and intelligent search for job shop machining.

PubMed

Salehi, Mojtaba; Bahreininejad, Ardeshir

2011-08-01

Optimization of process planning is considered as the key technology for computer-aided process planning which is a rather complex and difficult procedure. A good process plan of a part is built up based on two elements: (1) the optimized sequence of the operations of the part; and (2) the optimized selection of the machine, cutting tool and Tool Access Direction (TAD) for each operation. In the present work, the process planning is divided into preliminary planning, and secondary/detailed planning. In the preliminary stage, based on the analysis of order and clustering constraints as a compulsive constraint aggregation in operation sequencing and using an intelligent searching strategy, the feasible sequences are generated. Then, in the detailed planning stage, using the genetic algorithm which prunes the initial feasible sequences, the optimized operation sequence and the optimized selection of the machine, cutting tool and TAD for each operation based on optimization constraints as an additive constraint aggregation are obtained. The main contribution of this work is the optimization of sequence of the operations of the part, and optimization of machine selection, cutting tool and TAD for each operation using the intelligent search and genetic algorithm simultaneously.

Optimization process planning using hybrid genetic algorithm and intelligent search for job shop machining

PubMed Central

Salehi, Mojtaba

2010-01-01

Optimization of process planning is considered as the key technology for computer-aided process planning which is a rather complex and difficult procedure. A good process plan of a part is built up based on two elements: (1) the optimized sequence of the operations of the part; and (2) the optimized selection of the machine, cutting tool and Tool Access Direction (TAD) for each operation. In the present work, the process planning is divided into preliminary planning, and secondary/detailed planning. In the preliminary stage, based on the analysis of order and clustering constraints as a compulsive constraint aggregation in operation sequencing and using an intelligent searching strategy, the feasible sequences are generated. Then, in the detailed planning stage, using the genetic algorithm which prunes the initial feasible sequences, the optimized operation sequence and the optimized selection of the machine, cutting tool and TAD for each operation based on optimization constraints as an additive constraint aggregation are obtained. The main contribution of this work is the optimization of sequence of the operations of the part, and optimization of machine selection, cutting tool and TAD for each operation using the intelligent search and genetic algorithm simultaneously. PMID:21845020

Learning by subtraction: Hippocampal activity and effects of ethanol during the acquisition and performance of response sequences.

PubMed

Ketchum, Myles J; Weyand, Theodore G; Weed, Peter F; Winsauer, Peter J

2016-05-01

Learning is believed to be reflected in the activity of the hippocampus. However, neural correlates of learning have been difficult to characterize because hippocampal activity is integrated with ongoing behavior. To address this issue, male rats (n = 5) implanted with electrodes (n = 14) in the CA1 subfield responded during two tasks within a single test session. In one task, subjects acquired a new 3-response sequence (acquisition), whereas in the other task, subjects completed a well-rehearsed 3-response sequence (performance). Both tasks though could be completed using an identical response topography and used the same sensory stimuli and schedule of reinforcement. More important, comparing neural patterns during sequence acquisition to those during sequence performance allows for a subtractive approach whereby activity associated with learning could potentially be dissociated from the activity associated with ongoing behavior. At sites where CA1 activity was closely associated with behavior, the patterns of activity were differentially modulated by key position and the serial position of a response within the schedule of reinforcement. Temporal shifts between peak activity and responding on particular keys also occurred during sequence acquisition, but not during sequence performance. Ethanol disrupted CA1 activity while producing rate-decreasing effects in both tasks and error-increasing effects that were more selective for sequence acquisition than sequence performance. Ethanol also produced alterations in the magnitude of modulations and temporal pattern of CA1 activity, although these effects were not selective for sequence acquisition. Similar to ethanol, hippocampal micro-stimulation decreased response rate in both tasks and selectively increased the percentage of errors during sequence acquisition, and provided a more direct demonstration of hippocampal involvement during sequence acquisition. Together, these results strongly support the notion that ethanol disrupts sequence acquisition by disrupting hippocampal activity and that the hippocampus is necessary for the conditioned associations required for sequence acquisition. © 2015 Wiley Periodicals, Inc.

Learning by Subtraction: Hippocampal Activity and Effects of Ethanol during the Acquisition and Performance of Response Sequences

PubMed Central

Ketchum, Myles J.; Weyand, Theodore G.; Weed, Peter F.; Winsauer, Peter J.

2015-01-01

Learning is believed to be reflected in the activity of the hippocampus. However, neural correlates of learning have been difficult to characterize because hippocampal activity is integrated with ongoing behavior. To address this issue, male rats (n=5) implanted with electrodes (n=14) in the CA1 subfield responded during two tasks within a single test session. In one task, subjects acquired a new 3-response sequence (acquisition), whereas in the other task, subjects completed a well-rehearsed 3-response sequence (performance). Both tasks though could be completed using an identical response topography and used the same sensory stimuli and schedule of reinforcement. More important, comparing neural patterns during sequence acquisition to those during sequence performance allows for a subtractive approach whereby activity associated with learning could potentially be dissociated from the activity associated with ongoing behavior. At sites where CA1 activity was closely associated with behavior, the patterns of activity were differentially modulated by key position and the serial position of a response within the schedule of reinforcement. Temporal shifts between peak activity and responding on particular keys also occurred during sequence acquisition, but not during sequence performance. Ethanol disrupted CA1 activity while producing rate-decreasing effects in both tasks and error-increasing effects that were more selective for sequence acquisition than sequence performance. Ethanol also produced alterations in the magnitude of modulations and temporal pattern of CA1 activity, although these effects were not selective for sequence acquisition. Similar to ethanol, hippocampal micro-stimulation decreased response rate in both tasks and selectively increased the percentage of errors during sequence acquisition, and provided a more direct demonstration of hippocampal involvement during sequence acquisition. Together, these results strongly support the notion that ethanol disrupts sequence acquisition by disrupting hippocampal activity and that the hippocampus is necessary for the conditioned associations required for sequence acquisition. PMID:26482846

Biomimetic Artificial Epigenetic Code for Targeted Acetylation of Histones.

PubMed

Taniguchi, Junichi; Feng, Yihong; Pandian, Ganesh N; Hashiya, Fumitaka; Hidaka, Takuya; Hashiya, Kaori; Park, Soyoung; Bando, Toshikazu; Ito, Shinji; Sugiyama, Hiroshi

2018-06-13

While the central role of locus-specific acetylation of histone proteins in eukaryotic gene expression is well established, the availability of designer tools to regulate acetylation at particular nucleosome sites remains limited. Here, we develop a unique strategy to introduce acetylation by constructing a bifunctional molecule designated Bi-PIP. Bi-PIP has a P300/CBP-selective bromodomain inhibitor (Bi) as a P300/CBP recruiter and a pyrrole-imidazole polyamide (PIP) as a sequence-selective DNA binder. Biochemical assays verified that Bi-PIPs recruit P300 to the nucleosomes having their target DNA sequences and extensively accelerate acetylation. Bi-PIPs also activated transcription of genes that have corresponding cognate DNA sequences inside living cells. Our results demonstrate that Bi-PIPs could act as a synthetic programmable histone code of acetylation, which emulates the bromodomain-mediated natural propagation system of histone acetylation to activate gene expression in a sequence-selective manner.

CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites

PubMed Central

Naito, Yuki; Hino, Kimihiro; Bono, Hidemasa; Ui-Tei, Kumiko

2015-01-01

Summary: CRISPRdirect is a simple and functional web server for selecting rational CRISPR/Cas targets from an input sequence. The CRISPR/Cas system is a promising technique for genome engineering which allows target-specific cleavage of genomic DNA guided by Cas9 nuclease in complex with a guide RNA (gRNA), that complementarily binds to a ∼20 nt targeted sequence. The target sequence requirements are twofold. First, the 5′-NGG protospacer adjacent motif (PAM) sequence must be located adjacent to the target sequence. Second, the target sequence should be specific within the entire genome in order to avoid off-target editing. CRISPRdirect enables users to easily select rational target sequences with minimized off-target sites by performing exhaustive searches against genomic sequences. The server currently incorporates the genomic sequences of human, mouse, rat, marmoset, pig, chicken, frog, zebrafish, Ciona, fruit fly, silkworm, Caenorhabditis elegans, Arabidopsis, rice, Sorghum and budding yeast. Availability: Freely available at http://crispr.dbcls.jp/. Contact: y-naito@dbcls.rois.ac.jp Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25414360

Application of whole genome re-sequencing data in the development of diagnostic DNA markers tightly linked to a disease-resistance locus for marker-assisted selection in lupin (Lupinus angustifolius).

PubMed

Yang, Huaan; Jian, Jianbo; Li, Xuan; Renshaw, Daniel; Clements, Jonathan; Sweetingham, Mark W; Tan, Cong; Li, Chengdao

2015-09-02

Molecular marker-assisted breeding provides an efficient tool to develop improved crop varieties. A major challenge for the broad application of markers in marker-assisted selection is that the marker phenotypes must match plant phenotypes in a wide range of breeding germplasm. In this study, we used the legume crop species Lupinus angustifolius (lupin) to demonstrate the utility of whole genome sequencing and re-sequencing on the development of diagnostic markers for molecular plant breeding. Nine lupin cultivars released in Australia from 1973 to 2007 were subjected to whole genome re-sequencing. The re-sequencing data together with the reference genome sequence data were used in marker development, which revealed 180,596 to 795,735 SNP markers from pairwise comparisons among the cultivars. A total of 207,887 markers were anchored on the lupin genetic linkage map. Marker mining obtained an average of 387 SNP markers and 87 InDel markers for each of the 24 genome sequence assembly scaffolds bearing markers linked to 11 genes of agronomic interest. Using the R gene PhtjR conferring resistance to phomopsis stem blight disease as a test case, we discovered 17 candidate diagnostic markers by genotyping and selecting markers on a genetic linkage map. A further 243 candidate diagnostic markers were discovered by marker mining on a scaffold bearing non-diagnostic markers linked to the PhtjR gene. Nine out from the ten tested candidate diagnostic markers were confirmed as truly diagnostic on a broad range of commercial cultivars. Markers developed using these strategies meet the requirements for broad application in molecular plant breeding. We demonstrated that low-cost genome sequencing and re-sequencing data were sufficient and very effective in the development of diagnostic markers for marker-assisted selection. The strategies used in this study may be applied to any trait or plant species. Whole genome sequencing and re-sequencing provides a powerful tool to overcome current limitations in molecular plant breeding, which will enable plant breeders to precisely pyramid favourable genes to develop super crop varieties to meet future food demands.

Molecular and comparative analysis of Salmonella enterica Senftenberg from humans and animals using PFGE, MLST and NARMS.

PubMed

Stepan, Ryan M; Sherwood, Julie S; Petermann, Shana R; Logue, Catherine M

2011-06-27

Salmonella species are recognized worldwide as a significant cause of human and animal disease. In this study the molecular profiles and characteristics of Salmonella enterica Senftenberg isolated from human cases of illness and those recovered from healthy or diagnostic cases in animals were assessed. Included in the study was a comparison with our own sequenced strain of S. Senfteberg recovered from production turkeys in North Dakota. Isolates examined in this study were subjected to antimicrobial susceptibility profiling using the National Antimicrobial Resistance Monitoring System (NARMS) panel which tested susceptibility to 15 different antimicrobial agents. The molecular profiles of all isolates were determined using Pulsed Field Gel Electrophoresis (PFGE) and the sequence types of the strains were obtained using Multi-Locus Sequence Type (MLST) analysis based on amplification and sequence interrogation of seven housekeeping genes (aroC, dnaN, hemD, hisD, purE, sucA, and thrA). PFGE data was input into BioNumerics analysis software to generate a dendrogram of relatedness among the strains. The study found 93 profiles among 98 S. Senftenberg isolates tested and there were primarily two sequence types associated with humans and animals (ST185 and ST14) with overlap observed in all host types suggesting that the distribution of S. Senftenberg sequence types is not host dependent. Antimicrobial resistance was observed among the animal strains, however no resistance was detected in human isolates suggesting that animal husbandry has a significant influence on the selection and promotion of antimicrobial resistance. The data demonstrates the circulation of at least two strain types in both animal and human health suggesting that S. Senftenberg is relatively homogeneous in its distribution. The data generated in this study could be used towards defining a pathotype for this serovar.

Cultivation of Hard-To-Culture Subsurface Mercury-Resistant Bacteria and Discovery of New merA Gene Sequences▿

PubMed Central

Rasmussen, L. D.; Zawadsky, C.; Binnerup, S. J.; Øregaard, G.; Sørensen, S. J.; Kroer, N.

2008-01-01

Mercury-resistant bacteria may be important players in mercury biogeochemistry. To assess the potential for mercury reduction by two subsurface microbial communities, resistant subpopulations and their merA genes were characterized by a combined molecular and cultivation-dependent approach. The cultivation method simulated natural conditions by using polycarbonate membranes as a growth support and a nonsterile soil slurry as a culture medium. Resistant bacteria were pregrown to microcolony-forming units (mCFU) before being plated on standard medium. Compared to direct plating, culturability was increased up to 2,800 times and numbers of mCFU were similar to the total number of mercury-resistant bacteria in the soils. Denaturing gradient gel electrophoresis analysis of DNA extracted from membranes suggested stimulation of growth of hard-to-culture bacteria during the preincubation. A total of 25 different 16S rRNA gene sequences were observed, including Alpha-, Beta-, and Gammaproteobacteria; Actinobacteria; Firmicutes; and Bacteroidetes. The diversity of isolates obtained by direct plating included eight different 16S rRNA gene sequences (Alpha- and Betaproteobacteria and Actinobacteria). Partial sequencing of merA of selected isolates led to the discovery of new merA sequences. With phylum-specific merA primers, PCR products were obtained for Alpha- and Betaproteobacteria and Actinobacteria but not for Bacteroidetes and Firmicutes. The similarity to known sequences ranged between 89 and 95%. One of the sequences did not result in a match in the BLAST search. The results illustrate the power of integrating advanced cultivation methodology with molecular techniques for the characterization of the diversity of mercury-resistant populations and assessing the potential for mercury reduction in contaminated environments. PMID:18441111

Two alternative ways of start site selection in human norovirus reinitiation of translation.

PubMed

Luttermann, Christine; Meyers, Gregor

2014-04-25

The calicivirus minor capsid protein VP2 is expressed via termination/reinitiation. This process depends on an upstream sequence element denoted termination upstream ribosomal binding site (TURBS). We have shown for feline calicivirus and rabbit hemorrhagic disease virus that the TURBS contains three sequence motifs essential for reinitiation. Motif 1 is conserved among caliciviruses and is complementary to a sequence in the 18 S rRNA leading to the model that hybridization between motif 1 and 18 S rRNA tethers the post-termination ribosome to the mRNA. Motif 2 and motif 2* are proposed to establish a secondary structure positioning the ribosome relative to the start site of the terminal ORF. Here, we analyzed human norovirus (huNV) sequences for the presence and importance of these motifs. The three motifs were identified by sequence analyses in the region upstream of the VP2 start site, and we showed that these motifs are essential for reinitiation of huNV VP2 translation. More detailed analyses revealed that the site of reinitiation is not fixed to a single codon and does not need to be an AUG, even though this codon is clearly preferred. Interestingly, we were able to show that reinitiation can occur at AUG codons downstream of the canonical start/stop site in huNV and feline calicivirus but not in rabbit hemorrhagic disease virus. Although reinitiation at the original start site is independent of the Kozak context, downstream initiation exhibits requirements for start site sequence context known for linear scanning. These analyses on start codon recognition give a more detailed insight into this fascinating mechanism of gene expression.

Simulation-Based Evaluation of Learning Sequences for Instructional Technologies

ERIC Educational Resources Information Center

McEneaney, John E.

2016-01-01

Instructional technologies critically depend on systematic design, and learning hierarchies are a commonly advocated tool for designing instructional sequences. But hierarchies routinely allow numerous sequences and choosing an optimal sequence remains an unsolved problem. This study explores a simulation-based approach to modeling learning…

Phage display screening without repetitious selection rounds.

PubMed

't Hoen, Peter A C; Jirka, Silvana M G; Ten Broeke, Bradley R; Schultes, Erik A; Aguilera, Begoña; Pang, Kar Him; Heemskerk, Hans; Aartsma-Rus, Annemieke; van Ommen, Gertjan J; den Dunnen, Johan T

2012-02-15

Phage display screenings are frequently employed to identify high-affinity peptides or antibodies. Although successful, phage display is a laborious technology and is notorious for identification of false positive hits. To accelerate and improve the selection process, we have employed Illumina next generation sequencing to deeply characterize the Ph.D.-7 M13 peptide phage display library before and after several rounds of biopanning on KS483 osteoblast cells. Sequencing of the naive library after one round of amplification in bacteria identifies propagation advantage as an important source of false positive hits. Most important, our data show that deep sequencing of the phage pool after a first round of biopanning is already sufficient to identify positive phages. Whereas traditional sequencing of a limited number of clones after one or two rounds of selection is uninformative, the required additional rounds of biopanning are associated with the risk of losing promising clones propagating slower than nonbinding phages. Confocal and live cell imaging confirms that our screen successfully selected a peptide with very high binding and uptake in osteoblasts. We conclude that next generation sequencing can significantly empower phage display screenings by accelerating the finding of specific binders and restraining the number of false positive hits. Copyright © 2011 Elsevier Inc. All rights reserved.

Vertebrate codon bias indicates a highly GC-rich ancestral genome.

PubMed

Nabiyouni, Maryam; Prakash, Ashwin; Fedorov, Alexei

2013-04-25

Two factors are thought to have contributed to the origin of codon usage bias in eukaryotes: 1) genome-wide mutational forces that shape overall GC-content and create context-dependent nucleotide bias, and 2) positive selection for codons that maximize efficient and accurate translation. Particularly in vertebrates, these two explanations contradict each other and cloud the origin of codon bias in the taxon. On the one hand, mutational forces fail to explain GC-richness (~60%) of third codon positions, given the GC-poor overall genomic composition among vertebrates (~40%). On the other hand, positive selection cannot easily explain strict regularities in codon preferences. Large-scale bioinformatic assessment, of nucleotide composition of coding and non-coding sequences in vertebrates and other taxa, suggests a simple possible resolution for this contradiction. Specifically, we propose that the last common vertebrate ancestor had a GC-rich genome (~65% GC). The data suggest that whole-genome mutational bias is the major driving force for generating codon bias. As the bias becomes prominent, it begins to affect translation and can result in positive selection for optimal codons. The positive selection can, in turn, significantly modulate codon preferences. Copyright © 2013 Elsevier B.V. All rights reserved.

Phylogenetic analysis of Rutaceous plants based on single nucleotide polymorphism in chloroplast and nuclear gene sequences

USDA-ARS?s Scientific Manuscript database

The family Rutaceae encompasses several genera including the economically important genus Citrus. In this study, we selected 22 citrus relatives belonging to the various sub groups of Rutaceae and compared the sequences of three gene fragments. The accessions selected belong to the subfamily Rutoide...

In vitro optimization of truncated stem-loop II variants of the hammerhead ribozyme for cleavage in low concentrations of magnesium under non-turnover conditions.

PubMed Central

Zillmann, M; Limauro, S E; Goodchild, J

1997-01-01

By truncating helix II to two base pairs in a hammerhead ribozyme having long flanking sequences (greater than 30 bases), the rate of cleavage in 1 mM magnesium can be increased roughly 100-fold. Replacing most of the nucleotides in a typical stem-loop II with 1-4 randomized nucleotides gave an RNA library that, even before selection, was more active in 1 mM magnesium than the parent ribozyme, but considerably less active than the truncated stem-loop II ribozyme. A novel, multiround selection for intermolecular cleavage was exploited to optimize this library for cleavage in low concentrations of magnesium. After three rounds of selection at sequentially lower concentrations of magnesium, the library cleaved substrate RNA 20-fold faster than the initial pool and was cloned. This pool was heavily enriched for one particular sequence (5'-CGUG-3') that represented 16 of 52 isolates (the next most common sequence was represented only six times). This sequence also represented the most active sequence, exceeding the activity of the short helix II variant under the conditions of the selection, thereby demonstrating the effectiveness of the selection technique. Analysis of the cleavage rates of RNAs made from eight isolates having different four-base insert sequences allowed assignment of highly preferred bases at each position in the insert. Analysis of pool clones having insert of differing lengths showed that, in general, activity decreased as the length of the insert decreased from 4 to 1. This supports the suggested role of stem-loop II in stabilizing the non-Watson-Crick interactions between the conserved bases of the catalytic core. PMID:9214657

Photosensitization of DNA damage by a new cationic pyropheophorbide derivative: sequence-specific formation of a frank scission.

PubMed

Kanony, Claire; Fabiano-Tixier, Anne-Sylvie; Ravanat, Jean-Luc; Vicendo, Patricia; Paillous, Nicole

2003-06-01

Pyropheophorbides are red-absorbing porphyrin-like photosensitizers that may interact with DNA either by intercalation or by external binding with self-stacking according to the value of the nucleotide to chromophore molar ratio (N/C). This article reports on the nature and sequence selectivity of the DNA damage photoinduced by a water-soluble chlorhydrate of aminopyropheophorbide. First, this pyropheophorbide is shown to induce on irradiation the cleavage of phiX174 DNA by both Type-I and -II mechanisms, suggested by scavengers and D2O effects. These conclusions are then improved by sequencing experiments performed on a 20-mer oligodeoxynucleotide (ODN) irradiated at wavelengths >345 nm in the presence of the dye, N/C varying from 2.5 to 0.5. Oxidation of all guanine residues to the same extent is observed after piperidine treatment on both single- and double-stranded ODN. Moreover, unexpectedly, a remarkable sequence-selective cleavage occurring at a 5'-CG-3' site is detected before alkali treatment. This frank break is clearly predominant for a low nucleotide to chromophore molar ratio, corresponding to a self-stacking of the dye along the DNA helix. The electrophoretic properties of the band suggest that this lesion results from a sugar oxidation, which leads via a base release to a ribonolactone residue. The proposal is supported by high-performance liquid chromatography-matrix-assisted laser desorption-ionization mass spectrometry experiments that also reveal other sequence-selective frank scissions of lower intensity at 5'-GC-3' or other 5'-CG-3' sites. This sequence selectivity is discussed with regard to the binding selectivity of cationic porphyrins.

Selection and Trans-Species Polymorphism of Major Histocompatibility Complex Class II Genes in the Order Crocodylia

PubMed Central

Jaratlerdsiri, Weerachai; Isberg, Sally R.; Higgins, Damien P.; Miles, Lee G.; Gongora, Jaime

2014-01-01

Major Histocompatibility Complex (MHC) class II genes encode for molecules that aid in the presentation of antigens to helper T cells. MHC characterisation within and between major vertebrate taxa has shed light on the evolutionary mechanisms shaping the diversity within this genomic region, though little characterisation has been performed within the Order Crocodylia. Here we investigate the extent and effect of selective pressures and trans-species polymorphism on MHC class II α and β evolution among 20 extant species of Crocodylia. Selection detection analyses showed that diversifying selection influenced MHC class II β diversity, whilst diversity within MHC class II α is the result of strong purifying selection. Comparison of translated sequences between species revealed the presence of twelve trans-species polymorphisms, some of which appear to be specific to the genera Crocodylus and Caiman. Phylogenetic reconstruction clustered MHC class II α sequences into two major clades representing the families Crocodilidae and Alligatoridae. However, no further subdivision within these clades was evident and, based on the observation that most MHC class II α sequences shared the same trans-species polymorphisms, it is possible that they correspond to the same gene lineage across species. In contrast, phylogenetic analyses of MHC class II β sequences showed a mixture of subclades containing sequences from Crocodilidae and/or Alligatoridae, illustrating orthologous relationships among those genes. Interestingly, two of the subclades containing sequences from both Crocodilidae and Alligatoridae shared specific trans-species polymorphisms, suggesting that they may belong to ancient lineages pre-dating the divergence of these two families from the common ancestor 85–90 million years ago. The results presented herein provide an immunogenetic resource that may be used to further assess MHC diversity and functionality in Crocodylia. PMID:24503938