Spatialization in working memory is related to literacy and reading direction: Culture "literarily" directs our thoughts.

PubMed

Guida, Alessandro; Megreya, Ahmed M; Lavielle-Guida, Magali; Noël, Yvonnick; Mathy, Fabien; van Dijck, Jean-Philippe; Abrahamse, Elger

2018-06-01

The ability to maintain arbitrary sequences of items in the mind contributes to major cognitive faculties, such as language, reasoning, and episodic memory. Previous research suggests that serial order working memory is grounded in the brain's spatial attention system. In the present study, we show that the spatially defined mental organization of novel item sequences is related to literacy and varies as a function of reading/writing direction. Specifically, three groups (left-to-right Western readers, right-to-left Arabic readers, and Arabic-speaking illiterates) were asked to memorize random (and non-spatial) sequences of color patches and determine whether a subsequent probe was part of the memorized sequence (e.g., press left key) or not (e.g., press right key). The results showed that Western readers mentally organized the sequences from left to right, Arabic readers spontaneously used the opposite direction, and Arabic-speaking illiterates showed no systematic spatial organization. This finding suggests that cultural conventions shape one of the most "fluid" aspects of human cognition, namely, the spontaneous mental organization of novel non-spatial information. Copyright © 2018 Elsevier B.V. All rights reserved.

Evaluation of microbial community in hydrothermal field by direct DNA sequencing

NASA Astrophysics Data System (ADS)

Kawarabayasi, Y.; Maruyama, A.

2002-12-01

Many extremophiles have been discovered from terrestrial and marine hydrothermal fields. Some thermophiles can grow beyond 90°C in culture, while direct microscopic analysis occasionally indicates that microbes may survive in much hotter hydrothermal fluids. However, it is very difficult to isolate and cultivate such microbes from the environments, i.e., over 99% of total microbes remains undiscovered. Based on experiences of entire microbial genome analysis (Y.K.) and microbial community analysis (A.M.), we started to find out unique microbes/genes in hydrothermal fields through direct sequencing of environmental DNA fragments. At first, shotgun plasmid libraries were directly constructed with the DNA molecules prepared from mixed microbes collected by an in situ filtration system from low-temperature fluids at RM24 in the Southern East Pacific Rise (S-EPR). A gene amplification (PCR) technique was not used for preventing mutation in the process. The nucleotide sequences of 285 clones indicated that no sequence had identical data in public databases. Among 27 clones determined entire sequences, no ORF was identified on 14 clones like intron in Eukaryote. On four clones, tetra-nucleotide-long multiple tandem repetitive sequences were identified. This type of sequence was identified in some familiar disease in human. The result indicates that living/dead materials with eukaryotic features may exist in this low temperature field. Secondly, shotgun plasmid libraries were constructed from the environmental DNA prepared from Beppu hot springs. In randomly-selected 143 clones used for sequencing, no known sequence was identified. Unlike the clones in S-EPR library, clear ORFs were identified on all nine clones determined the entire sequence. It was found that one clone, H4052, contained the complete Aspartyl-tRNA synthetase. Phylogenetic analysis using amino acid sequences of this gene indicated that this gene was separated from other Euryarchaea before the differentiation of species. Thus, some novel archaeal species are expected to be in this field. The present direct cloning and sequencing technique is now opening a window to the new world in hydrothermal microbial community analysis.

Ultrasonic Multiple-Access Ranging System Using Spread Spectrum and MEMS Technology for Indoor Localization

PubMed Central

Segers, Laurent; Tiete, Jelmer; Braeken, An; Touhafi, Abdellah

2014-01-01

Indoor localization of persons and objects poses a great engineering challenge. Previously developed localization systems demonstrate the use of wideband techniques in ultrasound ranging systems. Direct sequence and frequency hopping spread spectrum ultrasound signals have been proven to achieve a high level of accuracy. A novel ranging method using the frequency hopping spread spectrum with finite impulse response filtering will be investigated and compared against the direct sequence spread spectrum. In the first setup, distances are estimated in a single-access environment, while in the second setup, two senders and one receiver are used. During the experiments, the micro-electromechanical systems are used as ultrasonic sensors, while the senders were implemented using field programmable gate arrays. Results show that in a single-access environment, the direct sequence spread spectrum method offers slightly better accuracy and precision performance compared to the frequency hopping spread spectrum. When two senders are used, measurements point out that the frequency hopping spread spectrum is more robust to near-far effects than the direct sequence spread spectrum. PMID:24553084

A simple procedure for parallel sequence analysis of both strands of 5'-labeled DNA.

PubMed

Razvi, F; Gargiulo, G; Worcel, A

1983-08-01

Ligation of a 5'-labeled DNA restriction fragment results in a circular DNA molecule carrying the two 32Ps at the reformed restriction site. Double digestions of the circular DNA with the original enzyme and a second restriction enzyme cleavage near the labeled site allows direct chemical sequencing of one 5'-labeled DNA strand. Similar double digestions, using an isoschizomer that cleaves differently at the 32P-labeled site, allows direct sequencing of the now 3'-labeled complementary DNA strand. It is possible to directly sequence both strands of cloned DNA inserts by using the above protocol and a multiple cloning site vector that provides the necessary restriction sites. The simultaneous and parallel visualization of both DNA strands eliminates sequence ambiguities. In addition, the labeled circular molecules are particularly useful for single-hit DNA cleavage studies and DNA footprint analysis. As an example, we show here an analysis of the micrococcal nuclease-induced breaks on the two strands of the somatic 5S RNA gene of Xenopus borealis, which suggests that the enzyme may recognize and cleave small AT-containing palindromes along the DNA helix.

Method for high-volume sequencing of nucleic acids: random and directed priming with libraries of oligonucleotides

DOEpatents

Studier, F. William

1995-04-18

Random and directed priming methods for determining nucleotide sequences by enzymatic sequencing techniques, using libraries of primers of lengths 8, 9 or 10 bases, are disclosed. These methods permit direct sequencing of nucleic acids as large as 45,000 base pairs or larger without the necessity for subcloning. Individual primers are used repeatedly to prime sequence reactions in many different nucleic acid molecules. Libraries containing as few as 10,000 octamers, 14,200 nonamers, or 44,000 decamers would have the capacity to determine the sequence of almost any cosmid DNA. Random priming with a fixed set of primers from a smaller library can also be used to initiate the sequencing of individual nucleic acid molecules, with the sequence being completed by directed priming with primers from the library. In contrast to random cloning techniques, a combined random and directed priming strategy is far more efficient.

Method for high-volume sequencing of nucleic acids: random and directed priming with libraries of oligonucleotides

DOEpatents

Studier, F.W.

1995-04-18

Random and directed priming methods for determining nucleotide sequences by enzymatic sequencing techniques, using libraries of primers of lengths 8, 9 or 10 bases, are disclosed. These methods permit direct sequencing of nucleic acids as large as 45,000 base pairs or larger without the necessity for subcloning. Individual primers are used repeatedly to prime sequence reactions in many different nucleic acid molecules. Libraries containing as few as 10,000 octamers, 14,200 nonamers, or 44,000 decamers would have the capacity to determine the sequence of almost any cosmid DNA. Random priming with a fixed set of primers from a smaller library can also be used to initiate the sequencing of individual nucleic acid molecules, with the sequence being completed by directed priming with primers from the library. In contrast to random cloning techniques, a combined random and directed priming strategy is far more efficient. 2 figs.

An investigation of application of the golden ratio and Fibonacci sequence in fashion design and pattern making

NASA Astrophysics Data System (ADS)

Kazlacheva, Z. I.

2017-10-01

The Golden ratio and Fibonacci sequence are used as proportions in design as symbols of beauty and harmony. That symbolism is a result of the strong connections in their mathematical nature. The Golden section is a number, introduced with Greek letter φ, which is found by dividing a line into two parts as the longer part divided by the smaller part is equal as the whole length of longer and smaller parts divided by the longer part. Fibonacci sequence is a series of numbers where every number is equal to the two numbers before it. An investigation of application of proportions based on the Golden ratio and Fibonacci sequence in the fashion design and pattern making of ladies’ clothing is the main aim of the paper. Based on the study it may be concluded that in fashion design and pattern making the Golden ratio and Fibonacci sequence can be used in creation of beautiful and harmonic forms directly or with the help of geometrical figures as: In directly use the Golden and Fibonacci numbers proportions can be in one and the same or different directions. In the application with the help of geometrical shapes the Golden and Fibonacci figures combine proportioning and form creation. The Golden and Fibonacci shapes can be used directly as forms or as frames of forms creation of elements and pieces. Its application can be in different directions and location according the bodice. The Golden section and Fibonacci sequence can combine proportions with other principles of design as symmetry, rhythm, etc.

Definition of Cis-Acting Elements Regulating Expression of the Drosophila Melanogaster Ninae Opsin Gene by Oligonucleotide-Directed Mutagenesis

PubMed Central

Mismer, D.; Rubin, G. M.

1989-01-01

We have analyzed the cis-acting regulatory sequences of the Rh1 (ninaE) gene in Drosophila melanogaster by P-element-mediated germline transformation of indicator genes transcribed from mutant ninaE promoter sequences. We have previously shown that a 200-bp region extending from -120 to +67 relative to the transcription start site is sufficient to obtain eye-specific expression from the ninaE promoter. In the present study, 22 different 4-13-bp sequences in the -120/+67 promoter region were altered by oligonucleotide-directed mutagenesis. Several of these sequences were found to be required for proper promoter function; two of these are conserved in the promoter of the homologous gene isolated from the related species Drosophila virilis. Alteration of a conserved 9-bp sequence results in aberrant, low level expression in the body. Alteration of a separate 11-bp sequence, found in the promoter regions of several photoreceptor-specific genes of Drosophila, results in an approximately 15-fold reduction in promoter efficiency but without apparent alteration of tissue-specificity. A protein factor capable of interacting with this 11-bp sequence has been detected by DNaseI footprinting in embryonic nuclear extracts. Finally, we have further characterized two separable enhancer sequences previously shown to be required for normal levels of expression from this promoter. PMID:2521839

MerCat: a versatile k-mer counter and diversity estimator for database-independent property analysis obtained from metagenomic and/or metatranscriptomic sequencing data

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

White, Richard A.; Panyala, Ajay R.; Glass, Kevin A.

MerCat is a parallel, highly scalable and modular property software package for robust analysis of features in next-generation sequencing data. MerCat inputs include assembled contigs and raw sequence reads from any platform resulting in feature abundance counts tables. MerCat allows for direct analysis of data properties without reference sequence database dependency commonly used by search tools such as BLAST and/or DIAMOND for compositional analysis of whole community shotgun sequencing (e.g. metagenomes and metatranscriptomes).

A Lossy Compression Technique Enabling Duplication-Aware Sequence Alignment

PubMed Central

Freschi, Valerio; Bogliolo, Alessandro

2012-01-01

In spite of the recognized importance of tandem duplications in genome evolution, commonly adopted sequence comparison algorithms do not take into account complex mutation events involving more than one residue at the time, since they are not compliant with the underlying assumption of statistical independence of adjacent residues. As a consequence, the presence of tandem repeats in sequences under comparison may impair the biological significance of the resulting alignment. Although solutions have been proposed, repeat-aware sequence alignment is still considered to be an open problem and new efficient and effective methods have been advocated. The present paper describes an alternative lossy compression scheme for genomic sequences which iteratively collapses repeats of increasing length. The resulting approximate representations do not contain tandem duplications, while retaining enough information for making their comparison even more significant than the edit distance between the original sequences. This allows us to exploit traditional alignment algorithms directly on the compressed sequences. Results confirm the validity of the proposed approach for the problem of duplication-aware sequence alignment. PMID:22518086

Advances in high throughput DNA sequence data compression.

PubMed

Sardaraz, Muhammad; Tahir, Muhammad; Ikram, Ataul Aziz

2016-06-01

Advances in high throughput sequencing technologies and reduction in cost of sequencing have led to exponential growth in high throughput DNA sequence data. This growth has posed challenges such as storage, retrieval, and transmission of sequencing data. Data compression is used to cope with these challenges. Various methods have been developed to compress genomic and sequencing data. In this article, we present a comprehensive review of compression methods for genome and reads compression. Algorithms are categorized as referential or reference free. Experimental results and comparative analysis of various methods for data compression are presented. Finally, key challenges and research directions in DNA sequence data compression are highlighted.

Mixed Sequence Reader: A Program for Analyzing DNA Sequences with Heterozygous Base Calling

PubMed Central

Chang, Chun-Tien; Tsai, Chi-Neu; Tang, Chuan Yi; Chen, Chun-Houh; Lian, Jang-Hau; Hu, Chi-Yu; Tsai, Chia-Lung; Chao, Angel; Lai, Chyong-Huey; Wang, Tzu-Hao; Lee, Yun-Shien

2012-01-01

The direct sequencing of PCR products generates heterozygous base-calling fluorescence chromatograms that are useful for identifying single-nucleotide polymorphisms (SNPs), insertion-deletions (indels), short tandem repeats (STRs), and paralogous genes. Indels and STRs can be easily detected using the currently available Indelligent or ShiftDetector programs, which do not search reference sequences. However, the detection of other genomic variants remains a challenge due to the lack of appropriate tools for heterozygous base-calling fluorescence chromatogram data analysis. In this study, we developed a free web-based program, Mixed Sequence Reader (MSR), which can directly analyze heterozygous base-calling fluorescence chromatogram data in .abi file format using comparisons with reference sequences. The heterozygous sequences are identified as two distinct sequences and aligned with reference sequences. Our results showed that MSR may be used to (i) physically locate indel and STR sequences and determine STR copy number by searching NCBI reference sequences; (ii) predict combinations of microsatellite patterns using the Federal Bureau of Investigation Combined DNA Index System (CODIS); (iii) determine human papilloma virus (HPV) genotypes by searching current viral databases in cases of double infections; (iv) estimate the copy number of paralogous genes, such as β-defensin 4 (DEFB4) and its paralog HSPDP3. PMID:22778697

Identification of the bacterial etiology of culture-negative endocarditis by amplification and sequencing of a small ribosomal RNA gene.

PubMed

Khulordava, Irakli; Miller, Geraldine; Haas, David; Li, Haijing; McKinsey, Joel; Vanderende, Daniel; Tang, Yi-Wei

2003-05-01

We report two cases of culture-negative bacterial endocarditis in which the organisms were identified by amplification and sequencing of the bacterial 16S rRNA gene. These results support an important role for polymerase chain reaction followed by direct sequencing to determine the etiology of culture-negative bacterial endocarditis and to guide appropriate antimicrobial therapy.

Sequences downstream of AAUAAA signals affect pre-mRNA cleavage and polyadenylation in vitro both directly and indirectly.

PubMed Central

Ryner, L C; Takagaki, Y; Manley, J L

1989-01-01

To investigate the role of sequences lying downstream of the conserved AAUAAA hexanucleotide in pre-mRNA cleavage and polyadenylation, deletions or substitutions were constructed in polyadenylation signals from simian virus 40 and adenovirus, and their effects were assayed in both crude and fractionated HeLa cell nuclear extracts. As expected, these sequences influenced the efficiency of both cleavage and polyadenylation as well as the accuracy of the cleavage reaction. Sequences near or upstream of the actual site of poly(A) addition appeared to specify a unique cleavage site, since their deletion resulted, in some cases, in heterogeneous cleavage. Furthermore, the sequences that allowed the simian virus 40 late pre-RNA to be cleaved preferentially by partially purified cleavage activity were also those at the cleavage site itself. Interestingly, sequences downstream of the cleavage site interacted with factors not directly involved in catalyzing cleavage and polyadenylation, since the effects of deletions were substantially diminished when partially purified components were used in assays. In addition, these sequences contained elements that could affect 3'-end formation both positively and negatively. Images PMID:2566911

Performance of Improved Channel Allocation for Multicarrier CDMA with Adaptive Frequency Hopping and Multiuser Detection

DTIC Science & Technology

2008-08-29

resulting in much larger system capacity than for a non-adaptive MC direct-sequence ( DS )- CDMA system. This conclusion is confirmed for realistic fading...system capacity than for a non-adaptive MC direct-sequence ( DS )- CDMA system. This conclusion is confirmed for realistic fading channels with correlated...transmission method exploits both frequency and multiuser diversity and improves on non-adaptive MC DS - CDMA sys- tems in [6]. In this paper, we focus on the

The Neandertal genome and ancient DNA authenticity

PubMed Central

Green, Richard E; Briggs, Adrian W; Krause, Johannes; Prüfer, Kay; Burbano, Hernán A; Siebauer, Michael; Lachmann, Michael; Pääbo, Svante

2009-01-01

Recent advances in high-thoughput DNA sequencing have made genome-scale analyses of genomes of extinct organisms possible. With these new opportunities come new difficulties in assessing the authenticity of the DNA sequences retrieved. We discuss how these difficulties can be addressed, particularly with regard to analyses of the Neandertal genome. We argue that only direct assays of DNA sequence positions in which Neandertals differ from all contemporary humans can serve as a reliable means to estimate human contamination. Indirect measures, such as the extent of DNA fragmentation, nucleotide misincorporations, or comparison of derived allele frequencies in different fragment size classes, are unreliable. Fortunately, interim approaches based on mtDNA differences between Neandertals and current humans, detection of male contamination through Y chromosomal sequences, and repeated sequencing from the same fossil to detect autosomal contamination allow initial large-scale sequencing of Neandertal genomes. This will result in the discovery of fixed differences in the nuclear genome between Neandertals and current humans that can serve as future direct assays for contamination. For analyses of other fossil hominins, which may become possible in the future, we suggest a similar ‘boot-strap' approach in which interim approaches are applied until sufficient data for more definitive direct assays are acquired. PMID:19661919

Restoration of distorted depth maps calculated from stereo sequences

NASA Technical Reports Server (NTRS)

Damour, Kevin; Kaufman, Howard

1991-01-01

A model-based Kalman estimator is developed for spatial-temporal filtering of noise and other degradations in velocity and depth maps derived from image sequences or cinema. As an illustration of the proposed procedures, edge information from image sequences of rigid objects is used in the processing of the velocity maps by selecting from a series of models for directional adaptive filtering. Adaptive filtering then allows for noise reduction while preserving sharpness in the velocity maps. Results from several synthetic and real image sequences are given.

Topological disposition of the sequences -QRKIVE- and -KETYY in native (Na sup + + K sup + )-ATPase

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

Bayer, R.

1990-03-06

The dispositions with respect to the plane of the membrane of lysine-905 in the internal sequence -EQRKIVE- and of lysine-1012 in the carboxy-terminal sequence -RRPGGWVEKETYY of the {alpha}-polypeptide of sodium and potassium ion activated adenosinetriphosphatase have been determined. These lysines are found in peptides released from the intact {alpha}-polypeptide by the extracellular protease from Staphylococcus aureus strain V8 and by trypsin, respectively. Synthetic peptides containing terminal sequences of these were used to prepare polyclonal antibodies, which were then used to prepare immunoadsorbents directed against the respective peptides. Sealed, right-side-out membrane vesicles containing native (Na{sup +} + K{sup +})-ATPase were labeledmore » with pyridoxal phosphate and sodium ({sup 3}H)borohydride in the absence or presence of saponin. The labeled {alpha}-polypeptide was isolated from these vesicles and digested with appropriate proteases. The incorporation of radioactivity into the peptides binding to the immunoadsorbent directed against the sequence pyrERXIVE increased 3-fold int the presence of saponin as a result of the increased accessibility of this portion of the protein to the reagent when the vesicles were breached by saponin; hence, this sequence is located on the cytoplasmic face of the membrane. It was inferred that the carboxy-terminal sequence -KETYY is on the extracytoplasmic face since the incorporation of radioactivity into peptides binding to the immunoadsorbent directed against the sequence -ETYY did not change when the vesicles were breached with saponin.« less

Anticipation measures of sequence learning: manual versus oculomotor versions of the serial reaction time task.

PubMed

Vakil, Eli; Bloch, Ayala; Cohen, Haggar

2017-03-01

The serial reaction time (SRT) task has generated a very large amount of research. Nevertheless the debate continues as to the exact cognitive processes underlying implicit sequence learning. Thus, the first goal of this study is to elucidate the underlying cognitive processes enabling sequence acquisition. We therefore compared reaction time (RT) in sequence learning in a standard manual activated (MA) to that in an ocular activated (OA) version of the task, within a single experimental setting. The second goal is to use eye movement measures to compare anticipation, as an additional indication of sequence learning, between the two versions of the SRT. Performance of the group given the MA version of the task (n = 29) was compared with that of the group given the OA version (n = 30). The results showed that although overall, RT was faster for the OA group, the rate of sequence learning was similar to that of the MA group performing the standard version of the SRT. Because the stimulus-response association is automatic and exists prior to training in the OA task, the decreased reaction time in this version of the task reflects a purer measure of the sequence learning that occurs in the SRT task. The results of this study show that eye tracking anticipation can be measured directly and can serve as a direct measure of sequence learning. Finally, using the OA version of the SRT to study sequence learning presents a significant methodological contribution by making sequence learning studies possible among populations that struggle to perform manual responses.

Asynchronous, Decentralized DS-CDMA Using Feedback-Controlled Spreading Sequences for Time-Dispersive Channels

NASA Astrophysics Data System (ADS)

Miyatake, Teruhiko; Chiba, Kazuki; Hamamura, Masanori; Tachikawa, Shin'ichi

We propose a novel asynchronous direct-sequence codedivision multiple access (DS-CDMA) using feedback-controlled spreading sequences (FCSSs) (FCSS/DS-CDMA). At the receiver of FCSS/DS-CDMA, the code-orthogonalizing filter (COF) produces a spreading sequence, and the receiver returns the spreading sequence to the transmitter. Then the transmitter uses the spreading sequence as its updated version. The performance of FCSS/DS-CDMA is evaluated over time-dispersive channels. The results indicate that FCSS/DS-CDMA greatly suppresses both the intersymbol interference (ISI) and multiple access interference (MAI) over time-invariant channels. FCSS/DS-CDMA is applicable to the decentralized multiple access.

BEAUTY: an enhanced BLAST-based search tool that integrates multiple biological information resources into sequence similarity search results.

PubMed

Worley, K C; Wiese, B A; Smith, R F

1995-09-01

BEAUTY (BLAST enhanced alignment utility) is an enhanced version of the NCBI's BLAST data base search tool that facilitates identification of the functions of matched sequences. We have created new data bases of conserved regions and functional domains for protein sequences in NCBI's Entrez data base, and BEAUTY allows this information to be incorporated directly into BLAST search results. A Conserved Regions Data Base, containing the locations of conserved regions within Entrez protein sequences, was constructed by (1) clustering the entire data base into families, (2) aligning each family using our PIMA multiple sequence alignment program, and (3) scanning the multiple alignments to locate the conserved regions within each aligned sequence. A separate Annotated Domains Data Base was constructed by extracting the locations of all annotated domains and sites from sequences represented in the Entrez, PROSITE, BLOCKS, and PRINTS data bases. BEAUTY performs a BLAST search of those Entrez sequences with conserved regions and/or annotated domains. BEAUTY then uses the information from the Conserved Regions and Annotated Domains data bases to generate, for each matched sequence, a schematic display that allows one to directly compare the relative locations of (1) the conserved regions, (2) annotated domains and sites, and (3) the locally aligned regions matched in the BLAST search. In addition, BEAUTY search results include World-Wide Web hypertext links to a number of external data bases that provide a variety of additional types of information on the function of matched sequences. This convenient integration of protein families, conserved regions, annotated domains, alignment displays, and World-Wide Web resources greatly enhances the biological informativeness of sequence similarity searches. BEAUTY searches can be performed remotely on our system using the "BCM Search Launcher" World-Wide Web pages (URL is < http:/ /gc.bcm.tmc.edu:8088/ search-launcher/launcher.html > ).

Structure-Function Analysis of Chloroplast Proteins via Random Mutagenesis Using Error-Prone PCR.

PubMed

Dumas, Louis; Zito, Francesca; Auroy, Pascaline; Johnson, Xenie; Peltier, Gilles; Alric, Jean

2018-06-01

Site-directed mutagenesis of chloroplast genes was developed three decades ago and has greatly advanced the field of photosynthesis research. Here, we describe a new approach for generating random chloroplast gene mutants that combines error-prone polymerase chain reaction of a gene of interest with chloroplast complementation of the knockout Chlamydomonas reinhardtii mutant. As a proof of concept, we targeted a 300-bp sequence of the petD gene that encodes subunit IV of the thylakoid membrane-bound cytochrome b 6 f complex. By sequencing chloroplast transformants, we revealed 149 mutations in the 300-bp target petD sequence that resulted in 92 amino acid substitutions in the 100-residue target subunit IV sequence. Our results show that this method is suited to the study of highly hydrophobic, multisubunit, and chloroplast-encoded proteins containing cofactors such as hemes, iron-sulfur clusters, and chlorophyll pigments. Moreover, we show that mutant screening and sequencing can be used to study photosynthetic mechanisms or to probe the mutational robustness of chloroplast-encoded proteins, and we propose that this method is a valuable tool for the directed evolution of enzymes in the chloroplast. © 2018 American Society of Plant Biologists. All rights reserved.

Turtle Graphics of Morphic Sequences

NASA Astrophysics Data System (ADS)

Zantema, Hans

2016-02-01

The simplest infinite sequences that are not ultimately periodic are pure morphic sequences: fixed points of particular morphisms mapping single symbols to strings of symbols. A basic way to visualize a sequence is by a turtle curve: for every alphabet symbol fix an angle, and then consecutively for all sequence elements draw a unit segment and turn the drawing direction by the corresponding angle. This paper investigates turtle curves of pure morphic sequences. In particular, criteria are given for turtle curves being finite (consisting of finitely many segments), and for being fractal or self-similar: it contains an up-scaled copy of itself. Also space-filling turtle curves are considered, and a turtle curve that is dense in the plane. As a particular result we give an exact relationship between the Koch curve and a turtle curve for the Thue-Morse sequence, where until now for such a result only approximations were known.

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

The World Health Organization Global Programme on AIDS proposal for standardization of HIV sequence nomenclature. WHO Network for HIV Isolation and Characterization.

PubMed

Korber, B T; Osmanov, S; Esparza, J; Myers, G

1994-11-01

The World Health Organization Global Programme on AIDS (WHO/GPA) is conducting a large-scale collaborative study of human immunodeficiency virus type 1 (HIV-1) variation, based in four potential vaccine-trial site countries: Brazil, Rwanda, Thailand, and Uganda. Through the course of this study, it was crucial to keep track of certain attributes of the samples from which the viral nucleotide sequences were derived (e.g., country of origin and viral culture characterization), so that meaningful sequence comparisons could be made. Here we describe a system developed in the context of the WHO/GPA study that summarizes such critical attributes by representing them as standardized characters directly incorporated into sequence names. This nomenclature allows linkage of clinical, phenotypic, and geographic information with molecular data. We propose that other investigators involved in human immunodeficiency virus (HIV) nucleotide sequencing efforts adopt a similar standardized sequence nomenclature to facilitate cross-study sequence comparison. HIV sequence data are being generated at an ever-increasing rate; directly coupled to this increase is our deepening understanding of biological parameters that influence or result from sequence variability. A standardized sequence nomenclature that includes relevant biological information would enable researchers to better utilize the growing body of sequence data, and enhance their ability to interpret the biological implications of their own data through facilitating comparisons with previously published work.

Human immunodeficiency virus type 1 LTR TATA and TAR region sequences required for transcriptional regulation.

PubMed Central

Garcia, J A; Harrich, D; Soultanakis, E; Wu, F; Mitsuyasu, R; Gaynor, R B

1989-01-01

The human immunodeficiency virus (HIV) type 1 LTR is regulated at the transcriptional level by both cellular and viral proteins. Using HeLa cell extracts, multiple regions of the HIV LTR were found to serve as binding sites for cellular proteins. An untranslated region binding protein UBP-1 has been purified and fractions containing this protein bind to both the TAR and TATA regions. To investigate the role of cellular proteins binding to both the TATA and TAR regions and their potential interaction with other HIV DNA binding proteins, oligonucleotide-directed mutagenesis of both these regions was performed followed by DNase I footprinting and transient expression assays. In the TATA region, two direct repeats TC/AAGC/AT/AGCTGC surround the TATA sequence. Mutagenesis of both of these direct repeats or of the TATA sequence interrupted binding over the TATA region on the coding strand, but only a mutation of the TATA sequence affected in vivo assays for tat-activation. In addition to TAR serving as the site of binding of cellular proteins, RNA transcribed from TAR is capable of forming a stable stem-loop structure. To determine the relative importance of DNA binding proteins as compared to secondary structure, oligonucleotide-directed mutations in the TAR region were studied. Local mutations that disrupted either the stem or loop structure were defective in gene expression. However, compensatory mutations which restored base pairing in the stem resulted in complete tat-activation. This indicated a significant role for the stem-loop structure in HIV gene expression. To determine the role of TAR binding proteins, mutations were constructed which extensively changed the primary structure of the TAR region, yet left stem base pairing, stem energy and the loop sequence intact. These mutations resulted in decreased protein binding to TAR DNA and defects in tat-activation, and revealed factor binding specifically to the loop DNA sequence. Further mutagenesis which inverted this stem and loop mutation relative to the HIV LTR mRNA start site resulted in even larger decreases in tat-activation. This suggests that multiple determinants, including protein binding, the loop sequence, and RNA or DNA secondary structure, are important in tat-activation and suggests that tat may interact with cellular proteins binding to DNA to increase HIV gene expression. Images PMID:2721501

Breaking Lander-Waterman’s Coverage Bound

PubMed Central

Nashta-ali, Damoun; Motahari, Seyed Abolfazl; Hosseinkhalaj, Babak

2016-01-01

Lander-Waterman’s coverage bound establishes the total number of reads required to cover the whole genome of size G bases. In fact, their bound is a direct consequence of the well-known solution to the coupon collector’s problem which proves that for such genome, the total number of bases to be sequenced should be O(G ln G). Although the result leads to a tight bound, it is based on a tacit assumption that the set of reads are first collected through a sequencing process and then are processed through a computation process, i.e., there are two different machines: one for sequencing and one for processing. In this paper, we present a significant improvement compared to Lander-Waterman’s result and prove that by combining the sequencing and computing processes, one can re-sequence the whole genome with as low as O(G) sequenced bases in total. Our approach also dramatically reduces the required computational power for the combined process. Simulation results are performed on real genomes with different sequencing error rates. The results support our theory predicting the log G improvement on coverage bound and corresponding reduction in the total number of bases required to be sequenced. PMID:27806058

Sequence analysis of dolphin ferritin H and L subunits and possible iron-dependent translational control of dolphin ferritin gene

PubMed Central

Takaesu, Azusa; Watanabe, Kiyotaka; Takai, Shinji; Sasaki, Yukako; Orino, Koichi

2008-01-01

Background Iron-storage protein, ferritin plays a central role in iron metabolism. Ferritin has dual function to store iron and segregate iron for protection of iron-catalyzed reactive oxygen species. Tissue ferritin is composed of two kinds of subunits (H: heavy chain or heart-type subunit; L: light chain or liver-type subunit). Ferritin gene expression is controlled at translational level in iron-dependent manner or at transcriptional level in iron-independent manner. However, sequencing analysis of marine mammalian ferritin subunits has not yet been performed fully. The purpose of this study is to reveal cDNA-derived amino acid sequences of cetacean ferritin H and L subunits, and demonstrate the possibility of expression of these subunits, especially H subunit, by iron. Methods Sequence analyses of cetacean ferritin H and L subunits were performed by direct sequencing of polymerase chain reaction (PCR) fragments from cDNAs generated via reverse transcription-PCR of leukocyte total RNA prepared from blood samples of six different dolphin species (Pseudorca crassidens, Lagenorhynchus obliquidens, Grampus griseus, Globicephala macrorhynchus, Tursiops truncatus, and Delphinapterus leucas). The putative iron-responsive element sequence in the 5'-untranslated region of the six different dolphin species was revealed by direct sequencing of PCR fragments obtained using leukocyte genomic DNA. Results Dolphin H and L subunits consist of 182 and 174 amino acids, respectively, and amino acid sequence identities of ferritin subunits among these dolphins are highly conserved (H: 99–100%, (99→98) ; L: 98–100%). The conserved 28 bp IRE sequence was located -144 bp upstream from the initiation codon in the six different dolphin species. Conclusion These results indicate that six different dolphin species have conserved ferritin sequences, and suggest that these genes are iron-dependently expressed. PMID:18954429

Transposon Tn10 contains two structural genes with opposite polarity between tetA and IS10R.

PubMed Central

Schollmeier, K; Hillen, W

1984-01-01

The nucleotide sequence of the central part of Tn10 has been determined from the rightmost HindIII site to IS10R. This sequence contains two open reading frames with opposite polarity. The in vivo transcription start points in this sequence have been determined by S1 mapping. These results define one minor and two major promoters. The transcription starts of the two major promoters are only 18 base pairs apart, and the transcripts show different polarity and overlap by 18 base pairs. The nucleotide sequence reveals two regions with palindromic symmetry which may serve as operators. Their possible involvement in the regulation of transcription of both genes is discussed. Taken together these results allow for a maximal coding capacity of 138 amino acids directed toward IS10R and 197 amino acids directed toward tetA. The possible function of these gene products is discussed. The accompanying article (Braus et al., J. Bacteriol. 160:504-509, 1984) presents evidence that these genes are expressed. Images PMID:6094471

PMS2 gene mutational analysis: direct cDNA sequencing to circumvent pseudogene interference.

PubMed

Wimmer, Katharina; Wernstedt, Annekatrin

2014-01-01

The presence of highly homologous pseudocopies can compromise the mutation analysis of a gene of interest. In particular, when using PCR-based strategies, pseudogene co-amplification has to be effectively prevented. This is often achieved by using primers designed to be parental gene specific according to the reference sequence and by applying stringent PCR conditions. However, there are cases in which this approach is of limited utility. For example, it has been shown that the PMS2 gene exchanges sequences with one of its pseudogenes, named PMS2CL. This results in functional PMS2 alleles containing pseudogene-derived sequences at their 3'-end and in nonfunctional PMS2CL pseudogene alleles that contain gene-derived sequences. Hence, the paralogues cannot be distinguished according to the reference sequence. This shortcoming can be effectively circumvented by using direct cDNA sequencing. This approach is based on the selective amplification of PMS2 transcripts in two overlapping 1.6-kb RT-PCR products. In addition to avoiding pseudogene co-amplification and allele dropout, this method has also the advantage that it allows to effectively identify deletions, splice mutations, and de novo retrotransposon insertions that escape the detection of most DNA-based mutation analysis protocols.

The complete chloroplast genome sequence of Gossypium hirsutum: organization and phylogenetic relationships to other angiosperms

PubMed Central

Lee, Seung-Bum; Kaittanis, Charalambos; Jansen, Robert K; Hostetler, Jessica B; Tallon, Luke J; Town, Christopher D; Daniell, Henry

2006-01-01

Background Cotton (Gossypium hirsutum) is the most important fiber crop grown in 90 countries. In 2004–2005, US farmers planted 79% of the 5.7-million hectares of nuclear transgenic cotton. Unfortunately, genetically modified cotton has the potential to hybridize with other cultivated and wild relatives, resulting in geographical restrictions to cultivation. However, chloroplast genetic engineering offers the possibility of containment because of maternal inheritance of transgenes. The complete chloroplast genome of cotton provides essential information required for genetic engineering. In addition, the sequence data were used to assess phylogenetic relationships among the major clades of rosids using cotton and 25 other completely sequenced angiosperm chloroplast genomes. Results The complete cotton chloroplast genome is 160,301 bp in length, with 112 unique genes and 19 duplicated genes within the IR, containing a total of 131 genes. There are four ribosomal RNAs, 30 distinct tRNA genes and 17 intron-containing genes. The gene order in cotton is identical to that of tobacco but lacks rpl22 and infA. There are 30 direct and 24 inverted repeats 30 bp or longer with a sequence identity ≥ 90%. Most of the direct repeats are within intergenic spacer regions, introns and a 72 bp-long direct repeat is within the psaA and psaB genes. Comparison of protein coding sequences with expressed sequence tags (ESTs) revealed nucleotide substitutions resulting in amino acid changes in ndhC, rpl23, rpl20, rps3 and clpP. Phylogenetic analysis of a data set including 61 protein-coding genes using both maximum likelihood and maximum parsimony were performed for 28 taxa, including cotton and five other angiosperm chloroplast genomes that were not included in any previous phylogenies. Conclusion Cotton chloroplast genome lacks rpl22 and infA and contains a number of dispersed direct and inverted repeats. RNA editing resulted in amino acid changes with significant impact on their hydropathy. Phylogenetic analysis provides strong support for the position of cotton in the Malvales in the eurosids II clade sister to Arabidopsis in the Brassicales. Furthermore, there is strong support for the placement of the Myrtales sister to the eurosid I clade, although expanded taxon sampling is needed to further test this relationship. PMID:16553962

Identification of presumed ancestral DNA sequences of phaseolin in Phaseolus vulgaris.

PubMed Central

Kami, J; Velásquez, V B; Debouck, D G; Gepts, P

1995-01-01

Common bean (Phaseolus vulgaris) consists of two major geographic gene pools, one distributed in Mexico, Central America, and Colombia and the other in the southern Andes (southern Peru, Bolivia, and Argentina). Amplification and sequencing of members of the multigene family coding for phaseolin, the major seed storage protein of the common bean, provide evidence for accumulation of tandem direct repeats in both introns and exons during evolution of the multigene family in this species. The presumed ancestral phaseolin sequences, without tandem repeats, were found in recently discovered but nearly extinct wild common bean populations of Ecuador and northern Peru that are intermediate between the two major gene pools of the species based on geographical and molecular arguments. Our results illustrate the usefulness of tandem direct repeats in establishing the polarity of DNA sequence divergence and therefore in proposing phylogenies. Images Fig. 1 Fig. 3 PMID:7862642

A paleomagnetic and paleointensity study on Pleistocene and Pliocene basaltic flows from the Djavakheti Highland (Southern Georgia, Caucasus)

NASA Astrophysics Data System (ADS)

Calvo-Rathert, Manuel; Goguitchaichvili, Avto; Bógalo, María-Felicidad; Vegas-Tubía, Néstor; Carrancho, Ángel; Sologashvili, Jemal

2011-08-01

New paleomagnetic, rock-magnetic and paleointensity results obtained on samples from 23 basaltic lava flows belonging to four different flow sequences (Mashavera, Kvemo Orozmani, Zemo Karabulaki and Diliska) of Pleistocene and Pliocene age from the eastern Djavakheti Highland, in southern Georgia, are presented. Radiometric dating of these sequences yields ages between 1.8 and 2.18 Ma for Mashavera, 2.07 and 2.58 Ma for Zemo-Karabulakhi and 2.12 and 3.27 for Diliska. No radiometric ages are available for the Kvemo Orozmani sequence, which is considered to be coeval to the Mashavera sequence. Rock-magnetic experiments including measurement of thermomagnetic, hysteresis and IRM-acquisition curves suggest low-Ti titanomagnetite as main carrier of remanence, although a lower Curie-temperature component was also observed in several cases. Reversible and non-reversible curves were recorded in thermomagnetic experiments. Paleomagnetic analysis generally indicated the presence of a single component (mainly in the Mashavera sequence), but also two more or less superimposed components in some other cases. In 21 sites a characteristic component could be determined and all except one were characterised by normal-polarity directions. Flows from the Mashavera sequence had a rather steep inclination (73.1°). Nevertheless, a mean paleomagnetic direction of all four sequences is obtained ( D = 8.5°, I = 60.8°, N = 4, α95 = 11.7°, k = 62.7) which agrees with the Plio-Quaternary directions obtained in previous studies in Georgia. The paleomagnetic pole obtained (latitude ϕ = 82.1°, longitude λ = 118.2°, A95 = 8.0°, k = 240.7) agrees with the pole values of both the 0 Ma and the 5 Ma windows of the synthetic Eurasian polar wander path from Besse and Courtillot (2002). In order to analyse the behaviour of secular variation, the scatter of paleosecular variation of virtual geomagnetic poles of both the Mashavera flow and all 18 studied flows of Pleistocene age was calculated. It could be observed that both data-sets seem to fit well the expected scatter at latitude 41°N. Paleointensity experiments were carried out with the Coe modification of the Thellier method. Twenty-five out of 84 samples (30%) provided reliable paleointensity results. These successful results were mainly obtained in the Mashavera sequence. Most flows yielded paleointensity results in the 30-45 μT range, in accordance with expected Pliocene to present day intensities. Two flows, however, located near the top of the Mashavera sequence yield high paleointensity values around 60 μT. Anomalous paleointensity results in the upper-lying Mashavera flows together with the steep inclinations observed in that sequence, could perhaps signal the near onset of the Olduvai-Matuyama reversal.

A teaching-learning sequence about weather map reading

NASA Astrophysics Data System (ADS)

Mandrikas, Achilleas; Stavrou, Dimitrios; Skordoulis, Constantine

2017-07-01

In this paper a teaching-learning sequence (TLS) introducing pre-service elementary teachers (PET) to weather map reading, with emphasis on wind assignment, is presented. The TLS includes activities about recognition of wind symbols, assignment of wind direction and wind speed on a weather map and identification of wind characteristics in a weather forecast. Sixty PET capabilities and difficulties in understanding weather maps were investigated, using inquiry-based learning activities. The results show that most PET became more capable of reading weather maps and assigning wind direction and speed on them. Our results also show that PET could be guided to understand meteorology concepts useful in everyday life and in teaching their future students.

Integrated circuit layer image segmentation

NASA Astrophysics Data System (ADS)

Masalskis, Giedrius; Petrauskas, Romas

2010-09-01

In this paper we present IC layer image segmentation techniques which are specifically created for precise metal layer feature extraction. During our research we used many samples of real-life de-processed IC metal layer images which were obtained using optical light microscope. We have created sequence of various image processing filters which provides segmentation results of good enough precision for our application. Filter sequences were fine tuned to provide best possible results depending on properties of IC manufacturing process and imaging technology. Proposed IC image segmentation filter sequences were experimentally tested and compared with conventional direct segmentation algorithms.

Representations of mechanical assembly sequences

NASA Technical Reports Server (NTRS)

Homem De Mello, Luiz S.; Sanderson, Arthur C.

1991-01-01

Five types of representations for assembly sequences are reviewed: the directed graph of feasible assembly sequences, the AND/OR graph of feasible assembly sequences, the set of establishment conditions, and two types of sets of precedence relationships. (precedence relationships between the establishment of one connection between parts and the establishment of another connection, and precedence relationships between the establishment of one connection and states of the assembly process). The mappings of one representation into the others are established. The correctness and completeness of these representations are established. The results presented are needed in the proof of correctness and completeness of algorithms for the generation of mechanical assembly sequences.

Integrated Georeferencing of Stereo Image Sequences Captured with a Stereovision Mobile Mapping System - Approaches and Practical Results

NASA Astrophysics Data System (ADS)

Eugster, H.; Huber, F.; Nebiker, S.; Gisi, A.

2012-07-01

Stereovision based mobile mapping systems enable the efficient capturing of directly georeferenced stereo pairs. With today's camera and onboard storage technologies imagery can be captured at high data rates resulting in dense stereo sequences. These georeferenced stereo sequences provide a highly detailed and accurate digital representation of the roadside environment which builds the foundation for a wide range of 3d mapping applications and image-based geo web-services. Georeferenced stereo images are ideally suited for the 3d mapping of street furniture and visible infrastructure objects, pavement inspection, asset management tasks or image based change detection. As in most mobile mapping systems, the georeferencing of the mapping sensors and observations - in our case of the imaging sensors - normally relies on direct georeferencing based on INS/GNSS navigation sensors. However, in urban canyons the achievable direct georeferencing accuracy of the dynamically captured stereo image sequences is often insufficient or at least degraded. Furthermore, many of the mentioned application scenarios require homogeneous georeferencing accuracy within a local reference frame over the entire mapping perimeter. To achieve these demands georeferencing approaches are presented and cost efficient workflows are discussed which allows validating and updating the INS/GNSS based trajectory with independently estimated positions in cases of prolonged GNSS signal outages in order to increase the georeferencing accuracy up to the project requirements.

Local alignment of two-base encoded DNA sequence

PubMed Central

Homer, Nils; Merriman, Barry; Nelson, Stanley F

2009-01-01

Background DNA sequence comparison is based on optimal local alignment of two sequences using a similarity score. However, some new DNA sequencing technologies do not directly measure the base sequence, but rather an encoded form, such as the two-base encoding considered here. In order to compare such data to a reference sequence, the data must be decoded into sequence. The decoding is deterministic, but the possibility of measurement errors requires searching among all possible error modes and resulting alignments to achieve an optimal balance of fewer errors versus greater sequence similarity. Results We present an extension of the standard dynamic programming method for local alignment, which simultaneously decodes the data and performs the alignment, maximizing a similarity score based on a weighted combination of errors and edits, and allowing an affine gap penalty. We also present simulations that demonstrate the performance characteristics of our two base encoded alignment method and contrast those with standard DNA sequence alignment under the same conditions. Conclusion The new local alignment algorithm for two-base encoded data has substantial power to properly detect and correct measurement errors while identifying underlying sequence variants, and facilitating genome re-sequencing efforts based on this form of sequence data. PMID:19508732

Real-time film recording from stroke-written CRT's

NASA Technical Reports Server (NTRS)

Hunt, R.; Grunwald, A. J.

1980-01-01

Real-time simulation studies often require motion-picture recording of events directly from stroke written cathode-ray tubes (CRT's). Difficulty presented is prevention of "flicker," which results from lack of synchronization between display sequence on CRT and shutter motion of camera. Programmable method has been devised for phasing display sequence to shutter motion, ensuring flicker-free recordings.

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

Sequencing actions: an information-search study of tradeoffs of priorities against spatiotemporal constraints.

PubMed

Gärling, T

1996-09-01

How people choose between sequences of actions was investigated in an everyday errand-planning task. In this task subjects chose the preferred sequence of performing a number of errands in a fictitious environment. Two experiments were conducted with undergraduate students serving as subjects. One group searched information about each alternative. The same information was directly available to another group. In Experiment 1 the results showed that for two errands subjects took into account all attributes describing the errands, thus suggesting a tradeoff between priority, wait time, and travel distance with priority being the most important. Consistent with this finding predominantly intraalternative information search was observed. These results were replicated in Experiment 2 for three errands. In addition choice outcomes, information search, and sequence of responding suggested that for more than two actions sequence choices are made in stages.

Face processing regions are sensitive to distinct aspects of temporal sequence in facial dynamics.

PubMed

Reinl, Maren; Bartels, Andreas

2014-11-15

Facial movement conveys important information for social interactions, yet its neural processing is poorly understood. Computational models propose that shape- and temporal sequence sensitive mechanisms interact in processing dynamic faces. While face processing regions are known to respond to facial movement, their sensitivity to particular temporal sequences has barely been studied. Here we used fMRI to examine the sensitivity of human face-processing regions to two aspects of directionality in facial movement trajectories. We presented genuine movie recordings of increasing and decreasing fear expressions, each of which were played in natural or reversed frame order. This two-by-two factorial design matched low-level visual properties, static content and motion energy within each factor, emotion-direction (increasing or decreasing emotion) and timeline (natural versus artificial). The results showed sensitivity for emotion-direction in FFA, which was timeline-dependent as it only occurred within the natural frame order, and sensitivity to timeline in the STS, which was emotion-direction-dependent as it only occurred for decreased fear. The occipital face area (OFA) was sensitive to the factor timeline. These findings reveal interacting temporal sequence sensitive mechanisms that are responsive to both ecological meaning and to prototypical unfolding of facial dynamics. These mechanisms are temporally directional, provide socially relevant information regarding emotional state or naturalness of behavior, and agree with predictions from modeling and predictive coding theory. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Sequence differences in the diagnostic region of the cysteine protease 8 gene of Tritrichomonas foetus parasites of cats and cattle.

PubMed

Sun, Zichen; Stack, Colin; Šlapeta, Jan

2012-05-25

In order to investigate the genetic variation between Tritrichomonas foetus from bovine and feline origins, cysteine protease 8 (CP8) coding sequence was selected as the polymorphic DNA marker. Direct sequencing of CP8 coding sequence of T. foetus from four feline isolates and two bovine isolates with polymerase chain reaction successfully revealed conserved nucleotide polymorphisms between feline and bovine isolates. These results provide useful information for CP8-based molecular differentiation of T. foetus genotypes. Copyright © 2011 Elsevier B.V. All rights reserved.

Direct repeat sequences are essential for function of the cis-acting locus of transfer (clt) of Streptomyces phaeochromogenes plasmid pJV1.

PubMed

Franco, Bernardo; González-Cerón, Gabriela; Servín-González, Luis

2003-11-01

The functionality of direct and inverted repeat sequences inside the cis acting locus of transfer (clt) of the Streptomyces plasmid pJV1 was determined by testing the effect of different deletions on plasmid transfer. The results show that the single most important element for pJV1 clt function is a series of evenly spaced 9 bp long direct repeats which match the consensus CCGCACA(C/G)(C/G), since their deletion caused a dramatic reduction in plasmid transfer. The presence of these repeats in the absence of any other clt sequences allowed plasmid transfer to occur at a frequency that was at least two orders of magnitude higher than that obtained in the complete absence of clt. A database search revealed regions with a similar organization, and in the same position, in Streptomyces plasmids pSN22 and pSLS, which have transfer proteins homologous to those of pJV1.

In situ multi-axial loading frame to probe elastomers using X-ray scattering.

PubMed

Pannier, Yannick; Proudhon, Henry; Mocuta, Cristian; Thiaudière, Dominique; Cantournet, Sabine

2011-11-01

An in situ tensile-shear loading device has been designed to study elastomer crystallization using synchrotron X-ray scattering at the Synchrotron Soleil on the DiffAbs beamline. Elastomer tape specimens of thickness 2 mm can be elongated by up to 500% in the longitudinal direction and sheared by up to 200% in the transverse direction. The device is fully automated and plugged into the TANGO control system of the beamline allowing synchronization between acquisition and loading sequences. Experimental results revealing the evolution of crystallization peaks under load are presented for several tension/shear loading sequences.

Microscopic models for bridging electrostatics and currents

NASA Astrophysics Data System (ADS)

Borghi, L.; DeAmbrosis, A.; Mascheretti, P.

2007-03-01

A teaching sequence based on the use of microscopic models to link electrostatic phenomena with direct currents is presented. The sequence, devised for high school students, was designed after initial work carried out with student teachers attending a school of specialization for teaching physics at high school, at the University of Pavia. The results obtained with them are briefly presented, because they directed our steps for the development of the teaching sequence. For both the design of the experiments and their interpretation, we drew inspiration from the original works of Alessandro Volta; in addition, a structural model based on the particular role of electrons as elementary charges both in electrostatic phenomena and in currents was proposed. The teaching sequence starts from experiments on charging objects by rubbing and by induction, and engages students in constructing microscopic models to interpret their observations. By using these models and by closely examining the ideas of tension and capacitance, the students acknowledge that a charging (or discharging) process is due to the motion of electrons that, albeit for short time intervals, represent a current. Finally, they are made to see that the same happens in transients of direct current circuits.

Colony-PCR Is a Rapid Method for DNA Amplification of Hyphomycetes

PubMed Central

Walch, Georg; Knapp, Maria; Rainer, Georg; Peintner, Ursula

2016-01-01

Fungal pure cultures identified with both classical morphological methods and through barcoding sequences are a basic requirement for reliable reference sequences in public databases. Improved techniques for an accelerated DNA barcode reference library construction will result in considerably improved sequence databases covering a wider taxonomic range. Fast, cheap, and reliable methods for obtaining DNA sequences from fungal isolates are, therefore, a valuable tool for the scientific community. Direct colony PCR was already successfully established for yeasts, but has not been evaluated for a wide range of anamorphic soil fungi up to now, and a direct amplification protocol for hyphomycetes without tissue pre-treatment has not been published so far. Here, we present a colony PCR technique directly from fungal hyphae without previous DNA extraction or other prior manipulation. Seven hundred eighty-eight fungal strains from 48 genera were tested with a success rate of 86%. PCR success varied considerably: DNA of fungi belonging to the genera Cladosporium, Geomyces, Fusarium, and Mortierella could be amplified with high success. DNA of soil-borne yeasts was always successfully amplified. Absidia, Mucor, Trichoderma, and Penicillium isolates had noticeably lower PCR success. PMID:29376929

Iterative pass optimization of sequence data

NASA Technical Reports Server (NTRS)

Wheeler, Ward C.

2003-01-01

The problem of determining the minimum-cost hypothetical ancestral sequences for a given cladogram is known to be NP-complete. This "tree alignment" problem has motivated the considerable effort placed in multiple sequence alignment procedures. Wheeler in 1996 proposed a heuristic method, direct optimization, to calculate cladogram costs without the intervention of multiple sequence alignment. This method, though more efficient in time and more effective in cladogram length than many alignment-based procedures, greedily optimizes nodes based on descendent information only. In their proposal of an exact multiple alignment solution, Sankoff et al. in 1976 described a heuristic procedure--the iterative improvement method--to create alignments at internal nodes by solving a series of median problems. The combination of a three-sequence direct optimization with iterative improvement and a branch-length-based cladogram cost procedure, provides an algorithm that frequently results in superior (i.e., lower) cladogram costs. This iterative pass optimization is both computation and memory intensive, but economies can be made to reduce this burden. An example in arthropod systematics is discussed. c2003 The Willi Hennig Society. Published by Elsevier Science (USA). All rights reserved.

How should Fitts' Law be applied to human-computer interaction?

NASA Technical Reports Server (NTRS)

Gillan, D. J.; Holden, K.; Adam, S.; Rudisill, M.; Magee, L.

1992-01-01

The paper challenges the notion that any Fitts' Law model can be applied generally to human-computer interaction, and proposes instead that applying Fitts' Law requires knowledge of the users' sequence of movements, direction of movement, and typical movement amplitudes as well as target sizes. Two experiments examined a text selection task with sequences of controlled movements (point-click and point-drag). For the point-click sequence, a Fitts' Law model that used the diagonal across the text object in the direction of pointing (rather than the horizontal extent of the text object) as the target size provided the best fit for the pointing time data, whereas for the point-drag sequence, a Fitts' Law model that used the vertical size of the text object as the target size gave the best fit. Dragging times were fitted well by Fitts' Law models that used either the vertical or horizontal size of the terminal character in the text object. Additional results of note were that pointing in the point-click sequence was consistently faster than in the point-drag sequence, and that pointing in either sequence was consistently faster than dragging. The discussion centres around the need to define task characteristics before applying Fitts' Law to an interface design or analysis, analyses of pointing and of dragging, and implications for interface design.

Stress Drop and Directivity Patterns Observed in Small-Magnitude (

NASA Astrophysics Data System (ADS)

Ruhl, C. J.; Hatch, R. L.; Abercrombie, R. E.; Smith, K.

2017-12-01

Recent improvements in seismic instrumentation and network coverage in the Reno, NV area have provided high-quality records of abundant microseismicity, including several swarms and clusters. Here, we discuss stress drop and directivity patterns of small-magnitude seismicity in the 2008 Mw4.9 Mogul earthquake swarm in Reno, NV and in the nearby region of an ML3.2 sequence near Virginia City, NV. In both sequences, double-difference relocated earthquakes cluster on multiple distinct structures consistent with focal mechanism and moment tensor fault plane solutions. Both sequences also show migration potentially related to fluid flow. We estimate corner frequency and stress drop using EGF-derived spectral ratios, convolving earthquake pairs (target*EGF) such that we preserve phase and recover source-time functions (STF) on a station-by-station basis. We then stack individual STFs per station for all EGF-target pairs per target earthquake, increasing the signal-to-noise of our results. By applying an azimuthal- and incidence-angle-dependent stretching factor to STFs in the time domain, we are able to invert for rupture directivity and velocity assuming both unilateral and bilateral rupture. Earthquakes in both sequences, some as low as ML2.1, show strong unilateral directivity consistent with independent fault plane solutions. We investigate and compare the relationship between rupture and migration directions on subfaults within each sequence. Average stress drops for both sequences are 4 MPa, but there is large variation in individual estimates for both sequences. Although this variation is not explained simply by any one parameter (e.g., depth), spatiotemporal variation in the Mogul swarm is distinct: coherent clusters of high and low stress drop earthquakes along the mainshock fault plane are seen, and high-stress-drop foreshocks correlate with an area of reduced aftershock productivity. These observations are best explained by a difference in rheology along the fault plane. The unprecedented detail achieved for these small magnitude earthquakes confirms that stress drop, when measured precisely, is a valuable observation of physically-meaningful fault zone properties and earthquake behavior.

Next-generation sequencing library preparation method for identification of RNA viruses on the Ion Torrent Sequencing Platform.

PubMed

Chen, Guiqian; Qiu, Yuan; Zhuang, Qingye; Wang, Suchun; Wang, Tong; Chen, Jiming; Wang, Kaicheng

2018-05-09

Next generation sequencing (NGS) is a powerful tool for the characterization, discovery, and molecular identification of RNA viruses. There were multiple NGS library preparation methods published for strand-specific RNA-seq, but some methods are not suitable for identifying and characterizing RNA viruses. In this study, we report a NGS library preparation method to identify RNA viruses using the Ion Torrent PGM platform. The NGS sequencing adapters were directly inserted into the sequencing library through reverse transcription and polymerase chain reaction, without fragmentation and ligation of nucleic acids. The results show that this method is simple to perform, able to identify multiple species of RNA viruses in clinical samples.

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).

The VirusBanker database uses a Java program to allow flexible searching through Bunyaviridae sequences

PubMed Central

Fourment, Mathieu; Gibbs, Mark J

2008-01-01

Background Viruses of the Bunyaviridae have segmented negative-stranded RNA genomes and several of them cause significant disease. Many partial sequences have been obtained from the segments so that GenBank searches give complex results. Sequence databases usually use HTML pages to mediate remote sorting, but this approach can be limiting and may discourage a user from exploring a database. Results The VirusBanker database contains Bunyaviridae sequences and alignments and is presented as two spreadsheets generated by a Java program that interacts with a MySQL database on a server. Sequences are displayed in rows and may be sorted using information that is displayed in columns and includes data relating to the segment, gene, protein, species, strain, sequence length, terminal sequence and date and country of isolation. Bunyaviridae sequences and alignments may be downloaded from the second spreadsheet with titles defined by the user from the columns, or viewed when passed directly to the sequence editor, Jalview. Conclusion VirusBanker allows large datasets of aligned nucleotide and protein sequences from the Bunyaviridae to be compiled and winnowed rapidly using criteria that are formulated heuristically. PMID:18251994

Short communication: novel truncating mutations in the CFTR gene causing a severe form of cystic fibrosis in Italian patients.

PubMed

Lenarduzzi, S; Morgutti, M; Crovella, S; Coiana, A; Rosatelli, M C

2014-11-14

Cystic fibrosis (CF) is a common recessive genetic disease caused by mutations in the gene encoding for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. More than 1800 different mutations have been described to date. Here, we report 3 novel mutations in CFTR in 3 Italian CF patients. To detect and identify 36 frequent mutations in Caucasians, we used the INNO-LiPA CFTR19 and INNO-LiPA CFTR17+Tn Update kits (Innogenetics; Ghent, Belgium). Our first analysis did not reveal both of the responsible mutations; thus, direct sequencing of the CFTR gene coding region was performed. The 3 patients were compound heterozygous. In one allele, the F508del (c.1521_1523delCTT, p.PHE508del) mutation in exon 11 was observed in each case. For the second allele, in patient No.1, direct sequencing revealed an 11-base pair deletion (GAGGCGATACT) in exon 14 (c.2236_2246del; pGlu746Alafs*29). In patient No. 2, direct sequencing revealed a nonsense mutation at nucleotide 3892 (c.3892G>T) in exon 24. In patient No. 3, direct sequencing revealed a deletion of cytosine in exon 27 (c.4296delC; p.Asn1432Lysfs*16). These 3 novel mutations indicate the production of a truncated protein, which consequently results in a non-functional polypeptide.

MicroRNA-200c Modulates the Expression of MUC4 and MUC16 by Directly Targeting Their Coding Sequences in Human Pancreatic Cancer

PubMed Central

Radhakrishnan, Prakash; Mohr, Ashley M.; Grandgenett, Paul M.; Steele, Maria M.; Batra, Surinder K.; Hollingsworth, Michael A.

2013-01-01

Transmembrane mucins, MUC4 and MUC16 are associated with tumor progression and metastatic potential in human pancreatic adenocarcinoma. We discovered that miR-200c interacts with specific sequences within the coding sequence of MUC4 and MUC16 mRNAs, and evaluated the regulatory nature of this association. Pancreatic cancer cell lines S2.028 and T3M-4 transfected with miR-200c showed a 4.18 and 8.50 fold down regulation of MUC4 mRNA, and 4.68 and 4.82 fold down regulation of MUC16 mRNA compared to mock-transfected cells, respectively. A significant reduction of glycoprotein expression was also observed. These results indicate that miR-200c overexpression regulates MUC4 and MUC16 mucins in pancreatic cancer cells by directly targeting the mRNA coding sequence of each, resulting in reduced levels of MUC4 and MUC16 mRNA and protein. These data suggest that, in addition to regulating proteins that modulate EMT, miR-200c influences expression of cell surface mucins in pancreatic cancer. PMID:24204560

MicroRNA-200c modulates the expression of MUC4 and MUC16 by directly targeting their coding sequences in human pancreatic cancer.

PubMed

Radhakrishnan, Prakash; Mohr, Ashley M; Grandgenett, Paul M; Steele, Maria M; Batra, Surinder K; Hollingsworth, Michael A

2013-01-01

Transmembrane mucins, MUC4 and MUC16 are associated with tumor progression and metastatic potential in human pancreatic adenocarcinoma. We discovered that miR-200c interacts with specific sequences within the coding sequence of MUC4 and MUC16 mRNAs, and evaluated the regulatory nature of this association. Pancreatic cancer cell lines S2.028 and T3M-4 transfected with miR-200c showed a 4.18 and 8.50 fold down regulation of MUC4 mRNA, and 4.68 and 4.82 fold down regulation of MUC16 mRNA compared to mock-transfected cells, respectively. A significant reduction of glycoprotein expression was also observed. These results indicate that miR-200c overexpression regulates MUC4 and MUC16 mucins in pancreatic cancer cells by directly targeting the mRNA coding sequence of each, resulting in reduced levels of MUC4 and MUC16 mRNA and protein. These data suggest that, in addition to regulating proteins that modulate EMT, miR-200c influences expression of cell surface mucins in pancreatic cancer.

Verona Coding Definitions of Emotional Sequences (VR-CoDES): Conceptual framework and future directions.

PubMed

Piccolo, Lidia Del; Finset, Arnstein; Mellblom, Anneli V; Figueiredo-Braga, Margarida; Korsvold, Live; Zhou, Yuefang; Zimmermann, Christa; Humphris, Gerald

2017-12-01

To discuss the theoretical and empirical framework of VR-CoDES and potential future direction in research based on the coding system. The paper is based on selective review of papers relevant to the construction and application of VR-CoDES. VR-CoDES system is rooted in patient-centered and biopsychosocial model of healthcare consultations and on a functional approach to emotion theory. According to the VR-CoDES, emotional interaction is studied in terms of sequences consisting of an eliciting event, an emotional expression by the patient and the immediate response by the clinician. The rationale for the emphasis on sequences, on detailed classification of cues and concerns, and on the choices of explicit vs. non-explicit responses and providing vs. reducing room for further disclosure, as basic categories of the clinician responses, is described. Results from research on VR-CoDES may help raise awareness of emotional sequences. Future directions in applying VR-CoDES in research may include studies on predicting patient and clinician behavior within the consultation, qualitative analyses of longer sequences including several VR-CoDES triads, and studies of effects of emotional communication on health outcomes. VR-CoDES may be applied to develop interventions to promote good handling of patients' emotions in healthcare encounters. Copyright © 2017 Elsevier B.V. All rights reserved.

An Internal Signal Sequence Directs Intramembrane Proteolysis of a Cellular Immunoglobulin Domain Protein*S⃞

PubMed Central

Robakis, Thalia; Bak, Beata; Lin, Shu-huei; Bernard, Daniel J.; Scheiffele, Peter

2008-01-01

Precursor proteolysis is a crucial mechanism for regulating protein structure and function. Signal peptidase (SP) is an enzyme with a well defined role in cleaving N-terminal signal sequences but no demonstrated function in the proteolysis of cellular precursor proteins. We provide evidence that SP mediates intraprotein cleavage of IgSF1, a large cellular Ig domain protein that is processed into two separate Ig domain proteins. In addition, our results suggest the involvement of signal peptide peptidase (SPP), an intramembrane protease, which acts on substrates that have been previously cleaved by SP. We show that IgSF1 is processed through sequential proteolysis by SP and SPP. Cleavage is directed by an internal signal sequence and generates two separate Ig domain proteins from a polytopic precursor. Our findings suggest that SP and SPP function are not restricted to N-terminal signal sequence cleavage but also contribute to the processing of cellular transmembrane proteins. PMID:18981173

Aircraft stress sequence development: A complex engineering process made simple

NASA Technical Reports Server (NTRS)

Schrader, K. H.; Butts, D. G.; Sparks, W. A.

1994-01-01

Development of stress sequences for critical aircraft structure requires flight measured usage data, known aircraft loads, and established relationships between aircraft flight loads and structural stresses. Resulting cycle-by-cycle stress sequences can be directly usable for crack growth analysis and coupon spectra tests. Often, an expert in loads and spectra development manipulates the usage data into a typical sequence of representative flight conditions for which loads and stresses are calculated. For a fighter/trainer type aircraft, this effort is repeated many times for each of the fatigue critical locations (FCL) resulting in expenditure of numerous engineering hours. The Aircraft Stress Sequence Computer Program (ACSTRSEQ), developed by Southwest Research Institute under contract to San Antonio Air Logistics Center, presents a unique approach for making complex technical computations in a simple, easy to use method. The program is written in Microsoft Visual Basic for the Microsoft Windows environment.

Metagenomic ventures into outer sequence space.

PubMed

Dutilh, Bas E

Sequencing DNA or RNA directly from the environment often results in many sequencing reads that have no homologs in the database. These are referred to as "unknowns," and reflect the vast unexplored microbial sequence space of our biosphere, also known as "biological dark matter." However, unknowns also exist because metagenomic datasets are not optimally mined. There is a pressure on researchers to publish and move on, and the unknown sequences are often left for what they are, and conclusions drawn based on reads with annotated homologs. This can cause abundant and widespread genomes to be overlooked, such as the recently discovered human gut bacteriophage crAssphage. The unknowns may be enriched for bacteriophage sequences, the most abundant and genetically diverse component of the biosphere and of sequence space. However, it remains an open question, what is the actual size of biological sequence space? The de novo assembly of shotgun metagenomes is the most powerful tool to address this question.

Evaluating the accuracy of SHAPE-directed RNA secondary structure predictions

PubMed Central

Sükösd, Zsuzsanna; Swenson, M. Shel; Kjems, Jørgen; Heitsch, Christine E.

2013-01-01

Recent advances in RNA structure determination include using data from high-throughput probing experiments to improve thermodynamic prediction accuracy. We evaluate the extent and nature of improvements in data-directed predictions for a diverse set of 16S/18S ribosomal sequences using a stochastic model of experimental SHAPE data. The average accuracy for 1000 data-directed predictions always improves over the original minimum free energy (MFE) structure. However, the amount of improvement varies with the sequence, exhibiting a correlation with MFE accuracy. Further analysis of this correlation shows that accurate MFE base pairs are typically preserved in a data-directed prediction, whereas inaccurate ones are not. Thus, the positive predictive value of common base pairs is consistently higher than the directed prediction accuracy. Finally, we confirm sequence dependencies in the directability of thermodynamic predictions and investigate the potential for greater accuracy improvements in the worst performing test sequence. PMID:23325843

Site directed recombination

DOEpatents

Jurka, Jerzy W.

1997-01-01

Enhanced homologous recombination is obtained by employing a consensus sequence which has been found to be associated with integration of repeat sequences, such as Alu and ID. The consensus sequence or sequence having a single transition mutation determines one site of a double break which allows for high efficiency of integration at the site. By introducing single or double stranded DNA having the consensus sequence flanking region joined to a sequence of interest, one can reproducibly direct integration of the sequence of interest at one or a limited number of sites. In this way, specific sites can be identified and homologous recombination achieved at the site by employing a second flanking sequence associated with a sequence proximal to the 3'-nick.

Parameter comparison of white matter diffusion tensor imaging (DTI) in rhesus macaques (Macaca mulatta)

PubMed Central

MO, Yin; CHAO, Fang; SONG, Ming; LIU, Ci-Rong; LIU, Hui-Lang; QIAN, Xi-Ying; ZHAO, Xu-Dong

2014-01-01

In this study, we analyzed diffusion tensor imaging (DTI) results of brain white matter in rhesus macaques (Macaca mulatta) with four different parameter settings and found that the sequence A (b=1 000 s/mm2, spatial resolution=1.25 mm×1.25 mm× 1.25 mm, numbers of direction=33, NSA=3) and B (b=800 s/mm2, spatial resolution=1.25 mm×1.25 mm×1.25 mm, numbers of direction=33, NSA=3) could accurately track coarse fibers. The fractional anisotropy (FA) derived from sequence C (b=1 000s/mm2, spatial resolution=0.55 mm×0.55 mm×2.5 mm, direction number=33, NSA=3) was too fuzzy to be used in tracking white matter fibers. By comparison, the high resolution and the FA with high contrast of gray matter and white matter derived from sequence D (b=800 s/mm2, spatial resolution=1.0 mm×1.0 mm ×1.0 mm, numbers of direction=33, NSA=3) qualified in its application in tracking both thick and thin fibers, making it an optimal DTI setting for rhesus macaques. PMID:24866488

A DS-UWB Cognitive Radio System Based on Bridge Function Smart Codes

NASA Astrophysics Data System (ADS)

Xu, Yafei; Hong, Sheng; Zhao, Guodong; Zhang, Fengyuan; di, Jinshan; Zhang, Qishan

This paper proposes a direct-sequence UWB Gaussian pulse of cognitive radio systems based on bridge function smart sequence matrix and the Gaussian pulse. As the system uses the spreading sequence code, that is the bridge function smart code sequence, the zero correlation zones (ZCZs) which the bridge function sequences' auto-correlation functions had, could reduce multipath fading of the pulse interference. The Modulated channel signal was sent into the IEEE 802.15.3a UWB channel. We analysis the ZCZs's inhibition to the interference multipath interference (MPI), as one of the main system sources interferences. The simulation in SIMULINK/MATLAB is described in detail. The result shows the system has better performance by comparison with that employing Walsh sequence square matrix, and it was verified by the formula in principle.

Genotyping of Leptospira directly in urine samples of cattle demonstrates a diversity of species and strains in Brazil.

PubMed

Hamond, C; Pestana, C P; Medeiros, M A; Lilenbaum, W

2016-01-01

The aim of this study was to identify Leptospira in urine samples of cattle by direct sequencing of the secY gene. The validity of this approach was assessed using ten Leptospira strains obtained from cattle in Brazil and 77 DNA samples previously extracted from cattle urine, that were positive by PCR for the genus-specific lipL32 gene of Leptospira. Direct sequencing identified 24 (31·1%) interpretable secY sequences and these were identical to those obtained from direct DNA sequencing of the urine samples from which they were recovered. Phylogenetic analyses identified four species: L. interrogans, L. borgpetersenii, L. noguchii, and L. santarosai with the most prevalent genotypes being associated with L. borgpetersenii. While direct sequencing cannot, as yet, replace culturing of leptospires, it is a valid additional tool for epidemiological studies. An unexpected finding from this study was the genetic diversity of Leptospira infecting Brazilian cattle.

Identification of a precursor genomic segment that provided a sequence unique to glycophorin B and E genes

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

Onda, M.; Kudo, S.; Fukuda, M.

Human glycophorin A, B, and E (GPA, GPB, and GPE) genes belong to a gene family located at the long arm of chromosome 4. These three genes are homologous from the 5'-flanking sequence to the Alu sequence, which is 1 kb downstream from the exon encoding the transmembrane domain. Analysis of the Alu sequence and flanking direct repeat sequences suggested that the GPA gene most closely resembles the ancestral gene, whereas the GPB and GPE gene arose by homologous recombination within the Alu sequence, acquiring 3' sequences from an unrelated precursor genomic segment. Here the authors describe the identification ofmore » this putative precursor genomic segment. A human genomic library was screened by using the sequence of the 3' region of the GPB gene as a probe. The genomic clones isolated were found to contain an Alu sequence that appeared to be involved in the recombination. Downstream from the Alu sequence, the nucleotide sequence of the precursor genomic segment is almost identical to that of the GPB or GPE gene. In contrast, the upstream sequence of the genomic segment differs entirely from that of the GPA, GPB, and GPE genes. Conservation of the direct repeats flanking the Alu sequence of the genomic segment strongly suggests that the sequence of this genomic segment has been maintained during evolution. This identified genomic segment was found to reside downstream from the GPA gene by both gene mapping and in situ chromosomal localization. The precursor genomic segment was also identified in the orangutan genome, which is known to lack GPB and GPE genes. These results indicate that one of the duplicated ancestral glycophorin genes acquired a unique 3' sequence by unequal crossing-over through its Alu sequence and the further downstream Alu sequence present in the duplicated gene. Further duplication and divergence of this gene yielded the GPB and GPE genes. 37 refs., 5 figs.« less

Direct detection of Mycobacterium tuberculosis rifampin resistance in bio-safe stained sputum smears.

PubMed

Lavania, Surabhi; Anthwal, Divya; Bhalla, Manpreet; Singh, Nagendra; Haldar, Sagarika; Tyagi, Jaya Sivaswami

2017-01-01

Direct smear microscopy of sputum forms the mainstay of TB diagnosis in resource-limited settings. Stained sputum smear slides can serve as a ready-made resource to transport sputum for molecular drug susceptibility testing. However, bio-safety is a major concern during transport of sputum/stained slides and for laboratory workers engaged in processing Mycobacterium tuberculosis infected sputum specimens. In this study, a bio-safe USP (Universal Sample Processing) concentration-based sputum processing method (Bio-safe method) was assessed on 87 M. tuberculosis culture positive sputum samples. Samples were processed for Ziehl-Neelsen (ZN) smear, liquid culture and DNA isolation. DNA isolated directly from sputum was subjected to an IS6110 PCR assay. Both sputum DNA and DNA extracted from bio-safe ZN concentrated smear slides were subjected to rpoB PCR and simultaneously assessed by DNA sequencing for determining rifampin (RIF) resistance. All sputum samples were rendered sterile by Bio-safe method. Bio-safe smears exhibited a 5% increment in positivity over direct smear with a 14% increment in smear grade status. All samples were positive for IS6110 and rpoB PCR. Thirty four percent samples were RIF resistant by rpoB PCR product sequencing. A 100% concordance (κ value = 1) was obtained between sequencing results derived from bio-safe smear slides and bio-safe sputum. This study demonstrates that Bio-safe method can address safety issues associated with sputum processing, provide an efficient alternative to sample transport in the form of bio-safe stained concentrated smear slides and can also provide information on drug (RIF) resistance by direct DNA sequencing.

Direct detection of Mycobacterium tuberculosis rifampin resistance in bio-safe stained sputum smears

PubMed Central

Lavania, Surabhi; Anthwal, Divya; Bhalla, Manpreet; Singh, Nagendra; Haldar, Sagarika; Tyagi, Jaya Sivaswami

2017-01-01

Direct smear microscopy of sputum forms the mainstay of TB diagnosis in resource-limited settings. Stained sputum smear slides can serve as a ready-made resource to transport sputum for molecular drug susceptibility testing. However, bio-safety is a major concern during transport of sputum/stained slides and for laboratory workers engaged in processing Mycobacterium tuberculosis infected sputum specimens. In this study, a bio-safe USP (Universal Sample Processing) concentration-based sputum processing method (Bio-safe method) was assessed on 87 M. tuberculosis culture positive sputum samples. Samples were processed for Ziehl-Neelsen (ZN) smear, liquid culture and DNA isolation. DNA isolated directly from sputum was subjected to an IS6110 PCR assay. Both sputum DNA and DNA extracted from bio-safe ZN concentrated smear slides were subjected to rpoB PCR and simultaneously assessed by DNA sequencing for determining rifampin (RIF) resistance. All sputum samples were rendered sterile by Bio-safe method. Bio-safe smears exhibited a 5% increment in positivity over direct smear with a 14% increment in smear grade status. All samples were positive for IS6110 and rpoB PCR. Thirty four percent samples were RIF resistant by rpoB PCR product sequencing. A 100% concordance (κ value = 1) was obtained between sequencing results derived from bio-safe smear slides and bio-safe sputum. This study demonstrates that Bio-safe method can address safety issues associated with sputum processing, provide an efficient alternative to sample transport in the form of bio-safe stained concentrated smear slides and can also provide information on drug (RIF) resistance by direct DNA sequencing. PMID:29216262

A novel progesterone receptor membrane component (PGRMC) in the human and swine parasite Taenia solium: implications to the host-parasite relationship.

PubMed

Aguilar-Díaz, Hugo; Nava-Castro, Karen E; Escobedo, Galileo; Domínguez-Ramírez, Lenin; García-Varela, Martín; Del Río-Araiza, Víctor H; Palacios-Arreola, Margarita I; Morales-Montor, Jorge

2018-03-09

We have previously reported that progesterone (P 4 ) has a direct in vitro effect on the scolex evagination and growth of Taenia solium cysticerci. Here, we explored the hypothesis that the P 4 direct effect on T. solium might be mediated by a novel steroid-binding parasite protein. By way of using immunofluorescent confocal microscopy, flow cytometry analysis, double-dimension electrophoresis analysis, and sequencing the corresponding protein spot, we detected a novel PGRMC in T. solium. Molecular modeling studies accompanied by computer docking using the sequenced protein, together with phylogenetic analysis and sequence alignment clearly demonstrated that T. solium PGRMC is from parasite origin. Our results show that P 4 in vitro increases parasite evagination and scolex size. Using immunofluorescent confocal microscopy, we detected that parasite cells showed expression of a P 4 -binding like protein exclusively located at the cysticercus subtegumental tissue. Presence of the P 4 -binding protein in cyst cells was also confirmed by flow cytometry. Double-dimension electrophoresis analysis, followed by sequencing the corresponding protein spot, revealed a protein that was previously reported in the T. solium genome belonging to a membrane-associated progesterone receptor component (PGRMC). Molecular modeling studies accompanied by computer docking using the sequenced protein showed that PGRMC is potentially able to bind steroid hormones such as progesterone, estradiol, testosterone and dihydrodrotestosterone with different affinities. Phylogenetic analysis and sequence alignment clearly demonstrated that T. solium PGRMC is related to a steroid-binding protein of Echinoccocus granulosus, both of them being nested within a cluster including similar proteins present in platyhelminths such as Schistocephalus solidus and Schistosoma haematobium. Progesterone may directly act upon T. solium cysticerci probably by binding to PGRMC. This research has implications in the field of host-parasite co-evolution as well as the sex-associated susceptibility to this infection. In a more practical matter, present results may contribute to the molecular design of new drugs with anti-parasite actions.

[The principle and application of the single-molecule real-time sequencing technology].

PubMed

Yanhu, Liu; Lu, Wang; Li, Yu

2015-03-01

Last decade witnessed the explosive development of the third-generation sequencing strategy, including single-molecule real-time sequencing (SMRT), true single-molecule sequencing (tSMSTM) and the single-molecule nanopore DNA sequencing. In this review, we summarize the principle, performance and application of the SMRT sequencing technology. Compared with the traditional Sanger method and the next-generation sequencing (NGS) technologies, the SMRT approach has several advantages, including long read length, high speed, PCR-free and the capability of direct detection of epigenetic modifications. However, the disadvantage of its low accuracy, most of which resulted from insertions and deletions, is also notable. So, the raw sequence data need to be corrected before assembly. Up to now, the SMRT is a good fit for applications in the de novo genomic sequencing and the high-quality assemblies of small genomes. In the future, it is expected to play an important role in epigenetics, transcriptomic sequencing, and assemblies of large genomes.

A direct method for computing extreme value (Gumbel) parameters for gapped biological sequence alignments.

PubMed

Quinn, Terrance; Sinkala, Zachariah

2014-01-01

We develop a general method for computing extreme value distribution (Gumbel, 1958) parameters for gapped alignments. Our approach uses mixture distribution theory to obtain associated BLOSUM matrices for gapped alignments, which in turn are used for determining significance of gapped alignment scores for pairs of biological sequences. We compare our results with parameters already obtained in the literature.

Extending earthquakes' reach through cascading.

PubMed

Marsan, David; Lengliné, Olivier

2008-02-22

Earthquakes, whatever their size, can trigger other earthquakes. Mainshocks cause aftershocks to occur, which in turn activate their own local aftershock sequences, resulting in a cascade of triggering that extends the reach of the initial mainshock. A long-lasting difficulty is to determine which earthquakes are connected, either directly or indirectly. Here we show that this causal structure can be found probabilistically, with no a priori model nor parameterization. Large regional earthquakes are found to have a short direct influence in comparison to the overall aftershock sequence duration. Relative to these large mainshocks, small earthquakes collectively have a greater effect on triggering. Hence, cascade triggering is a key component in earthquake interactions.

Pyrin gene and mutants thereof, which cause familial Mediterranean fever

DOEpatents

Kastner, Daniel L [Bethesda, MD; Aksentijevichh, Ivona [Bethesda, MD; Centola, Michael [Tacoma Park, MD; Deng, Zuoming [Gaithersburg, MD; Sood, Ramen [Rockville, MD; Collins, Francis S [Rockville, MD; Blake, Trevor [Laytonsville, MD; Liu, P Paul [Ellicott City, MD; Fischel-Ghodsian, Nathan [Los Angeles, CA; Gumucio, Deborah L [Ann Arbor, MI; Richards, Robert I [North Adelaide, AU; Ricke, Darrell O [San Diego, CA; Doggett, Norman A [Santa Cruz, NM; Pras, Mordechai [Tel-Hashomer, IL

2003-09-30

The invention provides the nucleic acid sequence encoding the protein associated with familial Mediterranean fever (FMF). The cDNA sequence is designated as MEFV. The invention is also directed towards fragments of the DNA sequence, as well as the corresponding sequence for the RNA transcript and fragments thereof. Another aspect of the invention provides the amino acid sequence for a protein (pyrin) associated with FMF. The invention is directed towards both the full length amino acid sequence, fusion proteins containing the amino acid sequence and fragments thereof. The invention is also directed towards mutants of the nucleic acid and amino acid sequences associated with FMF. In particular, the invention discloses three missense mutations, clustered in within about 40 to 50 amino acids, in the highly conserved rfp (B30.2) domain at the C-terminal of the protein. These mutants include M6801, M694V, K695R, and V726A. Additionally, the invention includes methods for diagnosing a patient at risk for having FMF and kits therefor.

Direct-Sequence Spread Spectrum System

DTIC Science & Technology

1990-06-01

by directly modulating a conventional narrowband frequency-modulated (FM) carrier by a high rate digital code. The direct modulation is binary phase ...specification of the DSSS system will not be developed. The results of the evaluation phase of this research will be compared against theoretical...spread spectrum is called binary phase -shift keying 19 (BPSK). BPSK is a modulation in which a binary Ŕ" represents a 0-degree relative phase

Library Design-Facilitated High-Throughput Sequencing of Synthetic Peptide Libraries.

PubMed

Vinogradov, Alexander A; Gates, Zachary P; Zhang, Chi; Quartararo, Anthony J; Halloran, Kathryn H; Pentelute, Bradley L

2017-11-13

A methodology to achieve high-throughput de novo sequencing of synthetic peptide mixtures is reported. The approach leverages shotgun nanoliquid chromatography coupled with tandem mass spectrometry-based de novo sequencing of library mixtures (up to 2000 peptides) as well as automated data analysis protocols to filter away incorrect assignments, noise, and synthetic side-products. For increasing the confidence in the sequencing results, mass spectrometry-friendly library designs were developed that enabled unambiguous decoding of up to 600 peptide sequences per hour while maintaining greater than 85% sequence identification rates in most cases. The reliability of the reported decoding strategy was additionally confirmed by matching fragmentation spectra for select authentic peptides identified from library sequencing samples. The methods reported here are directly applicable to screening techniques that yield mixtures of active compounds, including particle sorting of one-bead one-compound libraries and affinity enrichment of synthetic library mixtures performed in solution.

Single-cell template strand sequencing by Strand-seq enables the characterization of individual homologs.

PubMed

Sanders, Ashley D; Falconer, Ester; Hills, Mark; Spierings, Diana C J; Lansdorp, Peter M

2017-06-01

The ability to distinguish between genome sequences of homologous chromosomes in single cells is important for studies of copy-neutral genomic rearrangements (such as inversions and translocations), building chromosome-length haplotypes, refining genome assemblies, mapping sister chromatid exchange events and exploring cellular heterogeneity. Strand-seq is a single-cell sequencing technology that resolves the individual homologs within a cell by restricting sequence analysis to the DNA template strands used during DNA replication. This protocol, which takes up to 4 d to complete, relies on the directionality of DNA, in which each single strand of a DNA molecule is distinguished based on its 5'-3' orientation. Culturing cells in a thymidine analog for one round of cell division labels nascent DNA strands, allowing for their selective removal during genomic library construction. To preserve directionality of template strands, genomic preamplification is bypassed and labeled nascent strands are nicked and not amplified during library preparation. Each single-cell library is multiplexed for pooling and sequencing, and the resulting sequence data are aligned, mapping to either the minus or plus strand of the reference genome, to assign template strand states for each chromosome in the cell. The major adaptations to conventional single-cell sequencing protocols include harvesting of daughter cells after a single round of BrdU incorporation, bypassing of whole-genome amplification, and removal of the BrdU + strand during Strand-seq library preparation. By sequencing just template strands, the structure and identity of each homolog are preserved.

A New Method for Setting Calculation Sequence of Directional Relay Protection in Multi-Loop Networks

NASA Astrophysics Data System (ADS)

Haijun, Xiong; Qi, Zhang

2016-08-01

Workload of relay protection setting calculation in multi-loop networks may be reduced effectively by optimization setting calculation sequences. A new method of setting calculation sequences of directional distance relay protection in multi-loop networks based on minimum broken nodes cost vector (MBNCV) was proposed to solve the problem experienced in current methods. Existing methods based on minimum breakpoint set (MBPS) lead to more break edges when untying the loops in dependent relationships of relays leading to possibly more iterative calculation workloads in setting calculations. A model driven approach based on behavior trees (BT) was presented to improve adaptability of similar problems. After extending the BT model by adding real-time system characters, timed BT was derived and the dependency relationship in multi-loop networks was then modeled. The model was translated into communication sequence process (CSP) models and an optimization setting calculation sequence in multi-loop networks was finally calculated by tools. A 5-nodes multi-loop network was applied as an example to demonstrate effectiveness of the modeling and calculation method. Several examples were then calculated with results indicating the method effectively reduces the number of forced broken edges for protection setting calculation in multi-loop networks.

Accurate RNA consensus sequencing for high-fidelity detection of transcriptional mutagenesis-induced epimutations.

PubMed

Reid-Bayliss, Kate S; Loeb, Lawrence A

2017-08-29

Transcriptional mutagenesis (TM) due to misincorporation during RNA transcription can result in mutant RNAs, or epimutations, that generate proteins with altered properties. TM has long been hypothesized to play a role in aging, cancer, and viral and bacterial evolution. However, inadequate methodologies have limited progress in elucidating a causal association. We present a high-throughput, highly accurate RNA sequencing method to measure epimutations with single-molecule sensitivity. Accurate RNA consensus sequencing (ARC-seq) uniquely combines RNA barcoding and generation of multiple cDNA copies per RNA molecule to eliminate errors introduced during cDNA synthesis, PCR, and sequencing. The stringency of ARC-seq can be scaled to accommodate the quality of input RNAs. We apply ARC-seq to directly assess transcriptome-wide epimutations resulting from RNA polymerase mutants and oxidative stress.

Data Images and Other Graphical Displays for Directional Data

NASA Technical Reports Server (NTRS)

Morphet, Bill; Symanzik, Juergen

2005-01-01

Vectors, axes, and periodic phenomena have direction. Directional variation can be expressed as points on a unit circle and is the subject of circular statistics, a relatively new application of statistics. An overview of existing methods for the display of directional data is given. The data image for linear variables is reviewed, then extended to directional variables by displaying direction using a color scale composed of a sequence of four or more color gradients with continuity between sequences and ordered intuitively in a color wheel such that the color of the 0deg angle is the same as the color of the 360deg angle. Cross over, which arose in automating the summarization of historical wind data, and color discontinuity resulting from the use a single color gradient in computational fluid dynamics visualization are eliminated. The new method provides for simultaneous resolution of detail on a small scale and overall structure on a large scale. Example circular data images are given of a global view of average wind direction of El Nino periods, computed rocket motor internal combustion flow, a global view of direction of the horizontal component of earth's main magnetic field on 9/15/2004, and Space Shuttle solid rocket motor nozzle vectoring.

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

Performance Analysis of Direct-Sequence Code-Division Multiple-Access Communications with Asymmetric Quadrature Phase-Shift-Keying Modulation

NASA Technical Reports Server (NTRS)

Wang, C.-W.; Stark, W.

2005-01-01

This article considers a quaternary direct-sequence code-division multiple-access (DS-CDMA) communication system with asymmetric quadrature phase-shift-keying (AQPSK) modulation for unequal error protection (UEP) capability. Both time synchronous and asynchronous cases are investigated. An expression for the probability distribution of the multiple-access interference is derived. The exact bit-error performance and the approximate performance using a Gaussian approximation and random signature sequences are evaluated by extending the techniques used for uniform quadrature phase-shift-keying (QPSK) and binary phase-shift-keying (BPSK) DS-CDMA systems. Finally, a general system model with unequal user power and the near-far problem is considered and analyzed. The results show that, for a system with UEP capability, the less protected data bits are more sensitive to the near-far effect that occurs in a multiple-access environment than are the more protected bits.

Forensic strategy to ensure the quality of sequencing data of mitochondrial DNA in highly degraded samples.

PubMed

Adachi, Noboru; Umetsu, Kazuo; Shojo, Hideki

2014-01-01

Mitochondrial DNA (mtDNA) is widely used for DNA analysis of highly degraded samples because of its polymorphic nature and high number of copies in a cell. However, as endogenous mtDNA in deteriorated samples is scarce and highly fragmented, it is not easy to obtain reliable data. In the current study, we report the risks of direct sequencing mtDNA in highly degraded material, and suggest a strategy to ensure the quality of sequencing data. It was observed that direct sequencing data of the hypervariable segment (HVS) 1 by using primer sets that generate an amplicon of 407 bp (long-primer sets) was different from results obtained by using newly designed primer sets that produce an amplicon of 120-139 bp (mini-primer sets). The data aligned with the results of mini-primer sets analysis in an amplicon length-dependent manner; the shorter the amplicon, the more evident the endogenous sequence became. Coding region analysis using multiplex amplified product-length polymorphisms revealed the incongruence of single nucleotide polymorphisms between the coding region and HVS 1 caused by contamination with exogenous mtDNA. Although the sequencing data obtained using long-primer sets turned out to be erroneous, it was unambiguous and reproducible. These findings suggest that PCR primers that produce amplicons shorter than those currently recognized should be used for mtDNA analysis in highly degraded samples. Haplogroup motif analysis of the coding region and HVS should also be performed to improve the reliability of forensic mtDNA data. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

A CI-Independent Form of Replicative Inhibition: Turn Off of Early Replication of Bacteriophage Lambda

PubMed Central

Hayes, Sidney; Horbay, Monique A.; Hayes, Connie

2012-01-01

Several earlier studies have described an unusual exclusion phenotype exhibited by cells with plasmids carrying a portion of the replication region of phage lambda. Cells exhibiting this inhibition phenotype (IP) prevent the plating of homo-immune and hybrid hetero-immune lambdoid phages. We have attempted to define aspects of IP, and show that it is directed to repλ phages. IP was observed in cells with plasmids containing a λ DNA fragment including oop, encoding a short OOP micro RNA, and part of the lambda origin of replication, oriλ, defined by iteron sequences ITN1-4 and an adjacent high AT-rich sequence. Transcription of the intact oop sequence from its promoter, pO is required for IP, as are iterons ITN3–4, but not the high AT-rich portion of oriλ. The results suggest that IP silencing is directed to theta mode replication initiation from an infecting repλ genome, or an induced repλ prophage. Phage mutations suppressing IP, i.e., Sip, map within, or adjacent to cro or in O, or both. Our results for plasmid based IP suggest the hypothesis that there is a natural mechanism for silencing early theta-mode replication initiation, i.e. the buildup of λ genomes with oop + oriλ+ sequence. PMID:22590552

First detection of multiple knockdown resistance (kdr)-like mutations in voltage-gated sodium channel using three new genotyping methods in Anopheles sinensis from Guangxi Province, China.

PubMed

Tan, Wei L; Li, Chun X; Wang, Zhong M; Liu, Mei D; Dong, Yan D; Feng, Xiang Y; Wu, Zhi M; Guo, Xiao X; Xing, Dan; Zhang, Ying M; Wang, Zhong C; Zhao, Tong Y

2012-09-01

To investigate knockdown resistance (kdr)-like mutations associated with pyrethroid resistance in Anopheles sinensis (Wiedemann, 1828), from Guangxi province, southwest China, a segment of a sodium channel gene was sequenced and genotyped using three new genotyping assays. Direct sequencing revealed the presence of TTG-to-TCG and TG-to-TTT mutations at allele position L1014, which led to L1014S and L1014F substitutions in a few individual and two novel substitutions of N1013S and L1014W in two DNA templates. A low frequency of the kdr allele mostly in the heterozygous state of L1014S and L1014F was observed in this mosquito population. In this study, the genotyping of An. sinensis using three polymerase chain reaction-based methods generated consistent results, which agreed with the results of DNA sequencing. In total, 52 mosquitoes were genotyped using a direct sequencing assay. The number of mosquitoes and their genotypes were as follows: L/L = 24, L/S = 19, L/F = 8, and F/W = 1. The allelic frequency of L1014, 1014S, and 1014F were 72, 18, and 9%, respectively.

[Identification of a HPGD mutation in three families affected with primary hypertrophic osteoarthropathy].

PubMed

Zhang, Wanying; Wang, Tao; Huang, Shuaiwu; Zhao, Xiuli

2018-04-10

To detect mutation of HPGD gene among three pedigrees affected with primary hypertrophic osteoarthropathy (PHO) by DNA sequencing and high-resolution melting (HRM) analysis. Genomic DNA was extracted from peripheral blood samples collected from the pedigrees. PCR and direct sequencing were carried out to identify potential mutations of the HPGD gene. Amplicons containing the mutation spot were generated by nested PCR. The products were then subjected to HRM analysis using the HR-1 instrument. Direct sequencing was carried out in family members and healthy individuals to confirm the result of HRM analysis. A homozygous mutation c.310_311delCT was detected in 2 affected probands, while a heterozygous mutation c.310_311delCT was detected in the third proband. HRM analysis of the fragments encompassing HPGD exon 3 showed 3 curve patterns representing three different genotypes, i.e., the wild type, the c.310_311delCT homozygote, and the c.310_311delCT heterozygote. Result of DNA sequencing was consistent with that of the HRM analysis and phenotype of the subjects. The c.310_311delCT mutation may be the most prevalent mutation among Chinese population. HRM analysis has provided an optimized method for genetic testing of HPGD mutation for its simplicity, rapid turnover and high sensitivity.

Comparison of direct boiling method with commercial kits for extracting fecal microbiome DNA by Illumina sequencing of 16S rRNA tags.

PubMed

Peng, Xin; Yu, Ke-Qiang; Deng, Guan-Hua; Jiang, Yun-Xia; Wang, Yu; Zhang, Guo-Xia; Zhou, Hong-Wei

2013-12-01

Low cost and high throughput capacity are major advantages of using next generation sequencing (NGS) techniques to determine metagenomic 16S rRNA tag sequences. These methods have significantly changed our view of microorganisms in the fields of human health and environmental science. However, DNA extraction using commercial kits has shortcomings of high cost and time constraint. In the present study, we evaluated the determination of fecal microbiomes using a direct boiling method compared with 5 different commercial extraction methods, e.g., Qiagen and MO BIO kits. Principal coordinate analysis (PCoA) using UniFrac distances and clustering showed that direct boiling of a wide range of feces concentrations gave a similar pattern of bacterial communities as those obtained from most of the commercial kits, with the exception of the MO BIO method. Fecal concentration by boiling method affected the estimation of α-diversity indices, otherwise results were generally comparable between boiling and commercial methods. The operational taxonomic units (OTUs) determined through direct boiling showed highly consistent frequencies with those determined through most of the commercial methods. Even those for the MO BIO kit were also obtained by the direct boiling method with high confidence. The present study suggested that direct boiling could be used to determine the fecal microbiome and using this method would significantly reduce the cost and improve the efficiency of the sample preparation for studying gut microbiome diversity. © 2013 Elsevier B.V. All rights reserved.

Direct generation of all-optical random numbers from optical pulse amplitude chaos.

PubMed

Li, Pu; Wang, Yun-Cai; Wang, An-Bang; Yang, Ling-Zhen; Zhang, Ming-Jiang; Zhang, Jian-Zhong

2012-02-13

We propose and theoretically demonstrate an all-optical method for directly generating all-optical random numbers from pulse amplitude chaos produced by a mode-locked fiber ring laser. Under an appropriate pump intensity, the mode-locked laser can experience a quasi-periodic route to chaos. Such a chaos consists of a stream of pulses with a fixed repetition frequency but random intensities. In this method, we do not require sampling procedure and external triggered clocks but directly quantize the chaotic pulses stream into random number sequence via an all-optical flip-flop. Moreover, our simulation results show that the pulse amplitude chaos has no periodicity and possesses a highly symmetric distribution of amplitude. Thus, in theory, the obtained random number sequence without post-processing has a high-quality randomness verified by industry-standard statistical tests.

Associations of Perceived Norms With Intentions to Learn Genomic Sequencing Results: Roles for Attitudes and Ambivalence

PubMed Central

Reid, Allecia E.; Taber, Jennifer M.; Ferrer, Rebecca A.; Biesecker, Barbara B.; Lewis, Katie L.; Biesecker, Leslie G.; Klein, William M. P.

2018-01-01

Objective Genomic sequencing is becoming increasingly accessible, highlighting the need to understand the social and psychological factors that drive interest in receiving testing results. These decisions may depend on perceived descriptive norms (how most others behave) and injunctive norms (what is approved of by others). We predicted that descriptive norms would be directly associated with intentions to learn genomic sequencing results, whereas injunctive norms would be associated indirectly, via attitudes. These differential associations with intentions versus attitudes were hypothesized to be strongest when individuals held ambivalent attitudes toward obtaining results. Methods Participants enrolled in a genomic sequencing trial (n=372) reported intentions to learn medically actionable, non-medically actionable, and carrier sequencing results. Descriptive norms items referenced other study participants. Injunctive norms were analyzed separately for close friends and family members. Attitudes, attitudinal ambivalence, and sociodemographic covariates were also assessed. Results In structural equation models, both descriptive norms and friend injunctive norms were associated with intentions to receive all sequencing results (ps<.004). Attitudes consistently mediated all friend injunctive norms-intentions associations, but not the descriptive norms-intentions associations. Attitudinal ambivalence moderated the association between friend injunctive norms (p≤.001), but not descriptive norms (p=.16), and attitudes. Injunctive norms were significantly associated with attitudes when ambivalence was high, but were unrelated when ambivalence was low. Results replicated for family injunctive norms. Conclusions Descriptive and injunctive norms play roles in genomic sequencing decisions. Considering mediators and moderators of these processes enhances ability to optimize use of normative information to support informed decision making. PMID:29745680

An integrated semiconductor device enabling non-optical genome sequencing.

PubMed

Rothberg, Jonathan M; Hinz, Wolfgang; Rearick, Todd M; Schultz, Jonathan; Mileski, William; Davey, Mel; Leamon, John H; Johnson, Kim; Milgrew, Mark J; Edwards, Matthew; Hoon, Jeremy; Simons, Jan F; Marran, David; Myers, Jason W; Davidson, John F; Branting, Annika; Nobile, John R; Puc, Bernard P; Light, David; Clark, Travis A; Huber, Martin; Branciforte, Jeffrey T; Stoner, Isaac B; Cawley, Simon E; Lyons, Michael; Fu, Yutao; Homer, Nils; Sedova, Marina; Miao, Xin; Reed, Brian; Sabina, Jeffrey; Feierstein, Erika; Schorn, Michelle; Alanjary, Mohammad; Dimalanta, Eileen; Dressman, Devin; Kasinskas, Rachel; Sokolsky, Tanya; Fidanza, Jacqueline A; Namsaraev, Eugeni; McKernan, Kevin J; Williams, Alan; Roth, G Thomas; Bustillo, James

2011-07-20

The seminal importance of DNA sequencing to the life sciences, biotechnology and medicine has driven the search for more scalable and lower-cost solutions. Here we describe a DNA sequencing technology in which scalable, low-cost semiconductor manufacturing techniques are used to make an integrated circuit able to directly perform non-optical DNA sequencing of genomes. Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip. The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells, which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions. Use of the most widely used technology for constructing integrated circuits, the complementary metal-oxide semiconductor (CMOS) process, allows for low-cost, large-scale production and scaling of the device to higher densities and larger array sizes. We show the performance of the system by sequencing three bacterial genomes, its robustness and scalability by producing ion chips with up to 10 times as many sensors and sequencing a human genome.

Limited copy number - high resolution melting (LCN-HRM) enables the detection and identification by sequencing of low level mutations in cancer biopsies

PubMed Central

Do, Hongdo; Dobrovic, Alexander

2009-01-01

Background Mutation detection in clinical tumour samples is challenging when the proportion of tumour cells, and thus mutant alleles, is low. The limited sensitivity of conventional sequencing necessitates the adoption of more sensitive approaches. High resolution melting (HRM) is more sensitive than sequencing but identification of the mutation is desirable, particularly when it is important to discriminate false positives due to PCR errors or template degradation from true mutations. We thus developed limited copy number - high resolution melting (LCN-HRM) which applies limiting dilution to HRM. Multiple replicate reactions with a limited number of target sequences per reaction allow low level mutations to be detected. The dilutions used (based on Ct values) are chosen such that mutations, if present, can be detected by the direct sequencing of amplicons with aberrant melting patterns. Results Using cell lines heterozygous for mutations, we found that the mutations were not readily detected when they comprised 10% of total alleles (20% tumour cells) by sequencing, whereas they were readily detectable at 5% total alleles by standard HRM. LCN-HRM allowed these mutations to be identified by direct sequencing of those positive reactions. LCN-HRM was then used to review formalin-fixed paraffin-embedded (FFPE) clinical samples showing discordant findings between sequencing and HRM for KRAS exon 2 and EGFR exons 19 and 21. Both true mutations present at low levels and sequence changes due to artefacts were detected by LCN-HRM. The use of high fidelity polymerases showed that the majority of the artefacts were derived from the damaged template rather than replication errors during amplification. Conclusion LCN-HRM bridges the sensitivity gap between HRM and sequencing and is effective in distinguishing between artefacts and true mutations. PMID:19811662

Comparison of conventional MRI and MR arthrography in the evaluation of wrist ligament tears: A preliminary experience

PubMed Central

Pahwa, Shivani; Srivastava, Deep N; Sharma, Raju; Gamanagatti, Shivanand; Kotwal, Prakash P; Sharma, Vijay

2014-01-01

Aims: To compare conventional magnetic resonance imaging (MRI) and direct magnetic resonance (MR) arthrography in the evaluation of triangular fibrocartilage complex (TFCC) and intrinsic wrist ligament tears. Materials and Methods: T1-weighted, fat suppressed (FS) proton density plus T2-weighted (FS PD/T2), 3D multiple-echo data image combination (MEDIC) sequences and direct MR arthrography were performed in 53 patients with wrist pain. Images were evaluated for the presence and location of TFCC, scapholunate ligament (SLL) and lunatotriquetral ligament (LTL) tears, and imaging findings were compared with operative findings in 16 patients who underwent arthroscopy or open surgery (gold standard). Results: Sixteen patients underwent arthroscopy/open surgery: 12 TFCC tears were detected arthroscopically out of which 9 were detected on FS PD/T2 sequence, 10 on MEDIC sequence, and all 12 were detected on MR arthrography. The sensitivities of FS PD/T2, MEDIC sequences, and MR arthrography in the detection of TFCC tears were 75%, 83.3%, and 100%, respectively. Out of the eight arthroscopically confirmed SLL tears, three tears were detected on FS PD/T2 sequence, five on MEDIC sequence, and all eight were visualized on MR arthrography. The sensitivities of FS PD/T2, MEDIC sequences, and MR arthrography in detecting SLL tears were 37.5%, 62.5%, and 100%, respectively. One arthroscopically confirmed LTL tear was diagnosed on FS PD/T2 sequence, three on MEDIC sequence, and all five arthroscopically confirmed LTL tears were detected with MR arthrography. The sensitivities of PD, MEDIC sequences, and MR arthrography in detecting LTL tears were 20%, 40%, and 100%, respectively. Conclusions: MR arthrography is the most sensitive and specific imaging modality for the evaluation of wrist ligament tears. PMID:25114389

A TATA binding protein mutant with increased affinity for DNA directs transcription from a reversed TATA sequence in vivo.

PubMed

Spencer, J Vaughn; Arndt, Karen M

2002-12-01

The TATA-binding protein (TBP) nucleates the assembly and determines the position of the preinitiation complex at RNA polymerase II-transcribed genes. We investigated the importance of two conserved residues on the DNA binding surface of Saccharomyces cerevisiae TBP to DNA binding and sequence discrimination. Because they define a significant break in the twofold symmetry of the TBP-TATA interface, Ala100 and Pro191 have been proposed to be key determinants of TBP binding orientation and transcription directionality. In contrast to previous predictions, we found that substitution of an alanine for Pro191 did not allow recognition of a reversed TATA box in vivo; however, the reciprocal change, Ala100 to proline, resulted in efficient utilization of this and other variant TATA sequences. In vitro assays demonstrated that TBP mutants with the A100P and P191A substitutions have increased and decreased affinity for DNA, respectively. The TATA binding defect of TBP with the P191A mutation could be intragenically suppressed by the A100P substitution. Our results suggest that Ala100 and Pro191 are important for DNA binding and sequence recognition by TBP, that the naturally occurring asymmetry of Ala100 and Pro191 is not essential for function, and that a single amino acid change in TBP can lead to elevated DNA binding affinity and recognition of a reversed TATA sequence.

Transfer of movement sequences: bigger is better.

PubMed

Dean, Noah J; Kovacs, Attila J; Shea, Charles H

2008-02-01

Experiment 1 was conducted to determine if proportional transfer from "small to large" scale movements is as effective as transferring from "large to small." We hypothesize that the learning of larger scale movement will require the participant to learn to manage the generation, storage, and dissipation of forces better than when practicing smaller scale movements. Thus, we predict an advantage for transfer of larger scale movements to smaller scale movements relative to transfer from smaller to larger scale movements. Experiment 2 was conducted to determine if adding a load to a smaller scale movement would enhance later transfer to a larger scale movement sequence. It was hypothesized that the added load would require the participants to consider the dynamics of the movement to a greater extent than without the load. The results replicated earlier findings of effective transfer from large to small movements, but consistent with our hypothesis, transfer was less effective from small to large (Experiment 1). However, when a load was added during acquisition transfer from small to large was enhanced even though the load was removed during the transfer test. These results are consistent with the notion that the transfer asymmetry noted in Experiment 1 was due to factors related to movement dynamics that were enhanced during practice of the larger scale movement sequence, but not during the practice of the smaller scale movement sequence. The findings that the movement structure is unaffected by transfer direction but the movement dynamics are influenced by transfer direction is consistent with hierarchal models of sequence production.

DDM1 represses noncoding RNA expression and RNA-directed DNA methylation in heterochromatin.

PubMed

Tan, Feng; Lu, Yue; Jiang, Wei; Zhao, Yu; Wu, Tian; Zhang, Ruoyu; Zhou, Dao-Xiu

2018-05-24

Cytosine methylation of DNA, which occurs at CG, CHG, and CHH (H=A, C, or T) sequences in plants, is a hallmark for epigenetic repression of repetitive sequences. The chromatin remodeling factor DECREASE IN DNA METHYLATION1 (DDM1) is essential for DNA methylation, especially at CG and CHG sequences. However, its potential role in RNA-directed DNA methylation (RdDM) and in chromatin function is not completely understood in rice (Oryza sativa). In this work, we used high-throughput approaches to study the function of rice DDM1 (OsDDM1) in RdDM and the expression of non-coding RNA (ncRNA). We show that loss of function of OsDDM1 results in ectopic CHH methylation of transposable elements and repeats. The ectopic CHH methylation was dependent on rice DOMAINS REARRANGED METHYLTRANSFERASE2 (OsDRM2), a DNA methyltransferase involved in RdDM. Mutations in OsDDM1 lead to decreases of histone H3K9me2 and increases in the levels of heterochromatic small RNA (sRNA) and long noncoding RNA (lncRNA). In particular, OsDDM1 was found to be essential to repress transcription of the two repetitive sequences, Centromeric Retrotransposons of Rice1 (CRR1) and the dominant centromeric CentO repeats. These results suggest that OsDDM1 antagonizes RdDM at heterochromatin and represses tissue-specific expression of ncRNA from repetitive sequences in the rice genome. {copyright, serif} 2018 American Society of Plant Biologists. All rights reserved.

Nanoliter reactors improve multiple displacement amplification of genomes from single cells.

PubMed

Marcy, Yann; Ishoey, Thomas; Lasken, Roger S; Stockwell, Timothy B; Walenz, Brian P; Halpern, Aaron L; Beeson, Karen Y; Goldberg, Susanne M D; Quake, Stephen R

2007-09-01

Since only a small fraction of environmental bacteria are amenable to laboratory culture, there is great interest in genomic sequencing directly from single cells. Sufficient DNA for sequencing can be obtained from one cell by the Multiple Displacement Amplification (MDA) method, thereby eliminating the need to develop culture methods. Here we used a microfluidic device to isolate individual Escherichia coli and amplify genomic DNA by MDA in 60-nl reactions. Our results confirm a report that reduced MDA reaction volume lowers nonspecific synthesis that can result from contaminant DNA templates and unfavourable interaction between primers. The quality of the genome amplification was assessed by qPCR and compared favourably to single-cell amplifications performed in standard 50-microl volumes. Amplification bias was greatly reduced in nanoliter volumes, thereby providing a more even representation of all sequences. Single-cell amplicons from both microliter and nanoliter volumes provided high-quality sequence data by high-throughput pyrosequencing, thereby demonstrating a straightforward route to sequencing genomes from single cells.

Optimization of training sequence for DFT-spread DMT signal in optical access network with direct detection utilizing DML.

PubMed

Li, Fan; Li, Xinying; Yu, Jianjun; Chen, Lin

2014-09-22

We experimentally demonstrated the transmission of 79.86-Gb/s discrete-Fourier-transform spread 32 QAM discrete multi-tone (DFT-spread 32 QAM-DMT) signal over 20-km standard single-mode fiber (SSMF) utilizing directly modulated laser (DML). The experimental results show DFT-spread effectively reduces Peak-to-Average Power Ratio (PAPR) of DMT signal, and also well overcomes narrowband interference and high frequencies power attenuation. We compared different types of training sequence (TS) symbols and found that the optimized TS for channel estimation is the symbol with digital BPSK/QPSK modulation format due to its best performance against optical link noise during channel estimation.

Combining De Novo Peptide Sequencing Algorithms, A Synergistic Approach to Boost Both Identifications and Confidence in Bottom-up Proteomics.

PubMed

Blank-Landeshammer, Bernhard; Kollipara, Laxmikanth; Biß, Karsten; Pfenninger, Markus; Malchow, Sebastian; Shuvaev, Konstantin; Zahedi, René P; Sickmann, Albert

2017-09-01

Complex mass spectrometry based proteomics data sets are mostly analyzed by protein database searches. While this approach performs considerably well for sequenced organisms, direct inference of peptide sequences from tandem mass spectra, i.e., de novo peptide sequencing, oftentimes is the only way to obtain information when protein databases are absent. However, available algorithms suffer from drawbacks such as lack of validation and often high rates of false positive hits (FP). Here we present a simple method of combining results from commonly available de novo peptide sequencing algorithms, which in conjunction with minor tweaks in data acquisition ensues lower empirical FDR compared to the analysis using single algorithms. Results were validated using state-of-the art database search algorithms as well specifically synthesized reference peptides. Thus, we could increase the number of PSMs meeting a stringent FDR of 5% more than 3-fold compared to the single best de novo sequencing algorithm alone, accounting for an average of 11 120 PSMs (combined) instead of 3476 PSMs (alone) in triplicate 2 h LC-MS runs of tryptic HeLa digestion.

Integrating motion, illumination, and structure in video sequences with applications in illumination-invariant tracking.

PubMed

Xu, Yilei; Roy-Chowdhury, Amit K

2007-05-01

In this paper, we present a theory for combining the effects of motion, illumination, 3D structure, albedo, and camera parameters in a sequence of images obtained by a perspective camera. We show that the set of all Lambertian reflectance functions of a moving object, at any position, illuminated by arbitrarily distant light sources, lies "close" to a bilinear subspace consisting of nine illumination variables and six motion variables. This result implies that, given an arbitrary video sequence, it is possible to recover the 3D structure, motion, and illumination conditions simultaneously using the bilinear subspace formulation. The derivation builds upon existing work on linear subspace representations of reflectance by generalizing it to moving objects. Lighting can change slowly or suddenly, locally or globally, and can originate from a combination of point and extended sources. We experimentally compare the results of our theory with ground truth data and also provide results on real data by using video sequences of a 3D face and the entire human body with various combinations of motion and illumination directions. We also show results of our theory in estimating 3D motion and illumination model parameters from a video sequence.

The VirusBanker database uses a Java program to allow flexible searching through Bunyaviridae sequences.

PubMed

Fourment, Mathieu; Gibbs, Mark J

2008-02-05

Viruses of the Bunyaviridae have segmented negative-stranded RNA genomes and several of them cause significant disease. Many partial sequences have been obtained from the segments so that GenBank searches give complex results. Sequence databases usually use HTML pages to mediate remote sorting, but this approach can be limiting and may discourage a user from exploring a database. The VirusBanker database contains Bunyaviridae sequences and alignments and is presented as two spreadsheets generated by a Java program that interacts with a MySQL database on a server. Sequences are displayed in rows and may be sorted using information that is displayed in columns and includes data relating to the segment, gene, protein, species, strain, sequence length, terminal sequence and date and country of isolation. Bunyaviridae sequences and alignments may be downloaded from the second spreadsheet with titles defined by the user from the columns, or viewed when passed directly to the sequence editor, Jalview. VirusBanker allows large datasets of aligned nucleotide and protein sequences from the Bunyaviridae to be compiled and winnowed rapidly using criteria that are formulated heuristically.

Automated DNA mutation detection using universal conditions direct sequencing: application to ten muscular dystrophy genes

PubMed Central

2009-01-01

Background One of the most common and efficient methods for detecting mutations in genes is PCR amplification followed by direct sequencing. Until recently, the process of designing PCR assays has been to focus on individual assay parameters rather than concentrating on matching conditions for a set of assays. Primers for each individual assay were selected based on location and sequence concerns. The two primer sequences were then iteratively adjusted to make the individual assays work properly. This generally resulted in groups of assays with different annealing temperatures that required the use of multiple thermal cyclers or multiple passes in a single thermal cycler making diagnostic testing time-consuming, laborious and expensive. These factors have severely hampered diagnostic testing services, leaving many families without an answer for the exact cause of a familial genetic disease. A search of GeneTests for sequencing analysis of the entire coding sequence for genes that are known to cause muscular dystrophies returns only a small list of laboratories that perform comprehensive gene panels. The hypothesis for the study was that a complete set of universal assays can be designed to amplify and sequence any gene or family of genes using computer aided design tools. If true, this would allow automation and optimization of the mutation detection process resulting in reduced cost and increased throughput. Results An automated process has been developed for the detection of deletions, duplications/insertions and point mutations in any gene or family of genes and has been applied to ten genes known to bear mutations that cause muscular dystrophy: DMD; CAV3; CAPN3; FKRP; TRIM32; LMNA; SGCA; SGCB; SGCG; SGCD. Using this process, mutations have been found in five DMD patients and four LGMD patients (one in the FKRP gene, one in the CAV3 gene, and two likely causative heterozygous pairs of variations in the CAPN3 gene of two other patients). Methods and assay sequences are reported in this paper. Conclusion This automated process allows laboratories to discover DNA variations in a short time and at low cost. PMID:19835634

Molecular Diagnosis of Orthopedic-Device-Related Infection Directly from Sonication Fluid by Metagenomic Sequencing

PubMed Central

Sanderson, Nicholas D.; Atkins, Bridget L.; Brent, Andrew J.; Cole, Kevin; Foster, Dona; McNally, Martin A.; Oakley, Sarah; Peto, Leon; Taylor, Adrian; Peto, Tim E. A.; Crook, Derrick W.; Eyre, David W.

2017-01-01

ABSTRACT Culture of multiple periprosthetic tissue samples is the current gold standard for microbiological diagnosis of prosthetic joint infections (PJI). Additional diagnostic information may be obtained through culture of sonication fluid from explants. However, current techniques can have relatively low sensitivity, with prior antimicrobial therapy and infection by fastidious organisms influencing results. We assessed if metagenomic sequencing of total DNA extracts obtained direct from sonication fluid can provide an alternative rapid and sensitive tool for diagnosis of PJI. We compared metagenomic sequencing with standard aerobic and anaerobic culture in 97 sonication fluid samples from prosthetic joint and other orthopedic device infections. Reads from Illumina MiSeq sequencing were taxonomically classified using Kraken. Using 50 derivation samples, we determined optimal thresholds for the number and proportion of bacterial reads required to identify an infection and confirmed our findings in 47 independent validation samples. Compared to results from sonication fluid culture, the species-level sensitivity of metagenomic sequencing was 61/69 (88%; 95% confidence interval [CI], 77 to 94%; for derivation samples 35/38 [92%; 95% CI, 79 to 98%]; for validation samples, 26/31 [84%; 95% CI, 66 to 95%]), and genus-level sensitivity was 64/69 (93%; 95% CI, 84 to 98%). Species-level specificity, adjusting for plausible fastidious causes of infection, species found in concurrently obtained tissue samples, and prior antibiotics, was 85/97 (88%; 95% CI, 79 to 93%; for derivation samples, 43/50 [86%; 95% CI, 73 to 94%]; for validation samples, 42/47 [89%; 95% CI, 77 to 96%]). High levels of human DNA contamination were seen despite the use of laboratory methods to remove it. Rigorous laboratory good practice was required to minimize bacterial DNA contamination. We demonstrate that metagenomic sequencing can provide accurate diagnostic information in PJI. Our findings, combined with the increasing availability of portable, random-access sequencing technology, offer the potential to translate metagenomic sequencing into a rapid diagnostic tool in PJI. PMID:28490492

The rRNA evolution and procaryotic phylogeny

NASA Technical Reports Server (NTRS)

Fox, G. E.

1986-01-01

Studies of ribosomal RNA primary structure allow reconstruction of phylogenetic trees for prokaryotic organisms. Such studies reveal major dichotomy among the bacteria that separates them into eubacteria and archaebacteria. Both groupings are further segmented into several major divisions. The results obtained from 5S rRNA sequences are essentially the same as those obtained with the 16S rRNA data. In the case of Gram negative bacteria the ribosomal RNA sequencing results can also be directly compared with hybridization studies and cytochrome c sequencing studies. There is again excellent agreement among the several methods. It seems likely then that the overall picture of microbial phylogeny that is emerging from the RNA sequence studies is a good approximation of the true history of these organisms. The RNA data allow examination of the evolutionary process in a semi-quantitative way. The secondary structures of these RNAs are largely established. As a result it is possible to recognize examples of local structural evolution. Evolutionary pathways accounting for these events can be proposed and their probability can be assessed.

Direct typing of Canine parvovirus (CPV) from infected dog faeces by rapid mini sequencing technique.

PubMed

V, Pavana Jyothi; S, Akila; Selvan, Malini K; Naidu, Hariprasad; Raghunathan, Shwethaa; Kota, Sathish; Sundaram, R C Raja; Rana, Samir Kumar; Raj, G Dhinakar; Srinivasan, V A; Mohana Subramanian, B

2016-12-01

Canine parvovirus (CPV) is a non-enveloped single stranded DNA virus with an icosahedral capsid. Mini-sequencing based CPV typing was developed earlier to detect and differentiate all the CPV types and FPV in a single reaction. This technique was further evaluated in the present study by performing the mini-sequencing directly from fecal samples which avoided tedious virus isolation steps by cell culture system. Fecal swab samples were collected from 84 dogs with enteritis symptoms, suggestive of parvoviral infection from different locations across India. Seventy six of these samples were positive by PCR; the subsequent mini-sequencing reaction typed 74 of them as type 2a virus, and 2 samples as type 2b. Additionally, 25 of the positive samples were typed by cycle sequencing of PCR products. Direct CPV typing from fecal samples using mini-sequencing showed 100% correlation with CPV typing by cycle sequencing. Moreover, CPV typing was achieved by mini-sequencing even with faintly positive PCR amplicons which was not possible by cycle sequencing. Therefore, the mini-sequencing technique is recommended for regular epidemiological follow up of CPV types, since the technique is rapid, highly sensitive and high capacity method for CPV typing. Copyright © 2016. Published by Elsevier B.V.

Situation models and memory: the effects of temporal and causal information on recall sequence.

PubMed

Brownstein, Aaron L; Read, Stephen J

2007-10-01

Participants watched an episode of the television show Cheers on video and then reported free recall. Recall sequence followed the sequence of events in the story; if one concept was observed immediately after another, it was recalled immediately after it. We also made a causal network of the show's story and found that recall sequence followed causal links; effects were recalled immediately after their causes. Recall sequence was more likely to follow causal links than temporal sequence, and most likely to follow causal links that were temporally sequential. Results were similar at 10-minute and 1-week delayed recall. This is the most direct and detailed evidence reported on sequential effects in recall. The causal network also predicted probability of recall; concepts with more links and concepts on the main causal chain were most likely to be recalled. This extends the causal network model to more complex materials than previous research.

Assessment of a Pan-Dermatophyte Nested-PCR Compared with Conventional Methods for Direct Detection and Identification of Dermatophytosis Agents in Animals.

PubMed

Piri, Fahimeh; Zarei Mahmoudabadi, Ali; Ronagh, Ali; Ahmadi, Bahram; Makimura, Koichi; Rezaei-Matehkolaei, Ali

2018-06-26

Conventional direct microscopy with potassium hydroxide (KOH) and culture were found to lack the ability to establish a fast and specific diagnosis of dermatophytosis. A pan-dermatophyte nested-PCR assay was developed using a novel primer pair targeting the translation elongation factor 1-α (Tef-1α) sequences for direct detection and identification of most veterinary relevant dermatophytes in animal samples suspected to dermatophytosis. A total of 140 animal skin and hair samples were subjected to direct microscopy, culture, and ITS-RFLP/ITS-sequencing of culture isolates for the detection and identification of dermatophytosis agents. Nested-PCR sequencing was performed on all the extracted DNAs using a commercial kit after dissolving the specimens by mechanical beating. Nested-PCR was positive in 90% of samples, followed by direct microscopy (85.7%) and culture (75%). The degree of agreement between nested-PCR and direct microscopy (94.4%) was higher than with culture (83.3%). In 105 culture positive cases, the measures of agreement for the identification of dermatophytosis agents were as follows: 100% between nested-PCR sequencing and ITS-RFLP/ITS-sequencing and 63.8% between nested-PCR sequencing and culture. The developed nested-PCR was faster as well as more sensitive and specific than conventional methods for detection and identification of dermatophytes in clinical samples, which was particularly suitable for epidemiological studies. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Complete genomic sequence of an infectious pancreatic necrosis virus isolated from rainbow trout (Oncorhynchus mykiss) in China.

PubMed

Ji, Feng; Zhao, Jing-Zhuang; Liu, Miao; Lu, Tong-Yan; Liu, Hong-Bai; Yin, Jiasheng; Xu, Li-Ming

2017-04-01

Infectious pancreatic necrosis (IPN) is a significant disease of farmed salmonids resulting in direct economic losses due to high mortality in China. However, no gene sequence of any Chinese infectious pancreatic necrosis virus (IPNV) isolates was available. In the study, moribund rainbow trout fry samples were collected during an outbreak of IPN in Yunnan province of southwest China in 2013. An IPNV was isolated and tentatively named ChRtm213. We determined the full genome sequence of the IPNV ChRtm213 and compared it with previously identified IPNV sequences worldwide. The sequences of different structural and non-structural protein genes were compared to those of other aquatic birnaviruses sequenced to date. The results indicated that the complete genome sequence of ChRtm213 strain contains a segment A (3099 nucleotides) coding a polyprotein VP2-VP4-VP3, and a segment B (2789 nucleotides) coding a RNA-dependent RNA polymerase VP1. The phylogenetic analyses showed that ChRtm213 strain fell within genogroup 1, serotype A9 (Jasper), having similarities of 96.3% (segment A) and 97.3% (segment B) with the IPNV strain AM98 from Japan. The results suggest that the Chinese IPNV isolate has relative closer relationship with Japanese IPNV strains. The sequence of ChRtm213 was the first gene sequence of IPNV isolates in China. This study provided a robust reference for diagnosis and/or control of IPNV prevalent in China.

Generation of a Maize B Centromere Minimal Map Containing the Central Core Domain.

PubMed

Ellis, Nathanael A; Douglas, Ryan N; Jackson, Caroline E; Birchler, James A; Dawe, R Kelly

2015-10-28

The maize B centromere has been used as a model for centromere epigenetics and as the basis for building artificial chromosomes. However, there are no sequence resources for this important centromere. Here we used transposon display for the centromere-specific retroelement CRM2 to identify a collection of 40 sequence tags that flank CRM2 insertion points on the B chromosome. These were confirmed to lie within the centromere by assaying deletion breakpoints from centromere misdivision derivatives (intracentromere breakages caused by centromere fission). Markers were grouped together on the basis of their association with other markers in the misdivision series and assembled into a pseudocontig containing 10.1 kb of sequence. To identify sequences that interact directly with centromere proteins, we carried out chromatin immunoprecipitation using antibodies to centromeric histone H3 (CENH3), a defining feature of functional centromeric sequences. The CENH3 chromatin immunoprecipitation map was interpreted relative to the known transmission rates of centromere misdivision derivatives to identify a centromere core domain spanning 33 markers. A subset of seven markers was mapped in additional B centromere misdivision derivatives with the use of unique primer pairs. A derivative previously shown to have no canonical centromere sequences (Telo3-3) lacks these core markers. Our results provide a molecular map of the B chromosome centromere and identify key sequences within the map that interact directly with centromeric histone H3. Copyright © 2015 Ellis et al.

Phylogeny of sipunculan worms: A combined analysis of four gene regions and morphology.

PubMed

Schulze, Anja; Cutler, Edward B; Giribet, Gonzalo

2007-01-01

The intra-phyletic relationships of sipunculan worms were analyzed based on DNA sequence data from four gene regions and 58 morphological characters. Initially we analyzed the data under direct optimization using parsimony as optimality criterion. An implied alignment resulting from the direct optimization analysis was subsequently utilized to perform a Bayesian analysis with mixed models for the different data partitions. For this we applied a doublet model for the stem regions of the 18S rRNA. Both analyses support monophyly of Sipuncula and most of the same clades within the phylum. The analyses differ with respect to the relationships among the major groups but whereas the deep nodes in the direct optimization analysis generally show low jackknife support, they are supported by 100% posterior probability in the Bayesian analysis. Direct optimization has been useful for handling sequences of unequal length and generating conservative phylogenetic hypotheses whereas the Bayesian analysis under mixed models provided high resolution in the basal nodes of the tree.

Assessing the Relationship of Ancient and Modern Populations

PubMed Central

Schraiber, Joshua G.

2018-01-01

Genetic material sequenced from ancient samples is revolutionizing our understanding of the recent evolutionary past. However, ancient DNA is often degraded, resulting in low coverage, error-prone sequencing. Several solutions exist to this problem, ranging from simple approach, such as selecting a read at random for each site, to more complicated approaches involving genotype likelihoods. In this work, we present a novel method for assessing the relationship of an ancient sample with a modern population, while accounting for sequencing error and postmortem damage by analyzing raw reads from multiple ancient individuals simultaneously. We show that, when analyzing SNP data, it is better to sequence more ancient samples to low coverage: two samples sequenced to 0.5× coverage provide better resolution than a single sample sequenced to 2× coverage. We also examined the power to detect whether an ancient sample is directly ancestral to a modern population, finding that, with even a few high coverage individuals, even ancient samples that are very slightly diverged from the modern population can be detected with ease. When we applied our approach to European samples, we found that no ancient samples represent direct ancestors of modern Europeans. We also found that, as shown previously, the most ancient Europeans appear to have had the smallest effective population sizes, indicating a role for agriculture in modern population growth. PMID:29167200

Bypassing bacterial infection in phage display by sequencing DNA released from phage particles.

PubMed

Villequey, Camille; Kong, Xu-Dong; Heinis, Christian

2017-11-01

Phage display relies on a bacterial infection step in which the phage particles are replicated to perform multiple affinity selection rounds and to enable the identification of isolated clones by DNA sequencing. While this process is efficient for wild-type phage, the bacterial infection rate of phage with mutant or chemically modified coat proteins can be low. For example, a phage mutant with a disulfide-free p3 coat protein, used for the selection of bicyclic peptides, has a more than 100-fold reduced infection rate compared to the wild-type. A potential strategy for bypassing the bacterial infection step is to directly sequence DNA extracted from phage particles after a single round of phage panning using high-throughput sequencing. In this work, we have quantified the fraction of phage clones that can be identified by directly sequencing DNA from phage particles. The results show that the DNA of essentially all of the phage particles can be 'decoded', and that the sequence coverage for mutants equals that of amplified DNA extracted from cells infected with wild-type phage. This procedure is particularly attractive for selections with phage that have a compromised infection capacity, and it may allow phage display to be performed with particles that are not infective at all. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Diverse tulasnelloid fungi form mycorrhizas with epiphytic orchids in an Andean cloud forest.

PubMed

Suárez, Juan Pablo; Weiss, Michael; Abele, Andrea; Garnica, Sigisfredo; Oberwinkler, Franz; Kottke, Ingrid

2006-11-01

The mycorrhizal state of epiphytic orchids has been controversially discussed, and the state and mycobionts of the pleurothallid orchids, occurring abundantly and with a high number of species on stems of trees in the Andean cloud forest, were unknown. Root samples of 77 adult individuals of the epiphytic orchids Stelis hallii, S. superbiens, S. concinna and Pleurothallis lilijae were collected in a tropical mountain rainforest of southern Ecuador. Ultrastructural evidence of symbiotic interaction was combined with molecular sequencing of fungi directly from the mycorrhizas and isolation of mycobionts. Ultrastructural analyses displayed vital orchid mycorrhizas formed by fungi with an imperforate parenthesome and cell wall slime bodies typical for the genus Tulasnella. Three different Tulasnella isolates were obtained in pure culture. Phylogenetic analysis of nuclear rDNA sequences from coding regions of the ribosomal large subunit (nucLSU) and the 5.8S subunit, including parts of the internal transcribed spacers, obtained directly from the roots and from the fungal isolates, yielded seven distinct Tulasnella clades. Tulasnella mycobionts in Stelis concinna were restricted to two Tulasnella sequence types while the other orchids were associated with up to six Tulasnella sequence types. All Tulasnella sequences are new to science and distinct from known sequences of mycobionts of terrestrial orchids. The results indicate that tulasnelloid fungi, adapted to the conditions on tree stems, might be important for orchid growth and maintenance in the Andean cloud forest.

Rapid and accurate detection of KRAS mutations in colorectal cancers using the isothermal-based optical sensor for companion diagnostics

PubMed Central

Koo, Bonhan; Lee, Tae Yoon; Lee, Jeong Hoon; Shin, Yong; Lim, Seok-Byung

2017-01-01

Although KRAS mutational status testing is becoming a companion diagnostic tool for managing patients with colorectal cancer (CRC), there are still several difficulties when analyzing KRAS mutations using the existing assays, particularly with regard to low sensitivity, its time-consuming, and the need for large instruments. We developed a rapid, sensitive, and specific mutation detection assay based on the bio-photonic sensor termed ISAD (isothermal solid-phase amplification/detection), and used it to analyze KRAS gene mutations in human clinical samples. To validate the ISAD-KRAS assay for use in clinical diagnostics, we examined for hotspot KRAS mutations (codon 12 and codon 13) in 70 CRC specimens using PCR and direct sequencing methods. In a serial dilution study, ISAD-KRAS could detect mutations in a sample containing only 1% of the mutant allele in a mixture of wild-type DNA, whereas both PCR and direct sequencing methods could detect mutations in a sample containing approximately 30% of mutant cells. The results of the ISAD-KRAS assay from 70 clinical samples matched those from PCR and direct sequencing, except in 5 cases, wherein ISAD-KRAS could detect mutations that were not detected by PCR and direct sequencing. We also found that the sensitivity and specificity of ISAD-KRAS were 100% within 30 min. The ISAD-KRAS assay provides a rapid, highly sensitive, and label-free method for KRAS mutation testing, and can serve as a robust and near patient testing approach for the rapid detection of patients most likely to respond to anti-EGFR drugs. PMID:29137388

Direct aperture optimization: a turnkey solution for step-and-shoot IMRT.

PubMed

Shepard, D M; Earl, M A; Li, X A; Naqvi, S; Yu, C

2002-06-01

IMRT treatment plans for step-and-shoot delivery have traditionally been produced through the optimization of intensity distributions (or maps) for each beam angle. The optimization step is followed by the application of a leaf-sequencing algorithm that translates each intensity map into a set of deliverable aperture shapes. In this article, we introduce an automated planning system in which we bypass the traditional intensity optimization, and instead directly optimize the shapes and the weights of the apertures. We call this approach "direct aperture optimization." This technique allows the user to specify the maximum number of apertures per beam direction, and hence provides significant control over the complexity of the treatment delivery. This is possible because the machine dependent delivery constraints imposed by the MLC are enforced within the aperture optimization algorithm rather than in a separate leaf-sequencing step. The leaf settings and the aperture intensities are optimized simultaneously using a simulated annealing algorithm. We have tested direct aperture optimization on a variety of patient cases using the EGS4/BEAM Monte Carlo package for our dose calculation engine. The results demonstrate that direct aperture optimization can produce highly conformal step-and-shoot treatment plans using only three to five apertures per beam direction. As compared with traditional optimization strategies, our studies demonstrate that direct aperture optimization can result in a significant reduction in both the number of beam segments and the number of monitor units. Direct aperture optimization therefore produces highly efficient treatment deliveries that maintain the full dosimetric benefits of IMRT.

Next-generation Sequencing-based genomic profiling: Fostering innovation in cancer care?

PubMed

Fernandes, Gustavo S; Marques, Daniel F; Girardi, Daniel M; Braghiroli, Maria Ignez F; Coudry, Renata A; Meireles, Sibele I; Katz, Artur; Hoff, Paulo M

2017-10-01

With the development of next-generation sequencing (NGS) technologies, DNA sequencing has been increasingly utilized in clinical practice. Our goal was to investigate the impact of genomic evaluation on treatment decisions for heavily pretreated patients with metastatic cancer. We analyzed metastatic cancer patients from a single institution whose cancers had progressed after all available standard-of-care therapies and whose tumors underwent next-generation sequencing analysis. We determined the percentage of patients who received any therapy directed by the test, and its efficacy. From July 2013 to December 2015, 185 consecutive patients were tested using a commercially available next-generation sequencing-based test, and 157 patients were eligible. Sixty-six patients (42.0%) were female, and 91 (58.0%) were male. The mean age at diagnosis was 52.2 years, and the mean number of pre-test lines of systemic treatment was 2.7. One hundred and seventy-seven patients (95.6%) had at least one identified gene alteration. Twenty-four patients (15.2%) underwent systemic treatment directed by the test result. Of these, one patient had a complete response, four (16.7%) had partial responses, two (8.3%) had stable disease, and 17 (70.8%) had disease progression as the best result. The median progression-free survival time with matched therapy was 1.6 months, and the median overall survival was 10 months. We identified a high prevalence of gene alterations using an next-generation sequencing test. Although some benefit was associated with the matched therapy, most of the patients had disease progression as the best response, indicating the limited biological potential and unclear clinical relevance of this practice.

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

A MAP-based image interpolation method via Viterbi decoding of Markov chains of interpolation functions.

PubMed

Vedadi, Farhang; Shirani, Shahram

2014-01-01

A new method of image resolution up-conversion (image interpolation) based on maximum a posteriori sequence estimation is proposed. Instead of making a hard decision about the value of each missing pixel, we estimate the missing pixels in groups. At each missing pixel of the high resolution (HR) image, we consider an ensemble of candidate interpolation methods (interpolation functions). The interpolation functions are interpreted as states of a Markov model. In other words, the proposed method undergoes state transitions from one missing pixel position to the next. Accordingly, the interpolation problem is translated to the problem of estimating the optimal sequence of interpolation functions corresponding to the sequence of missing HR pixel positions. We derive a parameter-free probabilistic model for this to-be-estimated sequence of interpolation functions. Then, we solve the estimation problem using a trellis representation and the Viterbi algorithm. Using directional interpolation functions and sequence estimation techniques, we classify the new algorithm as an adaptive directional interpolation using soft-decision estimation techniques. Experimental results show that the proposed algorithm yields images with higher or comparable peak signal-to-noise ratios compared with some benchmark interpolation methods in the literature while being efficient in terms of implementation and complexity considerations.

Line scanning system for direct digital chemiluminescence imaging of DNA sequencing blots

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

Karger, A.E.; Weiss, R.; Gesteland, R.F.

A cryogenically cooled charge-coupled device (CCD) camera equipped with an area CCD array is used in a line scanning system for low-light-level imaging of chemiluminescent DNA sequencing blots. Operating the CCD camera in time-delayed integration (TDI) mode results in continuous data acquisition independent of the length of the CCD array. Scanning is possible with a resolution of 1.4 line pairs/mm at the 50% level of the modulation transfer function. High-sensitivity, low-light-level scanning of chemiluminescent direct-transfer electrophoresis (DTE) DNA sequencing blots is shown. The detection of DNA fragments on the blot involves DNA-DNA hybridization with oligonucleotide-alkaline phosphatase conjugate and 1,2-dioxetane-based chemiluminescence.more » The width of the scan allows the recording of up to four sequencing reactions (16 lanes) on one scan. The scan speed of 52 cm/h used for the sequencing blots corresponds to a data acquisition rate of 384 pixels/s. The chemiluminescence detection limit on the scanned images is 3.9 [times] 10[sup [minus]18] mol of plasmid DNA. A conditional median filter is described to remove spikes caused by cosmic ray events from the CCD images. 39 refs., 9 refs.« less

Cysteine-containing peptide tag for site-specific conjugation of proteins

DOEpatents

Backer, Marina V.; Backer, Joseph M.

2008-04-08

The present invention is directed to a biological conjugate, comprising: (a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising the polypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein; and (b) a binding moiety bound to the targeting moiety; the biological conjugate having a covalent bond between the thiol group of SEQ ID NO:2 and a functional group in the binding moiety. The present invention is directed to a biological conjugate, comprising: (a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising the polypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein; and (b) a binding moiety that comprises an adapter protein, the adapter protein having a thiol group; the biological conjugate having a disulfide bond between the thiol group of SEQ ID NO:2 and the thiol group of the adapter protein. The present invention is also directed to biological sequences employed in the above biological conjugates, as well as pharmaceutical preparations and methods using the above biological conjugates.

Cysteine-containing peptide tag for site-specific conjugation of proteins

DOEpatents

Backer, Marina V.; Backer, Joseph M.

2010-10-05

The present invention is directed to a biological conjugate, comprising: (a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising the polypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein; and (b) a binding moiety bound to the targeting moiety; the biological conjugate having a covalent bond between the thiol group of SEQ ID NO:2 and a functional group in the binding moiety. The present invention is directed to a biological conjugate, comprising: (a) a targeting moiety comprising a polypeptide having an amino acid sequence comprising the polypeptide sequence of SEQ ID NO:2 and the polypeptide sequence of a selected targeting protein; and (b) a binding moiety that comprises an adapter protein, the adapter protein having a thiol group; the biological conjugate having a disulfide bond between the thiol group of SEQ ID NO:2 and the thiol group of the adapter protein. The present invention is also directed to biological sequences employed in the above biological conjugates, as well as pharmaceutical preparations and methods using the above biological conjugates.

Dual Priming Oligonucleotides for Broad-Range Amplification of the Bacterial 16S rRNA Gene Directly from Human Clinical Specimens

PubMed Central

Simmon, Keith; Karaca, Dilek; Langeland, Nina; Wiker, Harald G.

2012-01-01

Broad-range amplification and sequencing of the bacterial 16S rRNA gene directly from clinical specimens are offered as a diagnostic service in many laboratories. One major pitfall is primer cross-reactivity with human DNA which will result in mixed chromatograms. Mixed chromatograms will complicate subsequent sequence analysis and impede identification. In SYBR green real-time PCR assays, it can also affect crossing threshold values and consequently the status of a specimen as positive or negative. We evaluated two conventional primer pairs in common use and a new primer pair based on the dual priming oligonucleotide (DPO) principle. Cross-reactivity was observed when both conventional primer pairs were used, resulting in interpretation difficulties. No cross-reactivity was observed using the DPOs even in specimens with a high ratio of human to bacterial DNA. In addition to reducing cross-reactivity, the DPO principle also offers a high degree of flexibility in the design of primers and should be considered for any PCR assay intended for detection and identification of pathogens directly from human clinical specimens. PMID:22278843

Pitfalls and caveats in BRCA sequencing.

PubMed

Bellosillo, Beatriz; Tusquets, Ignacio

2006-01-01

Between 5 and 10% of breast cancer cases are considered to result from hereditary predisposition. Germ-line mutations in BRCA1 and BRCA2 are responsible for an inherited predisposition of breast and ovarian cancer. Direct nucleotide sequencing is considered the gold standard technique for mutation detection for genes such as BRCA1 and BRCA2. In many laboratories that analyze BRCA1 and BRCA2, previous to direct sequencing, screening techniques to identify sequence variants in the PCR amplicons are performed. The mutations detected in these genes may be frameshift mutations (insertions or deletions), nonsense mutations, or missense mutations. The clinical interpretation of the mutation as the cause of the disease may be difficult to establish in the case of missense mutations. Only in 30-70% of the families in which a hereditary component is suspected, a mutation in BRCA1 and/or BRCA2 is detected. Negative results may be due to: wrong selection of the proband; mutations in the regulatory portion of the genes; gene silencing due to epigenetic phenomena; or large genomic rearrangements that produce deletions of whole exons. Another possibility that explains the lack of detection of alterations in BRCA1 or BRCA2 is the presence of mutations in undiscovered genes or in genes that interact with BRCA1 and/or BRCA2, which may be low-penetrance genes, like CHEK2.

On the nature of phase attraction in sensorimotor synchronization with interleaved auditory sequences.

PubMed

Repp, Bruno H

2004-10-01

In a task that requires in-phase synchronization of finger taps with an isochronous sequence of target tones that is interleaved with a sequence of distractor tones at various fixed phase relationships, the taps tend to be attracted to the distractor tones, especially when the distractor tones closely precede the target tones [Repp, B. H. (2003a). Phase attraction in sensorimotor synchronization with auditory sequences: Effects of single and periodic distractors on synchronization accuracy. Journal of Experimental Psychology: Human Perception and Performance, 29, 290-309]. The present research addressed two related questions about this distractor effect: (1) Is it a function of the absolute temporal separation or of the relative phase of the two stimulus sequences? (2) Is it the result of perceptual grouping (integration) of target and distractor tones or of simultaneous attraction to two independent sequences? In three experiments, distractor effects were compared across two different sequence rates. The results suggest that absolute temporal separation, not relative phase, is the critical variable. Experiment 3 also included an anti-phase tapping task that addressed the second question directly. The results suggest that the attraction of taps to distractor tones is caused mainly by temporal integration of target and distractor tones within a fixed window of 100-150 ms duration, with the earlier-occurring tone being weighted more strongly than the later-occurring one.

BCM Search Launcher--an integrated interface to molecular biology data base search and analysis services available on the World Wide Web.

PubMed

Smith, R F; Wiese, B A; Wojzynski, M K; Davison, D B; Worley, K C

1996-05-01

The BCM Search Launcher is an integrated set of World Wide Web (WWW) pages that organize molecular biology-related search and analysis services available on the WWW by function, and provide a single point of entry for related searches. The Protein Sequence Search Page, for example, provides a single sequence entry form for submitting sequences to WWW servers that offer remote access to a variety of different protein sequence search tools, including BLAST, FASTA, Smith-Waterman, BEAUTY, PROSITE, and BLOCKS searches. Other Launch pages provide access to (1) nucleic acid sequence searches, (2) multiple and pair-wise sequence alignments, (3) gene feature searches, (4) protein secondary structure prediction, and (5) miscellaneous sequence utilities (e.g., six-frame translation). The BCM Search Launcher also provides a mechanism to extend the utility of other WWW services by adding supplementary hypertext links to results returned by remote servers. For example, links to the NCBI's Entrez data base and to the Sequence Retrieval System (SRS) are added to search results returned by the NCBI's WWW BLAST server. These links provide easy access to auxiliary information, such as Medline abstracts, that can be extremely helpful when analyzing BLAST data base hits. For new or infrequent users of sequence data base search tools, we have preset the default search parameters to provide the most informative first-pass sequence analysis possible. We have also developed a batch client interface for Unix and Macintosh computers that allows multiple input sequences to be searched automatically as a background task, with the results returned as individual HTML documents directly to the user's system. The BCM Search Launcher and batch client are available on the WWW at URL http:@gc.bcm.tmc.edu:8088/search-launcher.html.

nbCNV: a multi-constrained optimization model for discovering copy number variants in single-cell sequencing data.

PubMed

Zhang, Changsheng; Cai, Hongmin; Huang, Jingying; Song, Yan

2016-09-17

Variations in DNA copy number have an important contribution to the development of several diseases, including autism, schizophrenia and cancer. Single-cell sequencing technology allows the dissection of genomic heterogeneity at the single-cell level, thereby providing important evolutionary information about cancer cells. In contrast to traditional bulk sequencing, single-cell sequencing requires the amplification of the whole genome of a single cell to accumulate enough samples for sequencing. However, the amplification process inevitably introduces amplification bias, resulting in an over-dispersing portion of the sequencing data. Recent study has manifested that the over-dispersed portion of the single-cell sequencing data could be well modelled by negative binomial distributions. We developed a read-depth based method, nbCNV to detect the copy number variants (CNVs). The nbCNV method uses two constraints-sparsity and smoothness to fit the CNV patterns under the assumption that the read signals are negatively binomially distributed. The problem of CNV detection was formulated as a quadratic optimization problem, and was solved by an efficient numerical solution based on the classical alternating direction minimization method. Extensive experiments to compare nbCNV with existing benchmark models were conducted on both simulated data and empirical single-cell sequencing data. The results of those experiments demonstrate that nbCNV achieves superior performance and high robustness for the detection of CNVs in single-cell sequencing data.

High-capacity quantum secure direct communication using hyper-entanglement of photonic qubits

NASA Astrophysics Data System (ADS)

Cai, Jiarui; Pan, Ziwen; Wang, Tie-Jun; Wang, Sihai; Wang, Chuan

2016-11-01

Hyper-entanglement is a system constituted by photons entangled in multiple degrees of freedom (DOF), being considered as a promising way of increasing channel capacity and guaranteeing powerful eavesdropping safeguard. In this work, we propose a coding scheme based on a 3-particle hyper-entanglement of polarization and orbital angular momentum (OAM) system and its application as a quantum secure direct communication (QSDC) protocol. The OAM values are specially encoded by Fibonacci sequence and the polarization carries information by defined unitary operations. The internal relations of the secret message enhances security due to principle of quantum mechanics and Fibonacci sequence. We also discuss the coding capacity and security property along with some simulation results to show its superiority and extensibility.

A Unified Theoretical Framework for Cognitive Sequencing.

PubMed

Savalia, Tejas; Shukla, Anuj; Bapi, Raju S

2016-01-01

The capacity to sequence information is central to human performance. Sequencing ability forms the foundation stone for higher order cognition related to language and goal-directed planning. Information related to the order of items, their timing, chunking and hierarchical organization are important aspects in sequencing. Past research on sequencing has emphasized two distinct and independent dichotomies: implicit vs. explicit and goal-directed vs. habits. We propose a theoretical framework unifying these two streams. Our proposal relies on brain's ability to implicitly extract statistical regularities from the stream of stimuli and with attentional engagement organizing sequences explicitly and hierarchically. Similarly, sequences that need to be assembled purposively to accomplish a goal require engagement of attentional processes. With repetition, these goal-directed plans become habits with concomitant disengagement of attention. Thus, attention and awareness play a crucial role in the implicit-to-explicit transition as well as in how goal-directed plans become automatic habits. Cortico-subcortical loops basal ganglia-frontal cortex and hippocampus-frontal cortex loops mediate the transition process. We show how the computational principles of model-free and model-based learning paradigms, along with a pivotal role for attention and awareness, offer a unifying framework for these two dichotomies. Based on this framework, we make testable predictions related to the potential influence of response-to-stimulus interval (RSI) on developing awareness in implicit learning tasks.

A Unified Theoretical Framework for Cognitive Sequencing

PubMed Central

Savalia, Tejas; Shukla, Anuj; Bapi, Raju S.

2016-01-01

The capacity to sequence information is central to human performance. Sequencing ability forms the foundation stone for higher order cognition related to language and goal-directed planning. Information related to the order of items, their timing, chunking and hierarchical organization are important aspects in sequencing. Past research on sequencing has emphasized two distinct and independent dichotomies: implicit vs. explicit and goal-directed vs. habits. We propose a theoretical framework unifying these two streams. Our proposal relies on brain's ability to implicitly extract statistical regularities from the stream of stimuli and with attentional engagement organizing sequences explicitly and hierarchically. Similarly, sequences that need to be assembled purposively to accomplish a goal require engagement of attentional processes. With repetition, these goal-directed plans become habits with concomitant disengagement of attention. Thus, attention and awareness play a crucial role in the implicit-to-explicit transition as well as in how goal-directed plans become automatic habits. Cortico-subcortical loops basal ganglia-frontal cortex and hippocampus-frontal cortex loops mediate the transition process. We show how the computational principles of model-free and model-based learning paradigms, along with a pivotal role for attention and awareness, offer a unifying framework for these two dichotomies. Based on this framework, we make testable predictions related to the potential influence of response-to-stimulus interval (RSI) on developing awareness in implicit learning tasks. PMID:27917146

HLA-B*5808, a new HLA-B allele characterized by sequence based typing.

PubMed

Poli, F; Crespiatico, L; Frison, S; Longhi, E; Marlianici, E; Scalamogna, M

2003-12-01

This brief communication describes a new HLA-B allele (HLA-B*5808) detected in an Italian white volunteer bone marrow donor. With serology, this subject was typed as HLA-B15,17, whereas with molecular biology B*15, B*51, B*52 and/or B*58 could be assigned. In order to clarify the results, direct and cloning sequencing of exons 2, 3 and 4 were carried out. This new allele is identical to HLA-B*5801 in exon 2 except for a silent point mutation at nucleotide 141 where a C is substituted by a T; exons 3 and 4 are typical of HLA-B*51, B*52 and B*78. The peculiar sequence of B*5808 could explain the discrepancy between the serological and molecular typing results.

ddClone: joint statistical inference of clonal populations from single cell and bulk tumour sequencing data.

PubMed

Salehi, Sohrab; Steif, Adi; Roth, Andrew; Aparicio, Samuel; Bouchard-Côté, Alexandre; Shah, Sohrab P

2017-03-01

Next-generation sequencing (NGS) of bulk tumour tissue can identify constituent cell populations in cancers and measure their abundance. This requires computational deconvolution of allelic counts from somatic mutations, which may be incapable of fully resolving the underlying population structure. Single cell sequencing (SCS) is a more direct method, although its replacement of NGS is impeded by technical noise and sampling limitations. We propose ddClone, which analytically integrates NGS and SCS data, leveraging their complementary attributes through joint statistical inference. We show on real and simulated datasets that ddClone produces more accurate results than can be achieved by either method alone.

Alkahest NuclearBLAST : a user-friendly BLAST management and analysis system

PubMed Central

Diener, Stephen E; Houfek, Thomas D; Kalat, Sam E; Windham, DE; Burke, Mark; Opperman, Charles; Dean, Ralph A

2005-01-01

Background - Sequencing of EST and BAC end datasets is no longer limited to large research groups. Drops in per-base pricing have made high throughput sequencing accessible to individual investigators. However, there are few options available which provide a free and user-friendly solution to the BLAST result storage and data mining needs of biologists. Results - Here we describe NuclearBLAST, a batch BLAST analysis, storage and management system designed for the biologist. It is a wrapper for NCBI BLAST which provides a user-friendly web interface which includes a request wizard and the ability to view and mine the results. All BLAST results are stored in a MySQL database which allows for more advanced data-mining through supplied command-line utilities or direct database access. NuclearBLAST can be installed on a single machine or clustered amongst a number of machines to improve analysis throughput. NuclearBLAST provides a platform which eases data-mining of multiple BLAST results. With the supplied scripts, the program can export data into a spreadsheet-friendly format, automatically assign Gene Ontology terms to sequences and provide bi-directional best hits between two datasets. Users with SQL experience can use the database to ask even more complex questions and extract any subset of data they require. Conclusion - This tool provides a user-friendly interface for requesting, viewing and mining of BLAST results which makes the management and data-mining of large sets of BLAST analyses tractable to biologists. PMID:15958161

Two distinct sequences of blue straggler stars in the globular cluster M 30.

PubMed

Ferraro, F R; Beccari, G; Dalessandro, E; Lanzoni, B; Sills, A; Rood, R T; Pecci, F Fusi; Karakas, A I; Miocchi, P; Bovinelli, S

2009-12-24

Stars in globular clusters are generally believed to have all formed at the same time, early in the Galaxy's history. 'Blue stragglers' are stars massive enough that they should have evolved into white dwarfs long ago. Two possible mechanisms have been proposed for their formation: mass transfer between binary companions and stellar mergers resulting from direct collisions between two stars. Recently the binary explanation was claimed to be dominant. Here we report that there are two distinct parallel sequences of blue stragglers in M 30. This globular cluster is thought to have undergone 'core collapse', during which both the collision rate and the mass transfer activity in binary systems would have been enhanced. We suggest that the two observed sequences are a consequence of cluster core collapse, with the bluer population arising from direct stellar collisions and the redder one arising from the evolution of close binaries that are probably still experiencing an active phase of mass transfer.

Genome assembly from synthetic long read clouds

PubMed Central

Kuleshov, Volodymyr; Snyder, Michael P.; Batzoglou, Serafim

2016-01-01

Motivation: Despite rapid progress in sequencing technology, assembling de novo the genomes of new species as well as reconstructing complex metagenomes remains major technological challenges. New synthetic long read (SLR) technologies promise significant advances towards these goals; however, their applicability is limited by high sequencing requirements and the inability of current assembly paradigms to cope with combinations of short and long reads. Results: Here, we introduce Architect, a new de novo scaffolder aimed at SLR technologies. Unlike previous assembly strategies, Architect does not require a costly subassembly step; instead it assembles genomes directly from the SLR’s underlying short reads, which we refer to as read clouds. This enables a 4- to 20-fold reduction in sequencing requirements and a 5-fold increase in assembly contiguity on both genomic and metagenomic datasets relative to state-of-the-art assembly strategies aimed directly at fully subassembled long reads. Availability and Implementation: Our source code is freely available at https://github.com/kuleshov/architect. Contact: kuleshov@stanford.edu PMID:27307620

Flexible and polarization-controllable diffusion metasurface with optical transparency

NASA Astrophysics Data System (ADS)

Zhuang, Yaqiang; Wang, Guangming; Liang, Jiangang; Cai, Tong; Guo, Wenlong; Zhang, Qingfeng

2017-11-01

In this paper, a novel coding metasurface is proposed to realize polarization-controllable diffusion scattering. The anisotropic Jerusalem-cross unit cell is employed as the basic coding element due to its polarization-dependent phase response. The isotropic random coding sequence is firstly designed to obtain diffusion scattering, and the anisotropic random coding sequence is subsequently realized by adding different periodic coding sequences to the original isotropic one along different directions. For demonstration, we designed and fabricated a flexible polarization-controllable diffusion metasurface (PCDM) with both chessboard diffusion and hedge diffusion under different polarizations. The specular scattering reduction performance of the anisotropic metasurface is better than the isotropic one because the scattered energies are redirected away from the specular reflection direction. For potential applications, the flexible PCDM wrapped around a cylinder structure is investigated and tested for polarization-controllable diffusion scattering. The numerical and experimental results coincide well, indicating anisotropic low scatterings with comparable performances. This paper provides an alternative approach for designing high-performance, flexible, low-scattering platforms.

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.

Processing Motion Signals in Complex Environments

NASA Technical Reports Server (NTRS)

Verghese, Preeti

2000-01-01

Motion information is critical for human locomotion and scene segmentation. Currently we have excellent neurophysiological models that are able to predict human detection and discrimination of local signals. Local motion signals are insufficient by themselves to guide human locomotion and to provide information about depth, object boundaries and surface structure. My research is aimed at understanding the mechanisms underlying the combination of motion signals across space and time. A target moving on an extended trajectory amidst noise dots in Brownian motion is much more detectable than the sum of signals generated by independent motion energy units responding to the trajectory segments. This result suggests that facilitation occurs between motion units tuned to similar directions, lying along the trajectory path. We investigated whether the interaction between local motion units along the motion direction is mediated by contrast. One possibility is that contrast-driven signals from motion units early in the trajectory sequence are added to signals in subsequent units. If this were the case, then units later in the sequence would have a larger signal than those earlier in the sequence. To test this possibility, we compared contrast discrimination thresholds for the first and third patches of a triplet of sequentially presented Gabor patches, aligned along the motion direction. According to this simple additive model, contrast increment thresholds for the third patch should be higher than thresholds for the first patch.The lack of a measurable effect on contrast thresholds for these various manipulations suggests that the pooling of signals along a trajectory is not mediated by contrast-driven signals. Instead, these results are consistent with models that propose that the facilitation of trajectory signals is achieved by a second-level network that chooses the strongest local motion signals and combines them if they occur in a spatio-temporal sequence consistent with a trajectory. These results parallel the lack of increased apparent contrast along a static contour made up of similarly oriented elements.

Direct Detection of Drug-Resistant Hepatitis B Virus in Serum Using a Dendron-Modified Microarray

PubMed Central

Kim, Doo Hyun; Kang, Hong Seok; Hur, Seong-Suk; Sim, Seobo; Ahn, Sung Hyun; Park, Yong Kwang; Park, Eun-Sook; Lee, Ah Ram; Park, Soree; Kwon, So Young; Lee, Jeong-Hoon

2018-01-01

Background/Aims Direct sequencing is the gold standard for the detection of drug-resistance mutations in hepatitis B virus (HBV); however, this procedure is time-consuming, labor-intensive, and difficult to adapt to high-throughput screening. In this study, we aimed to develop a dendron-modified DNA microarray for the detection of genotypic resistance mutations and evaluate its efficiency. Methods The specificity, sensitivity, and selectivity of dendron-modified slides for the detection of representative drug-resistance mutations were evaluated and compared to those of conventional slides. The diagnostic accuracy was validated using sera obtained from 13 patients who developed viral breakthrough during lamivudine, adefovir, or entecavir therapy and compared with the accuracy of restriction fragment mass polymorphism and direct sequencing data. Results The dendron-modified slides significantly outperformed the conventional microarray slides and were able to detect HBV DNA at a very low level (1 copy/μL). Notably, HBV mutants could be detected in the chronic hepatitis B patient sera without virus purification. The validation of our data revealed that this technique is fully compatible with sequencing data of drug-resistant HBV. Conclusions We developed a novel diagnostic technique for the simultaneous detection of several drug-resistance mutations using a dendron-modified DNA microarray. This technique can be directly applied to sera from chronic hepatitis B patients who show resistance to several nucleos(t)ide analogues. PMID:29271185

Effectiveness of the standard and an alternative set of Streptococcus pneumoniae multi locus sequence typing primers.

PubMed

Adamiak, Paul; Vanderkooi, Otto G; Kellner, James D; Schryvers, Anthony B; Bettinger, Julie A; Alcantara, Joenel

2014-06-03

Multi-locus sequence typing (MLST) is a portable, broadly applicable method for classifying bacterial isolates at an intra-species level. This methodology provides clinical and scientific investigators with a standardized means of monitoring evolution within bacterial populations. MLST uses the DNA sequences from a set of genes such that each unique combination of sequences defines an isolate's sequence type. In order to reliably determine the sequence of a typing gene, matching sequence reads for both strands of the gene must be obtained. This study assesses the ability of both the standard, and an alternative set of, Streptococcus pneumoniae MLST primers to completely sequence, in both directions, the required typing alleles. The results demonstrated that for five (aroE, recP, spi, xpt, ddl) of the seven S. pneumoniae typing alleles, the standard primers were unable to obtain the complete forward and reverse sequences. This is due to the standard primers annealing too closely to the target regions, and current sequencing technology failing to sequence the bases that are too close to the primer. The alternative primer set described here, which includes a combination of primers proposed by the CDC and several designed as part of this study, addresses this limitation by annealing to highly conserved segments further from the target region. This primer set was subsequently employed to sequence type 105 S. pneumoniae isolates collected by the Canadian Immunization Monitoring Program ACTive (IMPACT) over a period of 18 years. The inability of several of the standard S. pneumoniae MLST primers to fully sequence the required region was consistently observed and is the result of a shift in sequencing technology occurring after the original primers were designed. The results presented here introduce clear documentation describing this phenomenon into the literature, and provide additional guidance, through the introduction of a widely validated set of alternative primers, to research groups seeking to undertake S. pneumoniae MLST based studies.

Non-Homologous End Joining and Homology Directed DNA Repair Frequency of Double-Stranded Breaks Introduced by Genome Editing Reagents.

PubMed

Zaboikin, Michail; Zaboikina, Tatiana; Freter, Carl; Srinivasakumar, Narasimhachar

2017-01-01

Genome editing using transcription-activator like effector nucleases or RNA guided nucleases allows one to precisely engineer desired changes within a given target sequence. The genome editing reagents introduce double stranded breaks (DSBs) at the target site which can then undergo DNA repair by non-homologous end joining (NHEJ) or homology directed recombination (HDR) when a template DNA molecule is available. NHEJ repair results in indel mutations at the target site. As PCR amplified products from mutant target regions are likely to exhibit different melting profiles than PCR products amplified from wild type target region, we designed a high resolution melting analysis (HRMA) for rapid identification of efficient genome editing reagents. We also designed TaqMan assays using probes situated across the cut site to discriminate wild type from mutant sequences present after genome editing. The experiments revealed that the sensitivity of the assays to detect NHEJ-mediated DNA repair could be enhanced by selection of transfected cells to reduce the contribution of unmodified genomic DNA from untransfected cells to the DNA melting profile. The presence of donor template DNA lacking the target sequence at the time of genome editing further enhanced the sensitivity of the assays for detection of mutant DNA molecules by excluding the wild-type sequences modified by HDR. A second TaqMan probe that bound to an adjacent site, outside of the primary target cut site, was used to directly determine the contribution of HDR to DNA repair in the presence of the donor template sequence. The TaqMan qPCR assay, designed to measure the contribution of NHEJ and HDR in DNA repair, corroborated the results from HRMA. The data indicated that genome editing reagents can produce DSBs at high efficiency in HEK293T cells but a significant proportion of these are likely masked by reversion to wild type as a result of HDR. Supplying a donor plasmid to provide a template for HDR (that eliminates a PCR amplifiable target) revealed these cryptic DSBs and facilitated the determination of the true efficacy of genome editing reagents. The results indicated that in HEK293T cells, approximately 40% of the DSBs introduced by genome editing, were available for participation in HDR.

Compressing DNA sequence databases with coil

PubMed Central

White, W Timothy J; Hendy, Michael D

2008-01-01

Background Publicly available DNA sequence databases such as GenBank are large, and are growing at an exponential rate. The sheer volume of data being dealt with presents serious storage and data communications problems. Currently, sequence data is usually kept in large "flat files," which are then compressed using standard Lempel-Ziv (gzip) compression – an approach which rarely achieves good compression ratios. While much research has been done on compressing individual DNA sequences, surprisingly little has focused on the compression of entire databases of such sequences. In this study we introduce the sequence database compression software coil. Results We have designed and implemented a portable software package, coil, for compressing and decompressing DNA sequence databases based on the idea of edit-tree coding. coil is geared towards achieving high compression ratios at the expense of execution time and memory usage during compression – the compression time represents a "one-off investment" whose cost is quickly amortised if the resulting compressed file is transmitted many times. Decompression requires little memory and is extremely fast. We demonstrate a 5% improvement in compression ratio over state-of-the-art general-purpose compression tools for a large GenBank database file containing Expressed Sequence Tag (EST) data. Finally, coil can efficiently encode incremental additions to a sequence database. Conclusion coil presents a compelling alternative to conventional compression of flat files for the storage and distribution of DNA sequence databases having a narrow distribution of sequence lengths, such as EST data. Increasing compression levels for databases having a wide distribution of sequence lengths is a direction for future work. PMID:18489794

No effects of transcranial DLPFC stimulation on implicit task sequence learning and consolidation.

PubMed

Savic, Branislav; Cazzoli, Dario; Müri, René; Meier, Beat

2017-08-29

Neurostimulation of the dorsolateral prefrontal cortex (DLPFC) can modulate performance in cognitive tasks. In a recent study, however, transcranial direct current stimulation (tDCS) of the DLPFC did not affect implicit task sequence learning and consolidation in a paradigm that involved bimanual responses. Because bimanual performance increases the coupling between homologous cortical areas of the hemispheres and left and right DLPFC were stimulated separately the null findings may have been due to the bimanual setup. The aim of the present study was to test the effect of neuro-stimulation on sequence learning in a uni-manual setup. For this purpose two experiments were conducted. In Experiment 1, the DLPFC was stimulated with tDCS. In Experiment 2 the DLPFC was stimulated with transcranial magnetic stimulation (TMS). In both experiments, consolidation was measured 24 hours later. The results showed that sequence learning was present in all conditions and sessions, but it was not influenced by stimulation. Likewise, consolidation of sequence learning was robust across sessions, but it was not influenced by stimulation. These results replicate and extend previous findings. They indicate that established tDCS and TMS protocols on the DLPFC do not influence implicit task sequence learning and consolidation.

Proliferating cell nuclear antigen (Pcna) as a direct downstream target gene of Hoxc8

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

Min, Hyehyun; Lee, Ji-Yeon; Bok, Jinwoong

2010-02-19

Hoxc8 is a member of Hox family transcription factors that play crucial roles in spatiotemporal body patterning during embryogenesis. Hox proteins contain a conserved 61 amino acid homeodomain, which is responsible for recognition and binding of the proteins onto Hox-specific DNA binding motifs and regulates expression of their target genes. Previously, using proteome analysis, we identified Proliferating cell nuclear antigen (Pcna) as one of the putative target genes of Hoxc8. Here, we asked whether Hoxc8 regulates Pcna expression by directly binding to the regulatory sequence of Pcna. In mouse embryos at embryonic day 11.5, the expression pattern of Pcna wasmore » similar to that of Hoxc8 along the anteroposterior body axis. Moreover, Pcna transcript levels as well as cell proliferation rate were increased by overexpression of Hoxc8 in C3H10T1/2 mouse embryonic fibroblast cells. Characterization of 2.3 kb genomic sequence upstream of Pcna coding region revealed that the upstream sequence contains several Hox core binding sequences and one Hox-Pbx binding sequence. Direct binding of Hoxc8 proteins to the Pcna regulatory sequence was verified by chromatin immunoprecipitation assay. Taken together, our data suggest that Pcna is a direct downstream target of Hoxc8.« less

Diagnostics for Yaws Eradication: Insights From Direct Next-Generation Sequencing of Cutaneous Strains of Treponema pallidum

PubMed Central

Marks, Michael; Fookes, Maria; Wagner, Josef; Butcher, Robert; Ghinai, Rosanna; Sokana, Oliver; Sarkodie, Yaw-Adu; Lukehart, Sheila A; Solomon, Anthony W; Mabey, David C W; Thomson, Nicholas

2018-01-01

Abstract Background Yaws-like chronic ulcers can be caused by Treponema pallidum subspecies pertenue, Haemophilus ducreyi, or other, still-undefined bacteria. To permit accurate evaluation of yaws elimination efforts, programmatic use of molecular diagnostics is required. The accuracy and sensitivity of current tools remain unclear because our understanding of T. pallidum diversity is limited by the low number of sequenced genomes. Methods We tested samples from patients with suspected yaws collected in the Solomon Islands and Ghana. All samples were from patients whose lesions had previously tested negative using the Centers for Disease Control and Prevention (CDC) diagnostic assay in widespread use. However, some of these patients had positive serological assays for yaws on blood. We used direct whole-genome sequencing to identify T. pallidum subsp pertenue strains missed by the current assay. Results From 45 Solomon Islands and 27 Ghanaian samples, 11 were positive for T. pallidum DNA using the species-wide quantitative polymerase chain reaction (PCR) assay, from which we obtained 6 previously undetected T. pallidum subsp pertenue whole-genome sequences. These show that Solomon Islands sequences represent distinct T. pallidum subsp pertenue clades. These isolates were invisible to the CDC diagnostic PCR assay, due to sequence variation in the primer binding site. Conclusions Our data double the number of published T. pallidum subsp pertenue genomes. We show that Solomon Islands strains are undetectable by the PCR used in many studies and by health ministries. This assay is therefore not adequate for the eradication program. Next-generation genome sequence data are essential for these efforts. PMID:29045605

Use of amplicon sequencing to improve sensitivity in PCR-based detection of microbial pathogen in environmental samples.

PubMed

Saingam, Prakit; Li, Bo; Yan, Tao

2018-06-01

DNA-based molecular detection of microbial pathogens in complex environments is still plagued by sensitivity, specificity and robustness issues. We propose to address these issues by viewing them as inadvertent consequences of requiring specific and adequate amplification (SAA) of target DNA molecules by current PCR methods. Using the invA gene of Salmonella as the model system, we investigated if next generation sequencing (NGS) can be used to directly detect target sequences in false-negative PCR reaction (PCR-NGS) in order to remove the SAA requirement from PCR. False-negative PCR and qPCR reactions were first created using serial dilutions of laboratory-prepared Salmonella genomic DNA and then analyzed directly by NGS. Target invA sequences were detected in all false-negative PCR and qPCR reactions, which lowered the method detection limits near the theoretical minimum of single gene copy detection. The capability of the PCR-NGS approach in correcting false negativity was further tested and confirmed under more environmentally relevant conditions using Salmonella-spiked stream water and sediment samples. Finally, the PCR-NGS approach was applied to ten urban stream water samples and detected invA sequences in eight samples that would be otherwise deemed Salmonella negative. Analysis of the non-target sequences in the false-negative reactions helped to identify primer dime-like short sequences as the main cause of the false negativity. Together, the results demonstrated that the PCR-NGS approach can significantly improve method sensitivity, correct false-negative detections, and enable sequence-based analysis for failure diagnostics in complex environmental samples. Copyright © 2018 Elsevier B.V. All rights reserved.

Transcription Factor Map Alignment of Promoter Regions

PubMed Central

Blanco, Enrique; Messeguer, Xavier; Smith, Temple F; Guigó, Roderic

2006-01-01

We address the problem of comparing and characterizing the promoter regions of genes with similar expression patterns. This remains a challenging problem in sequence analysis, because often the promoter regions of co-expressed genes do not show discernible sequence conservation. In our approach, thus, we have not directly compared the nucleotide sequence of promoters. Instead, we have obtained predictions of transcription factor binding sites, annotated the predicted sites with the labels of the corresponding binding factors, and aligned the resulting sequences of labels—to which we refer here as transcription factor maps (TF-maps). To obtain the global pairwise alignment of two TF-maps, we have adapted an algorithm initially developed to align restriction enzyme maps. We have optimized the parameters of the algorithm in a small, but well-curated, collection of human–mouse orthologous gene pairs. Results in this dataset, as well as in an independent much larger dataset from the CISRED database, indicate that TF-map alignments are able to uncover conserved regulatory elements, which cannot be detected by the typical sequence alignments. PMID:16733547

Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood

PubMed Central

Fan, H. Christina; Blumenfeld, Yair J.; Chitkara, Usha; Hudgins, Louanne; Quake, Stephen R.

2008-01-01

We directly sequenced cell-free DNA with high-throughput shotgun sequencing technology from plasma of pregnant women, obtaining, on average, 5 million sequence tags per patient sample. This enabled us to measure the over- and underrepresentation of chromosomes from an aneuploid fetus. The sequencing approach is polymorphism-independent and therefore universally applicable for the noninvasive detection of fetal aneuploidy. Using this method, we successfully identified all nine cases of trisomy 21 (Down syndrome), two cases of trisomy 18 (Edward syndrome), and one case of trisomy 13 (Patau syndrome) in a cohort of 18 normal and aneuploid pregnancies; trisomy was detected at gestational ages as early as the 14th week. Direct sequencing also allowed us to study the characteristics of cell-free plasma DNA, and we found evidence that this DNA is enriched for sequences from nucleosomes. PMID:18838674

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.

Histoimmunogenetics Markup Language 1.0: Reporting next generation sequencing-based HLA and KIR genotyping.

PubMed

Milius, Robert P; Heuer, Michael; Valiga, Daniel; Doroschak, Kathryn J; Kennedy, Caleb J; Bolon, Yung-Tsi; Schneider, Joel; Pollack, Jane; Kim, Hwa Ran; Cereb, Nezih; Hollenbach, Jill A; Mack, Steven J; Maiers, Martin

2015-12-01

We present an electronic format for exchanging data for HLA and KIR genotyping with extensions for next-generation sequencing (NGS). This format addresses NGS data exchange by refining the Histoimmunogenetics Markup Language (HML) to conform to the proposed Minimum Information for Reporting Immunogenomic NGS Genotyping (MIRING) reporting guidelines (miring.immunogenomics.org). Our refinements of HML include two major additions. First, NGS is supported by new XML structures to capture additional NGS data and metadata required to produce a genotyping result, including analysis-dependent (dynamic) and method-dependent (static) components. A full genotype, consensus sequence, and the surrounding metadata are included directly, while the raw sequence reads and platform documentation are externally referenced. Second, genotype ambiguity is fully represented by integrating Genotype List Strings, which use a hierarchical set of delimiters to represent allele and genotype ambiguity in a complete and accurate fashion. HML also continues to enable the transmission of legacy methods (e.g. site-specific oligonucleotide, sequence-specific priming, and Sequence Based Typing (SBT)), adding features such as allowing multiple group-specific sequencing primers, and fully leveraging techniques that combine multiple methods to obtain a single result, such as SBT integrated with NGS. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Multiplex sequence analysis demonstrates the competitive growth advantage of the A-to-G mutants of clarithromycin-resistant Helicobacter pylori.

PubMed

Wang, G; Rahman, M S; Humayun, M Z; Taylor, D E

1999-03-01

Clarithromycin resistance in Helicobacter pylori is due to point mutation within the 23S rRNA. We examined the growth rates of different types of site-directed mutants and demonstrated quantitatively the competitive growth advantage of A-to-G mutants over other types of mutants by a multiplex sequencing assay. The results provide a rational explanation of why A-to-G mutants are predominantly observed among clarithromycin-resistant clinical isolates.

Multiplex Sequence Analysis Demonstrates the Competitive Growth Advantage of the A-to-G Mutants of Clarithromycin-Resistant Helicobacter pylori

PubMed Central

Wang, Ge; Rahman, M. Sayeedur; Humayun, M. Zafri; Taylor, Diane E.

1999-01-01

Clarithromycin resistance in Helicobacter pylori is due to point mutation within the 23S rRNA. We examined the growth rates of different types of site-directed mutants and demonstrated quantitatively the competitive growth advantage of A-to-G mutants over other types of mutants by a multiplex sequencing assay. The results provide a rational explanation of why A-to-G mutants are predominantly observed among clarithromycin-resistant clinical isolates. PMID:10049289

Site-directed mutagenesis in Petunia × hybrida protoplast system using direct delivery of purified recombinant Cas9 ribonucleoproteins.

PubMed

Subburaj, Saminathan; Chung, Sung Jin; Lee, Choongil; Ryu, Seuk-Min; Kim, Duk Hyoung; Kim, Jin-Soo; Bae, Sangsu; Lee, Geung-Joo

2016-07-01

Site-directed mutagenesis of nitrate reductase genes using direct delivery of purified Cas9 protein preassembled with guide RNA produces mutations efficiently in Petunia × hybrida protoplast system. The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR associated endonuclease 9 (CRISPR/Cas9) system has been recently announced as a powerful molecular breeding tool for site-directed mutagenesis in higher plants. Here, we report a site-directed mutagenesis method targeting Petunia nitrate reductase (NR) gene locus. This method could create mutations efficiently using direct delivery of purified Cas9 protein and single guide RNA (sgRNA) into protoplast cells. After transient introduction of RNA-guided endonuclease (RGEN) ribonucleoproteins (RNPs) with different sgRNAs targeting NR genes, mutagenesis at the targeted loci was detected by T7E1 assay and confirmed by targeted deep sequencing. T7E1 assay showed that RGEN RNPs induced site-specific mutations at frequencies ranging from 2.4 to 21 % at four different sites (NR1, 2, 4 and 6) in the PhNR gene locus with average mutation efficiency of 14.9 ± 2.2 %. Targeted deep DNA sequencing revealed mutation rates of 5.3-17.8 % with average mutation rate of 11.5 ± 2 % at the same NR gene target sites in DNA fragments of analyzed protoplast transfectants. Further analysis from targeted deep sequencing showed that the average ratio of deletion to insertion produced collectively by the four NR-RGEN target sites (NR1, 2, 4, and 6) was about 63:37. Our results demonstrated that direct delivery of RGEN RNPs into protoplast cells of Petunia can be exploited as an efficient tool for site-directed mutagenesis of genes or genome editing in plant systems.

Laser mass spectrometry for DNA sequencing, disease diagnosis, and fingerprinting

NASA Astrophysics Data System (ADS)

Chen, C. H. Winston; Taranenko, N. I.; Zhu, Y. F.; Chung, C. N.; Allman, S. L.

1997-05-01

Since laser mass spectrometry has the potential for achieving very fast DNA analysis, we recently applied it to DNA sequencing, DNA typing for fingerprinting, and DNA screening for disease diagnosis. Two different approaches for sequencing DNA have been successfully demonstrated. One is to sequence DNA with DNA ladders produced from Sanger's enzymatic method. The other is to do direct sequencing without DNA ladders. The need for quick DNA typing for identification purposes is critical for forensic application. Our preliminary results indicate laser mass spectrometry can possible be used for rapid DNA fingerprinting applications at a much lower cost than gel electrophoresis. Population screening for certain genetic disease can be a very efficient step to reducing medical costs through prevention. Since laser mass spectrometry can provide very fast DNA analysis, we applied laser mass spectrometry to disease diagnosis. Clinical samples with both base deletion and point mutation have been tested with complete success.

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing.

PubMed

Stiffler, Michael A; Subramanian, Subu K; Salinas, Victor H; Ranganathan, Rama

2016-07-03

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 β-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to β-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

Mapping the Geometric Evolution of Protein Folding Motor.

PubMed

Jerath, Gaurav; Hazam, Prakash Kishore; Shekhar, Shashi; Ramakrishnan, Vibin

2016-01-01

Polypeptide chain has an invariant main-chain and a variant side-chain sequence. How the side-chain sequence determines fold in terms of its chemical constitution has been scrutinized extensively and verified periodically. However, a focussed investigation on the directive effect of side-chain geometry may provide important insights supplementing existing algorithms in mapping the geometrical evolution of protein chains and its structural preferences. Geometrically, folding of protein structure may be envisaged as the evolution of its geometric variables: ϕ, and ψ dihedral angles of polypeptide main-chain directed by χ1, and χ2 of side chain. In this work, protein molecule is metaphorically modelled as a machine with 4 rotors ϕ, ψ, χ1 and χ2, with its evolution to the functional fold is directed by combinations of its rotor directions. We observe that differential rotor motions lead to different secondary structure formations and the combinatorial pattern is unique and consistent for particular secondary structure type. Further, we found that combination of rotor geometries of each amino acid is unique which partly explains how different amino acid sequence combinations have unique structural evolution and functional adaptation. Quantification of these amino acid rotor preferences, resulted in the generation of 3 substitution matrices, which later on plugged in the BLAST tool, for evaluating their efficiency in aligning sequences. We have employed BLOSUM62 and PAM30 as standard for primary evaluation. Generation of substitution matrices is a logical extension of the conceptual framework we attempted to build during the development of this work. Optimization of matrices following the conventional routines and possible application with biologically relevant data sets are beyond the scope of this manuscript, though it is a part of the larger project design.

Compatibility of motion facilitates visuomotor synchronization.

PubMed

Hove, Michael J; Spivey, Michael J; Krumhansl, Carol L

2010-12-01

Prior research indicates that synchronized tapping performance is very poor with flashing visual stimuli compared with auditory stimuli. Three finger-tapping experiments compared flashing visual metronomes with visual metronomes containing a spatial component, either compatible, incompatible, or orthogonal to the tapping action. In Experiment 1, synchronization success rates increased dramatically for spatiotemporal sequences of both geometric and biological forms over flashing sequences. In Experiment 2, synchronization performance was best when target sequences and movements were directionally compatible (i.e., simultaneously down), followed by orthogonal stimuli, and was poorest for incompatible moving stimuli and flashing stimuli. In Experiment 3, synchronization performance was best with auditory sequences, followed by compatible moving stimuli, and was worst for flashing and fading stimuli. Results indicate that visuomotor synchronization improves dramatically with compatible spatial information. However, an auditory advantage in sensorimotor synchronization persists.

Frequency of the first feature in action sequences influences feature binding.

PubMed

Mattson, Paul S; Fournier, Lisa R; Behmer, Lawrence P

2012-10-01

We investigated whether binding among perception and action feature codes is a preliminary step toward creating a more durable memory trace of an action event. If so, increasing the frequency of a particular event (e.g., a stimulus requiring a movement with the left or right hand in an up or down direction) should increase the strength and speed of feature binding for this event. The results from two experiments, using a partial-repetition paradigm, confirmed that feature binding increased in strength and/or occurred earlier for a high-frequency (e.g., left hand moving up) than for a low-frequency (e.g., right hand moving down) event. Moreover, increasing the frequency of the first-specified feature in the action sequence alone (e.g., "left" hand) increased the strength and/or speed of action feature binding (e.g., between the "left" hand and movement in an "up" or "down" direction). The latter finding suggests an update to the theory of event coding, as not all features in the action sequence equally determine binding strength. We conclude that action planning involves serial binding of features in the order of action feature execution (i.e., associations among features are not bidirectional but are directional), which can lead to a more durable memory trace. This is consistent with physiological evidence suggesting that serial order is preserved in an action plan executed from memory and that the first feature in the action sequence may be critical in preserving this serial order.

Gene discovery in the hamster: a comparative genomics approach for gene annotation by sequencing of hamster testis cDNAs

PubMed Central

Oduru, Sreedhar; Campbell, Janee L; Karri, SriTulasi; Hendry, William J; Khan, Shafiq A; Williams, Simon C

2003-01-01

Background Complete genome annotation will likely be achieved through a combination of computer-based analysis of available genome sequences combined with direct experimental characterization of expressed regions of individual genomes. We have utilized a comparative genomics approach involving the sequencing of randomly selected hamster testis cDNAs to begin to identify genes not previously annotated on the human, mouse, rat and Fugu (pufferfish) genomes. Results 735 distinct sequences were analyzed for their relatedness to known sequences in public databases. Eight of these sequences were derived from previously unidentified genes and expression of these genes in testis was confirmed by Northern blotting. The genomic locations of each sequence were mapped in human, mouse, rat and pufferfish, where applicable, and the structure of their cognate genes was derived using computer-based predictions, genomic comparisons and analysis of uncharacterized cDNA sequences from human and macaque. Conclusion The use of a comparative genomics approach resulted in the identification of eight cDNAs that correspond to previously uncharacterized genes in the human genome. The proteins encoded by these genes included a new member of the kinesin superfamily, a SET/MYND-domain protein, and six proteins for which no specific function could be predicted. Each gene was expressed primarily in testis, suggesting that they may play roles in the development and/or function of testicular cells. PMID:12783626

Psychological and behavioural impact of returning personal results from whole-genome sequencing: the HealthSeq project.

PubMed

Sanderson, Saskia C; Linderman, Michael D; Suckiel, Sabrina A; Zinberg, Randi; Wasserstein, Melissa; Kasarskis, Andrew; Diaz, George A; Schadt, Eric E

2017-02-01

Providing ostensibly healthy individuals with personal results from whole-genome sequencing could lead to improved health and well-being via enhanced disease risk prediction, prevention, and diagnosis, but also poses practical and ethical challenges. Understanding how individuals react psychologically and behaviourally will be key in assessing the potential utility of personal whole-genome sequencing. We conducted an exploratory longitudinal cohort study in which quantitative surveys and in-depth qualitative interviews were conducted before and after personal results were returned to individuals who underwent whole-genome sequencing. The participants were offered a range of interpreted results, including Alzheimer's disease, type 2 diabetes, pharmacogenomics, rare disease-associated variants, and ancestry. They were also offered their raw data. Of the 35 participants at baseline, 29 (82.9%) completed the 6-month follow-up. In the quantitative surveys, test-related distress was low, although it was higher at 1-week than 6-month follow-up (Z=2.68, P=0.007). In the 6-month qualitative interviews, most participants felt happy or relieved about their results. A few were concerned, particularly about rare disease-associated variants and Alzheimer's disease results. Two of the 29 participants had sought clinical follow-up as a direct or indirect consequence of rare disease-associated variants results. Several had mentioned their results to their doctors. Some participants felt having their raw data might be medically useful to them in the future. The majority reported positive reactions to having their genomes sequenced, but there were notable exceptions to this. The impact and value of returning personal results from whole-genome sequencing when implemented on a larger scale remains to be seen.

Psychological and behavioural impact of returning personal results from whole-genome sequencing: the HealthSeq project

PubMed Central

Sanderson, Saskia C; Linderman, Michael D; Suckiel, Sabrina A; Zinberg, Randi; Wasserstein, Melissa; Kasarskis, Andrew; Diaz, George A; Schadt, Eric E

2017-01-01

Providing ostensibly healthy individuals with personal results from whole-genome sequencing could lead to improved health and well-being via enhanced disease risk prediction, prevention, and diagnosis, but also poses practical and ethical challenges. Understanding how individuals react psychologically and behaviourally will be key in assessing the potential utility of personal whole-genome sequencing. We conducted an exploratory longitudinal cohort study in which quantitative surveys and in-depth qualitative interviews were conducted before and after personal results were returned to individuals who underwent whole-genome sequencing. The participants were offered a range of interpreted results, including Alzheimer's disease, type 2 diabetes, pharmacogenomics, rare disease-associated variants, and ancestry. They were also offered their raw data. Of the 35 participants at baseline, 29 (82.9%) completed the 6-month follow-up. In the quantitative surveys, test-related distress was low, although it was higher at 1-week than 6-month follow-up (Z=2.68, P=0.007). In the 6-month qualitative interviews, most participants felt happy or relieved about their results. A few were concerned, particularly about rare disease-associated variants and Alzheimer's disease results. Two of the 29 participants had sought clinical follow-up as a direct or indirect consequence of rare disease-associated variants results. Several had mentioned their results to their doctors. Some participants felt having their raw data might be medically useful to them in the future. The majority reported positive reactions to having their genomes sequenced, but there were notable exceptions to this. The impact and value of returning personal results from whole-genome sequencing when implemented on a larger scale remains to be seen. PMID:28051073

Isolation and characterization of a water stress-specific genomic gene, pwsi 18, from rice.

PubMed

Joshee, N; Kisaka, H; Kitagawa, Y

1998-01-01

One of the water stress-specific cDNA clones of rice characterised previously, wsi18, was selected for further study. The wsi18 gene can be induced by water stress conditions such as mannitol, NaCl, and dryness, but not by ABA, cold, or heat. A genomic clone for wsi18, pwsi18, contained about 1.7 kbp of the 5' upstream sequence, two introns, and the full coding sequence. The 5'-upstream sequence of pwsi18 contained putative cis-acting elements, namely an ABA-responsive element (ABRE), three G-boxes, three E-boxes, a MEF-2 sequence, four direct and two inverted repeats, and four sequences similar to DRE, which is involved in the dehydration response of Arabidopsis genes. The gusA reporter gene under the control of the pwsi18 promoter showed transient expression in response to water stress. Deletion of the downstream DRE-like sequence between the distal G-boxes-2 and -3 resulted in rather low GUS expression.

Chiron: translating nanopore raw signal directly into nucleotide sequence using deep learning.

PubMed

Teng, Haotian; Cao, Minh Duc; Hall, Michael B; Duarte, Tania; Wang, Sheng; Coin, Lachlan J M

2018-05-01

Sequencing by translocating DNA fragments through an array of nanopores is a rapidly maturing technology that offers faster and cheaper sequencing than other approaches. However, accurately deciphering the DNA sequence from the noisy and complex electrical signal is challenging. Here, we report Chiron, the first deep learning model to achieve end-to-end basecalling and directly translate the raw signal to DNA sequence without the error-prone segmentation step. Trained with only a small set of 4,000 reads, we show that our model provides state-of-the-art basecalling accuracy, even on previously unseen species. Chiron achieves basecalling speeds of more than 2,000 bases per second using desktop computer graphics processing units.

Studies and simulations of the DigiCipher system

NASA Technical Reports Server (NTRS)

Sayood, K.; Chen, Y. C.; Kipp, G.

1993-01-01

During this period the development of simulators for the various high definition television (HDTV) systems proposed to the FCC was continued. The FCC has indicated that it wants the various proposers to collaborate on a single system. Based on all available information this system will look very much like the advanced digital television (ADTV) system with major contributions only from the DigiCipher system. The results of our simulations of the DigiCipher system are described. This simulator was tested using test sequences from the MPEG committee. The results are extrapolated to HDTV video sequences. Once again, some caveats are in order. The sequences used for testing the simulator and generating the results are those used for testing the MPEG algorithm. The sequences are of much lower resolution than the HDTV sequences would be, and therefore the extrapolations are not totally accurate. One would expect to get significantly higher compression in terms of bits per pixel with sequences that are of higher resolution. However, the simulator itself is a valid one, and should HDTV sequences become available, they could be used directly with the simulator. A brief overview of the DigiCipher system is given. Some coding results obtained using the simulator are looked at. These results are compared to those obtained using the ADTV system. These results are evaluated in the context of the CCSDS specifications and make some suggestions as to how the DigiCipher system could be implemented in the NASA network. Simulations such as the ones reported can be biased depending on the particular source sequence used. In order to get more complete information about the system one needs to obtain a reasonable set of models which mirror the various kinds of sources encountered during video coding. A set of models which can be used to effectively model the various possible scenarios is provided. As this is somewhat tangential to the other work reported, the results are included as an appendix.

TP53, PIK3CA, FBXW7 and KRAS Mutations in Esophageal Cancer Identified by Targeted Sequencing.

PubMed

Zheng, Huili; Wang, Yan; Tang, Chuanning; Jones, Lindsey; Ye, Hua; Zhang, Guangchun; Cao, Weihai; Li, Jingwen; Liu, Lifeng; Liu, Zhencong; Zhang, Chao; Lou, Feng; Liu, Zhiyuan; Li, Yangyang; Shi, Zhenfen; Zhang, Jingbo; Zhang, Dandan; Sun, Hong; Dong, Haichao; Dong, Zhishou; Guo, Baishuai; Yan, H E; Lu, Qingyu; Huang, Xue; Chen, Si-Yi

2016-01-01

Esophageal cancer (EC) is a common malignancy with significant morbidity and mortality. As individual cancers exhibit unique mutation patterns, identifying and characterizing gene mutations in EC that may serve as biomarkers might help predict patient outcome and guide treatment. Traditionally, personalized cancer DNA sequencing was impractical and expensive. Recent technological advancements have made targeted DNA sequencing more cost- and time-effective with reliable results. This technology may be useful for clinicians to direct patient treatment. The Ion PGM and AmpliSeq Cancer Panel was used to identify mutations at 737 hotspot loci of 45 cancer-related genes in 64 EC samples from Chinese patients. Frequent mutations were found in TP53 and less frequent mutations in PIK3CA, FBXW7 and KRAS. These results demonstrate that targeted sequencing can reliably identify mutations in individual tumors that make this technology a possibility for clinical use. Copyright© 2016, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

Detection and identification of cutaneous leishmaniasis isolates by culture, Polymerase chain reaction and sequence analyses in Syrian and Central Anatolia patients.

PubMed

Beyhan, Yunus E; Karakus, Mehmet; Karagoz, Alper; Mungan, Mesut; Ozkan, Aysegul T; Hokelek, Murat

2017-09-01

To characterize the cutaneous leishmaniasis (CL) isolates of Syrian and Central Anatolia patients at species levels. Methods: Skin scrapings of 3 patients (2 Syrian, 1 Turkish) were taken and examined by direct examination, culture in Novy-MacNeal-Nicole (NNN) medium, internal transcribed spacer polymerase chain reaction and sequence analysis (PCR). Results:According to microscopic examination, culture and PCR methods, 3 samples were detected positive. The sequencing results of all isolates in the study were identified as Leishmania tropica. The same genotypes were detected in the 3 isolates and nucleotide sequence submitted into GenBank with the accession number: KP689599. Conclusion: This finding could give information about the transmission of CL between Turkey and Syria. Because of the Syrian civil war, most of the Syrian citizens circulating in Turkey and different part of Europe, this can be increase the risk of spreading the disease. So, prevention measurements must be taken urgently.

A novel model for DNA sequence similarity analysis based on graph theory.

PubMed

Qi, Xingqin; Wu, Qin; Zhang, Yusen; Fuller, Eddie; Zhang, Cun-Quan

2011-01-01

Determination of sequence similarity is one of the major steps in computational phylogenetic studies. As we know, during evolutionary history, not only DNA mutations for individual nucleotide but also subsequent rearrangements occurred. It has been one of major tasks of computational biologists to develop novel mathematical descriptors for similarity analysis such that various mutation phenomena information would be involved simultaneously. In this paper, different from traditional methods (eg, nucleotide frequency, geometric representations) as bases for construction of mathematical descriptors, we construct novel mathematical descriptors based on graph theory. In particular, for each DNA sequence, we will set up a weighted directed graph. The adjacency matrix of the directed graph will be used to induce a representative vector for DNA sequence. This new approach measures similarity based on both ordering and frequency of nucleotides so that much more information is involved. As an application, the method is tested on a set of 0.9-kb mtDNA sequences of twelve different primate species. All output phylogenetic trees with various distance estimations have the same topology, and are generally consistent with the reported results from early studies, which proves the new method's efficiency; we also test the new method on a simulated data set, which shows our new method performs better than traditional global alignment method when subsequent rearrangements happen frequently during evolutionary history.

Development of positive control materials for DNA-based detection of cystic fibrosis: Cloning and sequencing of 31 mutations

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

Iovannisci, D.; Brown, C.; Winn-Deen, E.

1994-09-01

The cloning and sequencing of the gene associated with cystic fibrosis (CF) now provides the opportunity for earlier detection and carrier screening through DNA-based detection schemes. To date, over 300 mutations have been reported to the CF Consortium; however, only 30 mutations have been observed frequently enough world-wide to warrant routine screening. Many of these mutations are not available as cloned material or as established tissue culture cell lines to aid in the development of DNA-based detection assays. We have therefore cloned the 30 most frequently reported mutations, plus the mutation R347H due to its association with male infertility (31more » mutations, total). Two approaches were employed: direct PCR amplification, where mutations were available from patient sources, and site-directed PCR mutagenesis of normal genomic DNA to generate the remaining mutations. After amplification, products were cloned into a sequencing vector, bacterial transformants were screened by a novel method (PCR/oligonucleotide litigation assay/sequence-coded separation), and plamid DNA sequences determined by automated fluorescent methods on the Applied Biosystems 373A. Mixing of the clones allows the construction of artificial genotypes useful as positive control material for assay validation. A second round of mutagenesis, resulting in the construction of plasmids bearing multiple mutations, will be evaluated for their utility as reagent control materials in kit development.« less

Genomics approach to the environmental community of microorganisms

NASA Astrophysics Data System (ADS)

Kawarabayasi, Y.; Maruyama, A.

2004-12-01

It was indicated by microscopic observation or comparison of 16S rDNA sequence that many extremophiles were surviving in many hydrothermal environments. But it is generally said that over 99% of total microbes are now uncultivable. Thus, we planned to identify uncultivable microbes through direct sequencing of environmental DNA. At first, shotgun plasmid libraries were directly constructed with the DNA molecules prepared from mixed microbes collected from low-temperature hydrothermal water at RM24 in the Southern East Pacific Rise (S-EPR). It was shown that the sequences of some number of clones indicated the similar feature to the intron in eukaryote or tandem repetitive sequence identified in some human familiar diseases. The results indicated that many microorganisms with eukaryotic feature were dominant in low temperature water of S-EPR. Secondly, shotgun plasmid libraries were constructed from the environmental DNA prepared from Beppu hot springs. The ORFs were easily identified all clones determined entire sequence. Thus it can be said that hot springs is good resources for searching novel genes. At last, the mixed microbes isolated from Suiyo seamount were used for construction of shotgun library. The clones in this library contained the ORFs. From some clones in hot spring and Suiyo sample, aminoacyl-tRNA synthatase, which is generally present in all organisms, was isolated by similarity. The phylogenetic analysis of aminoacyl-tRNA synthetase identified indicated that novel and unidentified microorganisms should be present in hot spring or Suiyo seamount. The novel genes identified from Suiyo seamount were also utilized for expression in E. coli. Some gene products were successfully obtained from the E. coli cells as soluble proteins. Some protein indicated the thermostability up to 70_E#8249;C, meaning that the original host cell of this gene should be stable up to the same temperature. Our work indicates that environmental genomics, including the direct cloning, sequencing of environmental DNA and expression of gene identified, is powerful approach to collect novel uncultivable microbes or novel active genes.

Idiopathic slow transit constipation and megacolon are not associated with neurturin mutations.

PubMed

Chen, B; Knowles, C H; Scott, M; Anand, P; Williams, N S; Milbrandt, J; Tam, P K H

2002-10-01

Chronic idiopathic slow-transit constipation (ISTC) and idiopathic megacolon (IMC) are early-onset gastrointestinal motility disorders of unknown aetiology. The gene encoding the neurotrophic factor neurturin may be a candidate for these disorders, as neurturin-deficient mice have a similar enteric phenotype. In the present study, we tested this hypothesis. Genomic DNA from 26 cases of chronic idiopathic STC [with a family history of constipation in 15 (58%) and Hirschsprung's disease in two (8%)], and five cases of IMC [two familial (40%)] was screened by direct DNA sequencing using the fluorescent dideoxy terminator method. Results were compared with published sequence data and 24 control DNAs. Our results revealed several previously unreported common sequence polymorphisms, but overall frequencies were comparable between patients and controls. We conclude that mutation of neurturin is not a frequent cause of ISTC or IMC.

The complete mitochondrial genome of the Japanese ornamental koi carp (Cyprinus carpio) and its implication for the history of koi.

PubMed

Mabuchi, Kohji; Song, Hayeun

2014-02-01

Complete mitochondrial genome (mitogenome) sequences were determined for two individuals of Japanese ornamental koi carp. Interestingly, the obtained mitogenomes (16,581 bp) were both completely identical to the recently reported mitogenome of Oujiang color carp from China. Control region (CR) sequences in DNA database demonstrated that more than half (65%) of the koi carp individuals so far reported had partial or complete CR sequences identical to those from Oujiang color carp. These results might suggest that the Japanese koi carp has been originated from Chinese Oujiang color carp, contrary to the belief in Japan that the koi carps have been developed directly from carp stocks in Japan. In any case, the present results emphasize the importance of analyzing Oujiang color carp when studying the origin of koi carp.

Comparison of Direct Sequence Spread Spectrum Rake Receiver with a Maximum Ratio Combining Multicarrier Spread Spectrum Receiver

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

Daryl Leon Wasden; Hussein Moradi; Behrouz Farhang-Broujeny

2014-06-01

This paper presents a theoretical analysis of the performance of a filter bank-based multicarrier spread spectrum (FB-MC-SS) system. We consider an FB-MC-SS setup where each data symbol is spread across multiple subcarriers, but there is no spreading in time. The results are then compared with those of the well-known direct sequence spread spectrum (DS-SS) system with a rake receiver for its best performance. We compare the two systems when the channel noise is white. We prove that as the processing gains of the two systems tend to infinity both approach the same performance. However, numerical simulations show that, in practice,more » where processing gain is limited, FB-MC-SS outperforms DS-SS.« less

Automated DNA mutation detection using universal conditions direct sequencing: application to ten muscular dystrophy genes.

PubMed

Bennett, Richard R; Schneider, Hal E; Estrella, Elicia; Burgess, Stephanie; Cheng, Andrew S; Barrett, Caitlin; Lip, Va; Lai, Poh San; Shen, Yiping; Wu, Bai-Lin; Darras, Basil T; Beggs, Alan H; Kunkel, Louis M

2009-10-18

One of the most common and efficient methods for detecting mutations in genes is PCR amplification followed by direct sequencing. Until recently, the process of designing PCR assays has been to focus on individual assay parameters rather than concentrating on matching conditions for a set of assays. Primers for each individual assay were selected based on location and sequence concerns. The two primer sequences were then iteratively adjusted to make the individual assays work properly. This generally resulted in groups of assays with different annealing temperatures that required the use of multiple thermal cyclers or multiple passes in a single thermal cycler making diagnostic testing time-consuming, laborious and expensive.These factors have severely hampered diagnostic testing services, leaving many families without an answer for the exact cause of a familial genetic disease. A search of GeneTests for sequencing analysis of the entire coding sequence for genes that are known to cause muscular dystrophies returns only a small list of laboratories that perform comprehensive gene panels.The hypothesis for the study was that a complete set of universal assays can be designed to amplify and sequence any gene or family of genes using computer aided design tools. If true, this would allow automation and optimization of the mutation detection process resulting in reduced cost and increased throughput. An automated process has been developed for the detection of deletions, duplications/insertions and point mutations in any gene or family of genes and has been applied to ten genes known to bear mutations that cause muscular dystrophy: DMD; CAV3; CAPN3; FKRP; TRIM32; LMNA; SGCA; SGCB; SGCG; SGCD. Using this process, mutations have been found in five DMD patients and four LGMD patients (one in the FKRP gene, one in the CAV3 gene, and two likely causative heterozygous pairs of variations in the CAPN3 gene of two other patients). Methods and assay sequences are reported in this paper. This automated process allows laboratories to discover DNA variations in a short time and at low cost.

Direct PCR Offers a Fast and Reliable Alternative to Conventional DNA Isolation Methods for Gut Microbiomes.

PubMed

Videvall, Elin; Strandh, Maria; Engelbrecht, Anel; Cloete, Schalk; Cornwallis, Charlie K

2017-01-01

The gut microbiome of animals is emerging as an important factor influencing ecological and evolutionary processes. A major bottleneck in obtaining microbiome data from large numbers of samples is the time-consuming laboratory procedures required, specifically the isolation of DNA and generation of amplicon libraries. Recently, direct PCR kits have been developed that circumvent conventional DNA extraction steps, thereby streamlining the laboratory process by reducing preparation time and costs. However, the reliability and efficacy of direct PCR for measuring host microbiomes have not yet been investigated other than in humans with 454 sequencing. Here, we conduct a comprehensive evaluation of the microbial communities obtained with direct PCR and the widely used Mo Bio PowerSoil DNA extraction kit in five distinct gut sample types (ileum, cecum, colon, feces, and cloaca) from 20 juvenile ostriches, using 16S rRNA Illumina MiSeq sequencing. We found that direct PCR was highly comparable over a range of measures to the DNA extraction method in cecal, colon, and fecal samples. However, the two methods significantly differed in samples with comparably low bacterial biomass: cloacal and especially ileal samples. We also sequenced 100 replicate sample pairs to evaluate repeatability during both extraction and PCR stages and found that both methods were highly consistent for cecal, colon, and fecal samples ( r s > 0.7) but had low repeatability for cloacal ( r s = 0.39) and ileal ( r s = -0.24) samples. This study indicates that direct PCR provides a fast, cheap, and reliable alternative to conventional DNA extraction methods for retrieving 16S rRNA data, which can aid future gut microbiome studies. IMPORTANCE The microbial communities of animals can have large impacts on their hosts, and the number of studies using high-throughput sequencing to measure gut microbiomes is rapidly increasing. However, the library preparation procedure in microbiome research is both costly and time-consuming, especially for large numbers of samples. We investigated a cheaper and faster direct PCR method designed to bypass the DNA isolation steps during 16S rRNA library preparation and compared it with a standard DNA extraction method. We used both techniques on five different gut sample types collected from 20 juvenile ostriches and sequenced samples with Illumina MiSeq. The methods were highly comparable and highly repeatable in three sample types with high microbial biomass (cecum, colon, and feces), but larger differences and low repeatability were found in the microbiomes obtained from the ileum and cloaca. These results will help microbiome researchers assess library preparation procedures and plan their studies accordingly.

Dual signal amplification for highly sensitive electrochemical detection of uropathogens via enzyme-based catalytic target recycling.

PubMed

Su, Jiao; Zhang, Haijie; Jiang, Bingying; Zheng, Huzhi; Chai, Yaqin; Yuan, Ruo; Xiang, Yun

2011-11-15

We report an ultrasensitive electrochemical approach for the detection of uropathogen sequence-specific DNA target. The sensing strategy involves a dual signal amplification process, which combines the signal enhancement by the enzymatic target recycling technique with the sensitivity improvement by the quantum dot (QD) layer-by-layer (LBL) assembled labels. The enzyme-based catalytic target DNA recycling process results in the use of each target DNA sequence for multiple times and leads to direct amplification of the analytical signal. Moreover, the LBL assembled QD labels can further enhance the sensitivity of the sensing system. The coupling of these two effective signal amplification strategies thus leads to low femtomolar (5fM) detection of the target DNA sequences. The proposed strategy also shows excellent discrimination between the target DNA and the single-base mismatch sequences. The advantageous intrinsic sequence-independent property of exonuclease III over other sequence-dependent enzymes makes our new dual signal amplification system a general sensing platform for monitoring ultralow level of various types of target DNA sequences. Copyright © 2011 Elsevier B.V. All rights reserved.

Response and recovery lessons from the 2010-2011 earthquake sequence in Canterbury, New Zealand

USGS Publications Warehouse

Pierepiekarz, Mark; Johnston, David; Berryman, Kelvin; Hare, John; Gomberg, Joan S.; Williams, Robert A.; Weaver, Craig S.

2014-01-01

The impacts and opportunities that result when low-probability moderate earthquakes strike an urban area similar to many throughout the US were vividly conveyed in a one-day workshop in which social and Earth scientists, public officials, engineers, and an emergency manager shared their experiences of the earthquake sequence that struck the city of Christchurch and surrounding Canterbury region of New Zealand in 2010-2011. Without question, the earthquake sequence has had unprecedented impacts in all spheres on New Zealand society, locally to nationally--10% of the country's population was directly impacted and losses total 8-10% of their GDP. The following paragraphs present a few lessons from Christchurch.

Transcriptional regulation of human eosinophil RNases by an evolutionary- conserved sequence motif in primate genome

PubMed Central

Wang, Hsiu-Yu; Chang, Hao-Teng; Pai, Tun-Wen; Wu, Chung-I; Lee, Yuan-Hung; Chang, Yen-Hsin; Tai, Hsiu-Ling; Tang, Chuan-Yi; Chou, Wei-Yao; Chang, Margaret Dah-Tsyr

2007-01-01

Background Human eosinophil-derived neurotoxin (edn) and eosinophil cationic protein (ecp) are members of a subfamily of primate ribonuclease (rnase) genes. Although they are generated by gene duplication event, distinct edn and ecp expression profile in various tissues have been reported. Results In this study, we obtained the upstream promoter sequences of several representative primate eosinophil rnases. Bioinformatic analysis revealed the presence of a shared 34-nucleotide (nt) sequence stretch located at -81 to -48 in all edn promoters and macaque ecp promoter. Such a unique sequence motif constituted a region essential for transactivation of human edn in hepatocellular carcinoma cells. Gel electrophoretic mobility shift assay, transient transfection and scanning mutagenesis experiments allowed us to identify binding sites for two transcription factors, Myc-associated zinc finger protein (MAZ) and SV-40 protein-1 (Sp1), within the 34-nt segment. Subsequent in vitro and in vivo binding assays demonstrated a direct molecular interaction between this 34-nt region and MAZ and Sp1. Interestingly, overexpression of MAZ and Sp1 respectively repressed and enhanced edn promoter activity. The regulatory transactivation motif was mapped to the evolutionarily conserved -74/-65 region of the edn promoter, which was guanidine-rich and critical for recognition by both transcription factors. Conclusion Our results provide the first direct evidence that MAZ and Sp1 play important roles on the transcriptional activation of the human edn promoter through specific binding to a 34-nt segment present in representative primate eosinophil rnase promoters. PMID:17927842

Inference of Markovian properties of molecular sequences from NGS data and applications to comparative genomics.

PubMed

Ren, Jie; Song, Kai; Deng, Minghua; Reinert, Gesine; Cannon, Charles H; Sun, Fengzhu

2016-04-01

Next-generation sequencing (NGS) technologies generate large amounts of short read data for many different organisms. The fact that NGS reads are generally short makes it challenging to assemble the reads and reconstruct the original genome sequence. For clustering genomes using such NGS data, word-count based alignment-free sequence comparison is a promising approach, but for this approach, the underlying expected word counts are essential.A plausible model for this underlying distribution of word counts is given through modeling the DNA sequence as a Markov chain (MC). For single long sequences, efficient statistics are available to estimate the order of MCs and the transition probability matrix for the sequences. As NGS data do not provide a single long sequence, inference methods on Markovian properties of sequences based on single long sequences cannot be directly used for NGS short read data. Here we derive a normal approximation for such word counts. We also show that the traditional Chi-square statistic has an approximate gamma distribution ,: using the Lander-Waterman model for physical mapping. We propose several methods to estimate the order of the MC based on NGS reads and evaluate those using simulations. We illustrate the applications of our results by clustering genomic sequences of several vertebrate and tree species based on NGS reads using alignment-free sequence dissimilarity measures. We find that the estimated order of the MC has a considerable effect on the clustering results ,: and that the clustering results that use a N: MC of the estimated order give a plausible clustering of the species. Our implementation of the statistics developed here is available as R package 'NGS.MC' at http://www-rcf.usc.edu/∼fsun/Programs/NGS-MC/NGS-MC.html fsun@usc.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.

[Identification of hepatitis B virus YMDD point mutation using peptide nucleic acid clamping PCR].

PubMed

Zhang, Yingying; He, Haitang; Yang, Jie; Hou, Jinlin

2013-06-01

To establish a peptide nucleic acid clamping PCR assay for detecting hepatitis B virus (HBV) drug resistance mutation. RtM204I (ATT) mutant, rtM204V (GTG) mutant and rtM204 (ATG) wild-type plasmids mixed at different ratios were detected for mutations by PNA clamping PCR assay and direct sequencing, and the sensitivity and specificity of the two methods were compared. Serum samples from 85 patients with chronic HBV infection were detected for drug resistance using the two methods. The sensitivity of PNA-PCR assay was 0.001% in a 10(5)-fold excess of wild-type HBV DNA with a detection limit of 10(1) copies. The sensitivity of direct sequencing was 10% with a detection limit of 10(4) copies. Mutants were detected in 73 of the 85 serum samples (85.9%), including YIDD in 40 samples, YVDD in 23 samples, and YIDD+YVDD in 10 samples. The agreement of PNA-PCR assay with direct sequencing was only 40% (34/85, YIDD in 21 samples, YVDD in 11 samples, and YIDD+YVDD in 2 samples). Neither of the two methods yielded positive results for the negative control samples, suggesting their good specificity. PNA-PCR assay appears to be a more sensitive and rapid assay for detection of HBV genotypic resistance.

A comparison of RNA with DNA in template-directed synthesis

NASA Technical Reports Server (NTRS)

Zielinski, M.; Kozlov, I. A.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

2000-01-01

Nonenzymatic template-directed copying of RNA sequences rich in cytidylic acid using nucleoside 5'-(2-methylimidazol-1-yl phosphates) as substrates is substantially more efficient than the copying of corresponding DNA sequences. However, many sequences cannot be copied, and the prospect of replication in this system is remote, even for RNA. Surprisingly, wobble-pairing leads to much more efficient incorporation of G opposite U on RNA templates than of G opposite T on DNA templates.

Sequence periodicity in nucleosomal DNA and intrinsic curvature

PubMed Central

2010-01-01

Background Most eukaryotic DNA contained in the nucleus is packaged by wrapping DNA around histone octamers. Histones are ubiquitous and bind most regions of chromosomal DNA. In order to achieve smooth wrapping of the DNA around the histone octamer, the DNA duplex should be able to deform and should possess intrinsic curvature. The deformability of DNA is a result of the non-parallelness of base pair stacks. The stacking interaction between base pairs is sequence dependent. The higher the stacking energy the more rigid the DNA helix, thus it is natural to expect that sequences that are involved in wrapping around the histone octamer should be unstacked and possess intrinsic curvature. Intrinsic curvature has been shown to be dictated by the periodic recurrence of certain dinucleotides. Several genome-wide studies directed towards mapping of nucleosome positions have revealed periodicity associated with certain stretches of sequences. In the current study, these sequences have been analyzed with a view to understand their sequence-dependent structures. Results Higher order DNA structures and the distribution of molecular bend loci associated with 146 base nucleosome core DNA sequence from C. elegans and chicken have been analyzed using the theoretical model for DNA curvature. The curvature dispersion calculated by cyclically permuting the sequences revealed that the molecular bend loci were delocalized throughout the nucleosome core region and had varying degrees of intrinsic curvature. Conclusions The higher order structures associated with nucleosomes of C.elegans and chicken calculated from the sequences revealed heterogeneity with respect to the deviation of the DNA axis. The results points to the possibility of context dependent curvature of varying degrees to be associated with nucleosomal DNA. PMID:20487515

Rabies in the arctic fox population, Svalbard, Norway.

PubMed

Mørk, Torill; Bohlin, Jon; Fuglei, Eva; Åsbakk, Kjetil; Tryland, Morten

2011-10-01

Arctic foxes, 620 that were trapped and 22 found dead on Svalbard, Norway (1996-2004), as well as 10 foxes trapped in Nenets, North-West Russia (1999), were tested for rabies virus antigen in brain tissue by standard direct fluorescent antibody test. Rabies antigen was found in two foxes from Svalbard and in three from Russia. Blood samples from 515 of the fox carcasses were screened for rabies antibodies with negative result. Our results, together with a previous screening (1980-1989, n=817) indicate that the prevalence of rabies in Svalbard has remained low or that the virus has not been enzootic in the arctic fox population since the first reported outbreak in 1980. Brain tissues from four arctic foxes (one from Svalbard, three from Russia) in which rabies virus antigen was detected were further analyzed by reverse-transcriptase polymerase chain reaction direct amplicon sequencing and phylogenetic analysis. Sequences were compared to corresponding sequences from rabies virus isolates from other arctic regions. The Svalbard isolate and two of the Russian isolates were identical (310 nucleotides), whereas the third Russian isolate differed in six nucleotide positions. However, when translated into amino acid sequences, none of these substitutions produced changes in the amino acid sequence. These findings suggest that the spread of rabies virus to Svalbard was likely due to migration of arctic foxes over sea ice from Russia to Svalbard. Furthermore, when compared to other Arctic rabies virus isolates, a high degree of homology was found, suggesting a high contact rate between arctic fox populations from different arctic regions. The high degree of homology also indicates that other, and more variable, regions of the genome than this part of the nucleoprotein gene should be used to distinguish Arctic rabies virus isolates for epidemiologic purposes.

A DNA Barcoding Method to Discriminate between the Model Plant Brachypodium distachyon and Its Close Relatives B. stacei and B. hybridum (Poaceae)

PubMed Central

López-Alvarez, Diana; López-Herranz, Maria Luisa; Betekhtin, Alexander; Catalán, Pilar

2012-01-01

Background Brachypodium distachyon s. l. has been widely investigated across the world as a model plant for temperate cereals and biofuel grasses. However, this annual plant shows three cytotypes that have been recently recognized as three independent species, the diploids B. distachyon (2n = 10) and B. stacei (2n = 20) and their derived allotetraploid B. hybridum (2n = 30). Methodology/Principal Findings We propose a DNA barcoding approach that consists of a rapid, accurate and automatable species identification method using the standard DNA sequences of complementary plastid (trnLF) and nuclear (ITS, GI) loci. The highly homogenous but largely divergent B. distachyon and B. stacei diploids could be easily distinguished (100% identification success) using direct trnLF (2.4%), ITS (5.5%) or GI (3.8%) sequence divergence. By contrast, B. hybridum could only be unambiguously identified through the use of combined trnLF+ITS sequences (90% of identification success) or by cloned GI sequences (96.7%) that showed 5.4% (ITS) and 4% (GI) rate divergence between the two parental sequences found in the allopolyploid. Conclusion/Significance Our data provide an unbiased and effective barcode to differentiate these three closely-related species from one another. This procedure overcomes the taxonomic uncertainty generated from methods based on morphology or flow cytometry identifications that have resulted in some misclassifications of the model plant and its allies. Our study also demonstrates that the allotetraploid B. hybridum has resulted from bi-directional crosses of B. distachyon and B. stacei plants acting either as maternal or paternal parents. PMID:23240000

The DynaMine webserver: predicting protein dynamics from sequence.

PubMed

Cilia, Elisa; Pancsa, Rita; Tompa, Peter; Lenaerts, Tom; Vranken, Wim F

2014-07-01

Protein dynamics are important for understanding protein function. Unfortunately, accurate protein dynamics information is difficult to obtain: here we present the DynaMine webserver, which provides predictions for the fast backbone movements of proteins directly from their amino-acid sequence. DynaMine rapidly produces a profile describing the statistical potential for such movements at residue-level resolution. The predicted values have meaning on an absolute scale and go beyond the traditional binary classification of residues as ordered or disordered, thus allowing for direct dynamics comparisons between protein regions. Through this webserver, we provide molecular biologists with an efficient and easy to use tool for predicting the dynamical characteristics of any protein of interest, even in the absence of experimental observations. The prediction results are visualized and can be directly downloaded. The DynaMine webserver, including instructive examples describing the meaning of the profiles, is available at http://dynamine.ibsquare.be. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Metagenomic insight into methanogenic reactors promoting direct interspecies electron transfer via granular activated carbon.

PubMed

Park, Jeong-Hoon; Park, Jong-Hun; Je Seong, Hoon; Sul, Woo Jun; Jin, Kang-Hyun; Park, Hee-Deung

2018-07-01

To provide insight into direct interspecies electron transfer via granular activated carbon (GAC), the effect of GAC supplementation on anaerobic digestion was evaluated. Compared to control samples, the GAC supplementation increased the total amount of methane production and its production rate by 31% and 72%, respectively. 16S rDNA sequencing analysis revealed a shift in the archaeal community composition; the Methanosarcina proportion decreased 17%, while the Methanosaeta proportion increased 5.6%. Metagenomic analyses based on shotgun sequencing demonstrated that the abundance of pilA and omcS genes belonging to Geobacter species decreased 69.4% and 29.4%, respectively. Furthermore, the analyses suggested a carbon dioxide reduction pathway rather than an acetate decarboxylation pathway for methane formation. Taken together, these results suggest that GAC improved methane production performance by shifting the microbial community and altering functional genes associated with direct interspecies electron transfer via conductive materials. Copyright © 2018 Elsevier Ltd. All rights reserved.

OTG-snpcaller: An Optimized Pipeline Based on TMAP and GATK for SNP Calling from Ion Torrent Data

PubMed Central

Huang, Wenpan; Xi, Feng; Lin, Lin; Zhi, Qihuan; Zhang, Wenwei; Tang, Y. Tom; Geng, Chunyu; Lu, Zhiyuan; Xu, Xun

2014-01-01

Because the new Proton platform from Life Technologies produced markedly different data from those of the Illumina platform, the conventional Illumina data analysis pipeline could not be used directly. We developed an optimized SNP calling method using TMAP and GATK (OTG-snpcaller). This method combined our own optimized processes, Remove Duplicates According to AS Tag (RDAST) and Alignment Optimize Structure (AOS), together with TMAP and GATK, to call SNPs from Proton data. We sequenced four sets of exomes captured by Agilent SureSelect and NimbleGen SeqCap EZ Kit, using Life Technology’s Ion Proton sequencer. Then we applied OTG-snpcaller and compared our results with the results from Torrent Variants Caller. The results indicated that OTG-snpcaller can reduce both false positive and false negative rates. Moreover, we compared our results with Illumina results generated by GATK best practices, and we found that the results of these two platforms were comparable. The good performance in variant calling using GATK best practices can be primarily attributed to the high quality of the Illumina sequences. PMID:24824529

OTG-snpcaller: an optimized pipeline based on TMAP and GATK for SNP calling from ion torrent data.

PubMed

Zhu, Pengyuan; He, Lingyu; Li, Yaqiao; Huang, Wenpan; Xi, Feng; Lin, Lin; Zhi, Qihuan; Zhang, Wenwei; Tang, Y Tom; Geng, Chunyu; Lu, Zhiyuan; Xu, Xun

2014-01-01

Because the new Proton platform from Life Technologies produced markedly different data from those of the Illumina platform, the conventional Illumina data analysis pipeline could not be used directly. We developed an optimized SNP calling method using TMAP and GATK (OTG-snpcaller). This method combined our own optimized processes, Remove Duplicates According to AS Tag (RDAST) and Alignment Optimize Structure (AOS), together with TMAP and GATK, to call SNPs from Proton data. We sequenced four sets of exomes captured by Agilent SureSelect and NimbleGen SeqCap EZ Kit, using Life Technology's Ion Proton sequencer. Then we applied OTG-snpcaller and compared our results with the results from Torrent Variants Caller. The results indicated that OTG-snpcaller can reduce both false positive and false negative rates. Moreover, we compared our results with Illumina results generated by GATK best practices, and we found that the results of these two platforms were comparable. The good performance in variant calling using GATK best practices can be primarily attributed to the high quality of the Illumina sequences.

Direct sequencing of hepatitis A virus and norovirus RT-PCR products from environmentally contaminated oyster using M13-tailed primers.

PubMed

Williams-Woods, Jacquelina; González-Escalona, Narjol; Burkhardt, William

2011-12-01

Human norovirus (HuNoV) and hepatitis A (HAV) are recognized as leading causes of non-bacterial foodborne associated illnesses in the United States. DNA sequencing is generally considered the standard for accurate viral genotyping in support of epidemiological investigations. Due to the genetic diversity of noroviruses (NoV), degenerate primer sets are often used in conventional reverse transcription (RT) PCR and real-time RT-quantitative PCR (RT-qPCR) for the detection of these viruses and cDNA fragments are generally cloned prior to sequencing. HAV detection methods that are sensitive and specific for real-time RT-qPCR yields small fragments sizes of 89-150bp, which can be difficult to sequence. In order to overcome these obstacles, norovirus and HAV primers were tailed with M13 forward and reverse primers. This modification increases the sequenced product size and allows for direct sequencing of the amplicons utilizing complementary M13 primers. HuNoV and HAV cDNA products from environmentally contaminated oysters were analyzed using this method. Alignments of the sequenced samples revealed ≥95% nucleotide identities. Tailing NoV and HAV primers with M13 sequence increases the cDNA product size, offers an alternative to cloning, and allows for rapid, accurate and direct sequencing of cDNA products produced by conventional or real time RT-qPCR assays. Published by Elsevier B.V.

Macaque Monkeys Discriminate Pitch Relationships

ERIC Educational Resources Information Center

Brosch, Michael; Selezneva, Elena; Bucks, Cornelia; Scheich, Henning

2004-01-01

This study demonstrates that non-human primates can categorize the direction of the pitch change of tones in a sequence. Two "Macaca fascicularis" were trained in a positive-reinforcement behavioral paradigm in which they listened to sequences of a variable number of different acoustic items. The training of discriminating pitch direction was…

Structural Heterogeneity of Doubly-Charged Peptide b-Ions

NASA Astrophysics Data System (ADS)

Li, Xiaojuan; Huang, Yiqun; O'Connor, Peter B.; Lin, Cheng

2011-02-01

Performing collisionally activated dissociation (CAD) and electron capture dissociation (ECD) in tandem has shown great promise in providing comprehensive sequence information that was otherwise unobtainable by using either fragmentation method alone or in duet. However, the general applicability of this MS3 approach in peptide sequencing may be undermined by the formation of non-direct sequence ions, as sometimes observed under CAD, particularly when multiple stages of CAD are involved. In this study, varied-sized doubly-charged b-ions from three tachykinin peptides were investigated by ECD. Sequence scrambling was observed in ECD of all b-ions from neurokinin A (HKTDSFVGLM-NH2), suggesting the presence of N- and C-termini linked macro-cyclic conformers. On the contrary, none of the b-ions from eledoisin (pEPSKDAFIGLM-NH2) produced non-direct sequence ions under ECD, as it does not contain a free N-terminal amino group. ECD of several b-ions from Substance P (RPKPQQFFGLM-NH2) showed series of cm-Lys fragment ions which suggested that the macro-cyclic structure may also be formed by connecting the C-terminal carbonyl group and the ɛ-amino group of the lysine side chain. Theoretical investigation of selected Substance P b-ions revealed several low energy conformers, including both linear oxazolones and macro-ring structures, in corroboration with the experimental observation. This study showed that a b-ion may exist as a mixture of several forms, with their propensities influenced by its N-terminus, length, and certain side-chain groups. Further, the presence of several macro-cyclic structures may result in erroneous sequence assignment when the combined CAD and ECD methods are used in peptide sequencing.

Structural Heterogeneity of Doubly-Charged Peptide b-Ions

PubMed Central

Li, Xiaojuan; Huang, Yiqun; O’Connor, Peter B.; Lin, Cheng

2011-01-01

Performing collisionally activated dissociation (CAD) and electron capture dissociation (ECD) in tandem has shown great promise in providing comprehensive sequence information that was otherwise unobtainable by using either fragmentation method alone or in duet. However, the general applicability of this MS3 approach in peptide sequencing may be undermined by the formation of non-direct sequence ions, as sometimes observed under CAD, particularly when multiple stages of CAD are involved. In this study, varied-sized doubly-charged b-ions from three tachykinin peptides were investigated by ECD. Sequence scrambling was observed in ECD of all b-ions from neurokinin A (HKTDSFVGLM-NH2), suggesting the presence of N- and C-termini linked macro-cyclic conformers. On the contrary, none of the b-ions from eledoisin (pEPSKDAFIGLM-NH2) produced non-direct sequence ions under ECD, as it does not contain a free N-terminal amino group. ECD of several b-ions from Substance P (RPKPQQFFGLM-NH2) showed series of cm-Lys fragment ions which suggested that the macro-cyclic structure may also be formed by connecting the C-terminal carbonyl group and the ε-amino group of the lysine side chain. Theoretical investigation of selected Substance P b-ions revealed several low energy conformers, including both linear oxazolones and macro-ring structures, in corroboration with the experimental observation. This study showed that a b-ion may exist as a mixture of several forms, with their propensities influenced by its N-terminus, length, and certain side-chain groups. Further, the presence of several macro-cyclic structures may result in erroneous sequence assignment when the combined CAD and ECD methods are used in peptide sequencing. PMID:21472584

Search-based optimization

NASA Technical Reports Server (NTRS)

Wheeler, Ward C.

2003-01-01

The problem of determining the minimum cost hypothetical ancestral sequences for a given cladogram is known to be NP-complete (Wang and Jiang, 1994). Traditionally, point estimations of hypothetical ancestral sequences have been used to gain heuristic, upper bounds on cladogram cost. These include procedures with such diverse approaches as non-additive optimization of multiple sequence alignment, direct optimization (Wheeler, 1996), and fixed-state character optimization (Wheeler, 1999). A method is proposed here which, by extending fixed-state character optimization, replaces the estimation process with a search. This form of optimization examines a diversity of potential state solutions for cost-efficient hypothetical ancestral sequences and can result in greatly more parsimonious cladograms. Additionally, such an approach can be applied to other NP-complete phylogenetic optimization problems such as genomic break-point analysis. c2003 The Willi Hennig Society. Published by Elsevier Science (USA). All rights reserved.

Comparison of performance of three commercial platforms for warfarin sensitivity genotyping.

PubMed

Babic, Nikolina; Haverfield, Eden V; Burrus, Julie A; Lozada, Anthony; Das, Soma; Yeo, Kiang-Teck J

2009-08-01

We performed a 3-way comparison on the Osmetech eSensor, AutoGenomics INFINITI, and a real-time PCR method (Paragonx reagents/Stratagene RT-PCR platform) for their FDA-cleared warfarin panels, and additional polymorphisms (CYP2C9*5, *6, and 11 and extended VKORC1 panels) where available. One hundred de-identified DNA samples were used in this IRB-approved study. Accuracy was determined by comparison of genotyping results across three platforms. Any discrepancy was resolved by bi-directional sequencing. The CYP4F2 on Osmetech was validated by bi-directional sequencing. Accuracies for CYP2C9*2 and *3 were 100% for all 3 platforms. VKORC1 3673 genotyping accuracies were 100% on eSensor and 97% on Infiniti. CYP2C9*5, *6 and *11 showed 100% concordance between eSensor and Infiniti. VKORC1 6484 and 9041 variants compared between ParagonDx and Infiniti analyzer were 100% (6484) and 99% (9041) concordant. CYP4F2 was 100% concordant with sequencing results. The time required to generate the results from automated DNA extraction-to-result was approximately 8h on Infiniti, and 4h on eSensor and ParagonDx, respectively. Overall, we observed excellent CYP2C9*2 and *3 genotyping accuracy for all three platforms. For VKORC1 3673 genotyping, eSensor demonstrated a slightly higher accuracy than the Infiniti, and CYP4F2 on Osmetech was 100% accurate.

Persistency of rupture directivity in moderate-magnitude earthquakes in Italy: Implications for seismic hazard

NASA Astrophysics Data System (ADS)

Rovelli, A.; Calderoni, G.

2012-12-01

A simple method based on the EGF deconvolution in the frequency domain is applied to detect the occurrence of unilateral ruptures in recent damaging earthquakes in Italy. The spectral ratio between event pairs with different magnitudes at individual stations shows large azimuthal variations above corner frequency when the target event is affected by source directivity and the EGF is not or vice versa. The analysis is applied to seismograms and accelerograms recorded during the seismic sequence following the 20 May 2012, Mw 5.6 main shock in Emilia, northern Italy, the 6 April 2009, Mw 6.1 earthquake of L'Aquila, central Italy, and the 26 September 1997, Mw 5.7 and 6.0 shocks in Umbria-Marche, central Italy. Events of each seismic sequence are selected as having consistent focal mechanisms, and the station selection obeys to the constraint of a similar source-to-receiver path for the event pairs. The analyzed data set of L'Aquila consists of 962 broad-band seismograms relative to 69 normal-faulting earthquakes (3.3 ≤ MW ≤ 6.1, according to Herrmann et al., 2011), stations are selected in the distance range 100 to 250 km to minimize differences in propagation paths. The seismogram analysis reveals that a strong along-strike (toward SE) source directivity characterized all of the three Mw > 5.0 shocks. Source directivity was also persistent up to the smallest magnitudes: 65% of earthquakes under study showed evidence of directivity toward SE whereas only one (Mw 3.7) event showed directivity in the opposite direction. Also the Mw 5.6 main shock of the 20 May 2012 in Emilia result in large azimuthal spectral variations indicating unilateral rupture propagation toward SE. According to the reconstructed geometry of the trust-fault plane, the inferred directivity direction suggests top-down rupture propagation. The analysis over the Emilia aftershock sequence is in progress. The third seismic sequence, dated 1997-1998, occurred in the northern Apennines and, similarly to L'Aquila faults, was characterized by normal-faulting earthquakes with strike substantially parallel to the Apennine trend. Although the amount of data is not as abundant as for the most recent earthquakes, the available data were already object of previous studies indicating unilateral rupture propagation in several of the strongest (5.5 < Mw < 6.0) shocks. We show that the effect of directivity is particularly significant in intermontane basins where long-period (T > 1 sec) ground motions are amplified by soft sediments and the combination of local amplification with source directivity causes exceedance of spectral ordinates at those periods up to more than 2 standard deviations from the expected values of commonly used GMPEs for soft sites. These results arise a concern in terms of seismic hazard because source directivity is found to be recurrent feature in the Apennines. Moreover, the predominant fault strike and intermontane basins are both aligned along the Apennine chain offering a condition potentially favorable to extra-amplifications at periods relevant to seismic risk.

Multiple Access Interference Reduction Using Received Response Code Sequence for DS-CDMA UWB System

NASA Astrophysics Data System (ADS)

Toh, Keat Beng; Tachikawa, Shin'ichi

This paper proposes a combination of novel Received Response (RR) sequence at the transmitter and a Matched Filter-RAKE (MF-RAKE) combining scheme receiver system for the Direct Sequence-Code Division Multiple Access Ultra Wideband (DS-CDMA UWB) multipath channel model. This paper also demonstrates the effectiveness of the RR sequence in Multiple Access Interference (MAI) reduction for the DS-CDMA UWB system. It suggests that by using conventional binary code sequence such as the M sequence or the Gold sequence, there is a possibility of generating extra MAI in the UWB system. Therefore, it is quite difficult to collect the energy efficiently although the RAKE reception method is applied at the receiver. The main purpose of the proposed system is to overcome the performance degradation for UWB transmission due to the occurrence of MAI during multiple accessing in the DS-CDMA UWB system. The proposed system improves the system performance by improving the RAKE reception performance using the RR sequence which can reduce the MAI effect significantly. Simulation results verify that significant improvement can be obtained by the proposed system in the UWB multipath channel models.

Molecular Evidence for a Natural Primary Triple Hybrid in Plants Revealed from Direct Sequencing

PubMed Central

Kaplan, Zdenek; Fehrer, Judith

2007-01-01

Background and Aims Molecular evidence for natural primary hybrids composed of three different plant species is very rarely reported. An investigation was therefore carried out into the origin and a possible scenario for the rise of a sterile plant clone showing a combination of diagnostic morphological features of three separate, well-defined Potamogeton species. Methods The combination of sequences from maternally inherited cytoplasmic (rpl20-rps12) and biparentally inherited nuclear ribosomal DNA (ITS) was used to identify the exact identity of the putative triple hybrid. Key Results Direct sequencing showed ITS variants of three parental taxa, P. gramineus, P. lucens and P. perfoliatus, whereas chloroplast DNA identified P. perfoliatus as the female parent. A scenario for the rise of the triple hybrid through a fertile binary hybrid P. gramineus × P. lucens crossed with P. perfoliatus is described. Conclusions Even though the triple hybrid is sterile, it possesses an efficient strategy for its existence and became locally successful even in the parental environment, perhaps as a result of heterosis. The population investigated is the only one known of this hybrid, P. × torssanderi, worldwide. Isozyme analysis indicated the colony to be genetically uniform. The plants studied represented a single clone that seems to have persisted at this site for a long time. PMID:17478544

Chromodomains direct integration of retrotransposons to heterochromatin

PubMed Central

Gao, Xiang; Hou, Yi; Ebina, Hirotaka; Levin, Henry L.; Voytas, Daniel F.

2008-01-01

The enrichment of mobile genetic elements in heterochromatin may be due, in part, to targeted integration. The chromoviruses are Ty3/gypsy retrotransposons with chromodomains at their integrase C termini. Chromodomains are logical determinants for targeting to heterochromatin, because the chromodomain of heterochromatin protein 1 (HP1) typically recognizes histone H3 K9 methylation, an epigenetic mark characteristic of heterochromatin. We describe three groups of chromoviruses based on amino acid sequence relationships of their integrase C termini. Genome sequence analysis indicates that representative chromoviruses from each group are enriched in gene-poor regions of the genome relative to other retrotransposons, and when fused to fluorescent marker proteins, the chromodomains target proteins to specific subnuclear foci coincident with heterochromatin. The chromodomain of the fungal element, MAGGY, interacts with histone H3 dimethyl- and trimethyl-K9, and when the MAGGY chromodomain is fused to integrase of the Schizosaccharomyces pombe Tf1 retrotransposon, new Tf1 insertions are directed to sites of H3 K9 methylation. Repetitive sequences such as transposable elements trigger the RNAi pathway resulting in their epigenetic modification. Our results suggest a dynamic interplay between retrotransposons and heterochromatin, wherein mobile elements recognize heterochromatin at the time of integration and then perpetuate the heterochromatic mark by triggering epigenetic modification. PMID:18256242

The first genome sequences of human bocaviruses from Vietnam

PubMed Central

Thanh, Tran Tan; Van, Hoang Minh Tu; Hong, Nguyen Thi Thu; Nhu, Le Nguyen Truc; Anh, Nguyen To; Tuan, Ha Manh; Hien, Ho Van; Tuong, Nguyen Manh; Kien, Trinh Trung; Khanh, Truong Huu; Nhan, Le Nguyen Thanh; Hung, Nguyen Thanh; Chau, Nguyen Van Vinh; Thwaites, Guy; van Doorn, H. Rogier; Tan, Le Van

2017-01-01

As part of an ongoing effort to generate complete genome sequences of hand, foot and mouth disease-causing enteroviruses directly from clinical specimens, two complete coding sequences and two partial genomic sequences of human bocavirus 1 (n=3) and 2 (n=1) were co-amplified and sequenced, representing the first genome sequences of human bocaviruses from Vietnam. The sequences may aid future study aiming at understanding the evolution of the virus. PMID:28090592

Generation of Tandem Direct Duplications by Reversed-Ends Transposition of Maize Ac Elements

PubMed Central

Peterson, Thomas

2013-01-01

Tandem direct duplications are a common feature of the genomes of eukaryotes ranging from yeast to human, where they comprise a significant fraction of copy number variations. The prevailing model for the formation of tandem direct duplications is non-allelic homologous recombination (NAHR). Here we report the isolation of a series of duplications and reciprocal deletions isolated de novo from a maize allele containing two Class II Ac/Ds transposons. The duplication/deletion structures suggest that they were generated by alternative transposition reactions involving the termini of two nearby transposable elements. The deletion/duplication breakpoint junctions contain 8 bp target site duplications characteristic of Ac/Ds transposition events, confirming their formation directly by an alternative transposition mechanism. Tandem direct duplications and reciprocal deletions were generated at a relatively high frequency (∼0.5 to 1%) in the materials examined here in which transposons are positioned nearby each other in appropriate orientation; frequencies would likely be much lower in other genotypes. To test whether this mechanism may have contributed to maize genome evolution, we analyzed sequences flanking Ac/Ds and other hAT family transposons and identified three small tandem direct duplications with the structural features predicted by the alternative transposition mechanism. Together these results show that some class II transposons are capable of directly inducing tandem sequence duplications, and that this activity has contributed to the evolution of the maize genome. PMID:23966872

Naturally occurring deletions/insertions in HBV core promoter tend to decrease in hepatitis B e antigen-positive chronic hepatitis B patients during antiviral therapy.

PubMed

Peng, Yaqin; Liu, Baoming; Hou, Jinlin; Sun, Jian; Hao, Ran; Xiang, Kuanhui; Yan, Ling; Zhang, Jiangbo; Zhuang, Hui; Li, Tong

2015-01-01

Mutations in HBV core promoter (CP) are suggested to affect viral replication and disease progression. We investigated CP deletion/insertion mutations (Del/Ins) in hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) patients before and during antiviral treatment. Direct and clone sequencings were used for detection of CP Del/Ins in 12 patients. The dynamic changes of CP Del/Ins were tracked in these cases until week 48 of treatment. The effects of Del/Ins on CP activities and hepatitis B X protein (HBx) were analysed using luciferase assay and sequence comparison, respectively. Furthermore, 292 untreated HBeAg-positive CHB cases were also analysed. Twelve cases with multi-peak PCR direct sequencing electropherograms at baseline were confirmed to have CP Del/Ins by clone sequencing, with detection rates varying from 14.8% to 93.3% of clones analysed. Follow-up studies showed the detection rates of CP Del/Ins in patients decreased from 100% (12/12) at baseline to 16.7% (2/12) at week 48 of treatment (P<0.001), in parallel with a decline in HBV DNA, hepatitis B surface antigen (HBsAg), alanine aminotransferase (ALT) and aspartate transaminase (AST) levels along with an increase in HBeAg loss. Luciferase assay results showed distinct promoter activities among Del/Ins-harbouring CP sequences. Importantly, 71.8% (148/206) of Del/Ins sequences potentially resulted in HBx carboxy-terminal truncations. CP Del/Ins mutations were also found in 27.4% (80/292) of untreated cases. Naturally occurring complex of CP Del/Ins mutants existed in untreated HBeAg-positive CHB patients. These mutations would affect HBV transcription activities and integrity of HBx, which might correlate with disease progression. Their prevalence decreases on antiviral therapy in parallel with the decline in HBV DNA, HBsAg and ALT and AST levels.

Extensive characterization of Tupaia belangeri neuropeptidome using an integrated mass spectrometric approach.

PubMed

Petruzziello, Filomena; Fouillen, Laetitia; Wadensten, Henrik; Kretz, Robert; Andren, Per E; Rainer, Gregor; Zhang, Xiaozhe

2012-02-03

Neuropeptidomics is used to characterize endogenous peptides in the brain of tree shrews (Tupaia belangeri). Tree shrews are small animals similar to rodents in size but close relatives of primates, and are excellent models for brain research. Currently, tree shrews have no complete proteome information available on which direct database search can be allowed for neuropeptide identification. To increase the capability in the identification of neuropeptides in tree shrews, we developed an integrated mass spectrometry (MS)-based approach that combines methods including data-dependent, directed, and targeted liquid chromatography (LC)-Fourier transform (FT)-tandem MS (MS/MS) analysis, database construction, de novo sequencing, precursor protein search, and homology analysis. Using this integrated approach, we identified 107 endogenous peptides that have sequences identical or similar to those from other mammalian species. High accuracy MS and tandem MS information, with BLAST analysis and chromatographic characteristics were used to confirm the sequences of all the identified peptides. Interestingly, further sequence homology analysis demonstrated that tree shrew peptides have a significantly higher degree of homology to equivalent sequences in humans than those in mice or rats, consistent with the close phylogenetic relationship between tree shrews and primates. Our results provide the first extensive characterization of the peptidome in tree shrews, which now permits characterization of their function in nervous and endocrine system. As the approach developed fully used the conservative properties of neuropeptides in evolution and the advantage of high accuracy MS, it can be portable for identification of neuropeptides in other species for which the fully sequenced genomes or proteomes are not available.

Newborn Screening in the Era of Precision Medicine.

PubMed

Yang, Lan; Chen, Jiajia; Shen, Bairong

2017-01-01

As newborn screening success stories gained general confirmation during the past 50 years, scientists quickly discovered diagnostic tests for a host of genetic disorders that could be treated at birth. Outstanding progress in sequencing technologies over the last two decades has made it possible to comprehensively profile newborn screening (NBS) and identify clinically relevant genomic alterations. With the rapid developments in whole-genome sequencing (WGS) and whole-exome sequencing (WES) recently, we can detect newborns at the genomic level and be able to direct the appropriate diagnosis to the different individuals at the appropriate time, which is also encompassed in the concept of precision medicine. Besides, we can develop novel interventions directed at the molecular characteristics of genetic diseases in newborns. The implementation of genomics in NBS programs would provide an effective premise for the identification of the majority of genetic aberrations and primarily help in accurate guidance in treatment and better prediction. However, there are some debate correlated with the widespread application of genome sequencing in NBS due to some major concerns such as clinical analysis, result interpretation, storage of sequencing data, and communication of clinically relevant mutations to pediatricians and parents, along with the ethical, legal, and social implications (so-called ELSI). This review is focused on these critical issues and concerns about the expanding role of genomics in NBS for precision medicine. If WGS or WES is to be incorporated into NBS practice, considerations about these challenges should be carefully regarded and tackled properly to adapt the requirement of genome sequencing in the era of precision medicine.

Single-molecule, full-length transcript sequencing provides insight into the extreme metabolism of the ruby-throated hummingbird Archilochus colubris.

PubMed

Workman, Rachael E; Myrka, Alexander M; Wong, G William; Tseng, Elizabeth; Welch, Kenneth C; Timp, Winston

2018-03-01

Hummingbirds oxidize ingested nectar sugars directly to fuel foraging but cannot sustain this fuel use during fasting periods, such as during the night or during long-distance migratory flights. Instead, fasting hummingbirds switch to oxidizing stored lipids that are derived from ingested sugars. The hummingbird liver plays a key role in moderating energy homeostasis and this remarkable capacity for fuel switching. Additionally, liver is the principle location of de novo lipogenesis, which can occur at exceptionally high rates, such as during premigratory fattening. Yet understanding how this tissue and whole organism moderates energy turnover is hampered by a lack of information regarding how relevant enzymes differ in sequence, expression, and regulation. We generated a de novo transcriptome of the hummingbird liver using PacBio full-length cDNA sequencing (Iso-Seq), yielding 8.6Gb of sequencing data, or 2.6M reads from 4 different size fractions. We analyzed data using the SMRTAnalysis v3.1 Iso-Seq pipeline, then clustered isoforms into gene families to generate de novo gene contigs using Cogent. We performed orthology analysis to identify closely related sequences between our transcriptome and other avian and human gene sets. Finally, we closely examined homology of critical lipid metabolism genes between our transcriptome data and avian and human genomes. We confirmed high levels of sequence divergence within hummingbird lipogenic enzymes, suggesting a high probability of adaptive divergent function in the hepatic lipogenic pathways. Our results leverage cutting-edge technology and a novel bioinformatics pipeline to provide a first direct look at the transcriptome of this incredible organism.

Impact of cultivation on characterisation of species composition of soil bacterial communities.

PubMed

McCaig, A E.; Grayston, S J.; Prosser, J I.; Glover, L A.

2001-03-01

The species composition of culturable bacteria in Scottish grassland soils was investigated using a combination of Biolog and 16S rDNA analysis for characterisation of isolates. The inclusion of a molecular approach allowed direct comparison of sequences from culturable bacteria with sequences obtained during analysis of DNA extracted directly from the same soil samples. Bacterial strains were isolated on Pseudomonas isolation agar (PIA), a selective medium, and on tryptone soya agar (TSA), a general laboratory medium. In total, 12 and 21 morphologically different bacterial cultures were isolated on PIA and TSA, respectively. Biolog and sequencing placed PIA isolates in the same taxonomic groups, the majority of cultures belonging to the Pseudomonas (sensu stricto) group. However, analysis of 16S rDNA sequences proved more efficient than Biolog for characterising TSA isolates due to limitations of the Microlog database for identifying environmental bacteria. In general, 16S rDNA sequences from TSA isolates showed high similarities to cultured species represented in sequence databases, although TSA-8 showed only 92.5% similarity to the nearest relative, Bacillus insolitus. In general, there was very little overlap between the culturable and uncultured bacterial communities, although two sequences, PIA-2 and TSA-13, showed >99% similarity to soil clones. A cloning step was included prior to sequence analysis of two isolates, TSA-5 and TSA-14, and analysis of several clones confirmed that these cultures comprised at least four and three sequence types, respectively. All isolate clones were most closely related to uncultured bacteria, with clone TSA-5.1 showing 99.8% similarity to a sequence amplified directly from the same soil sample. Interestingly, one clone, TSA-5.4, clustered within a novel group comprising only uncultured sequences. This group, which is associated with the novel, deep-branching Acidobacterium capsulatum lineage, also included clones isolated during direct analysis of the same soil and from a wide range of other sample types studied elsewhere. The study demonstrates the value of fine-scale molecular analysis for identification of laboratory isolates and indicates the culturability of approximately 1% of the total population but under a restricted range of media and cultivation conditions.

Sequence stratigraphic principles applied to the Miocene Hawthorn Group, west-central Florida

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

Norton, V.L.; Randazzo, A.F.

1993-03-01

Sequence boundaries for the Miocene Hawthorn Group in the ROMP 20 drill core from Osprey, Sarasota County, FL were generally delineated by lithologic variations recognized from core slabs, thin section analysis, and geophysical logs. At least six depositional sequences representing third order sea level fluctuations were identified. Depositional environments were determined on the basis of the characteristic lithologic constituents including rip-up clasts, pellets, fossils, laminations, burrow, degree of induration, and grain sorting. The sequence boundaries appear to have formed when the rate of the eustatic fall exceeded basin subsidence rates producing a relative sea level fall at a depositional shorelinemore » break. As a result of the basinward facies shift associated with this sequence type, peritidal facies may directly overlie deeper water facies. Subaerial exposure and erosion can be expected. The sequence of facies representing progressively deeper water depositional environments, followed by a progressive shallowing, were present between bounding surfaces. Among the six sequences recognized, four were clearly delineated as representative of regression, subaerial exposure, and subsequent transgression. Two sequences were less clearly defined and probably represent transitional facies which had exposure surfaces developed. Comparison of the petrologically established sequence stratigraphy with published sea level curves resulted in a strong correlation between the number of sequences recognized and the number of coastal on-lap/off-lap cycles depicted for the early to middle Miocene. This correlation suggests that petrologic examination of core slabs, with supplemental thin section data, can provide useful information regarding the recognition of stratigraphic sequences and relative sea level fluctuations, particularly, in situations where seismic data may not be available.« less

The murine Cd48 gene: allelic polymorphism in the IgV-like region.

PubMed

Cabrero, J G; Freeman, G J; Reiser, H

1998-12-01

The murine CD48 molecule is a member of the immunoglobulin superfamily which regulates the activation of T lymphocytes. prior cloning experiments using mRNA from two different mouse strains had yielded discrepant sequences within the IgV-like domain of murine CD48. To resolve this issue, we have directly sequenced genomic DNA of 10 laboratory strains and two inbred strains of wild origin. The results of our analysis reveal an allelic polymorphism within the IgV-like domain of murine CD48.

Influence of stacking sequence on scattering characteristics of the fundamental anti-symmetric Lamb wave at through holes in composite laminates.

PubMed

Veidt, Martin; Ng, Ching-Tai

2011-03-01

This paper investigates the scattering characteristics of the fundamental anti-symmetric (A(0)) Lamb wave at through holes in composite laminates. Three-dimensional (3D) finite element (FE) simulations and experimental measurements are used to study the physical phenomenon. Unidirectional, bidirectional, and quasi-isotropic composite laminates are considered in the study. The influence of different hole diameter to wavelength aspect ratios and different stacking sequences on wave scattering characteristics are investigated. The results show that amplitudes and directivity distribution of the scattered Lamb wave depend on these parameters. In the case of quasi-isotropic composite laminates, the scattering directivity patterns are dominated by the fiber orientation of the outer layers and are quite different for composite laminates with the same number of laminae but different stacking sequence. The study provides improved physical insight into the scattering phenomena at through holes in composite laminates, which is essential to develop, validate, and optimize guided wave damage detection and characterization techniques. © 2011 Acoustical Society of America

In vivo evolution of antimicrobial resistance in a series of Staphylococcus aureus patient isolates: the entire picture or a cautionary tale?

PubMed Central

van Hal, Sebastiaan J.; Steen, Jason A.; Espedido, Björn A.; Grimmond, Sean M.; Cooper, Matthew A.; Holden, Matthew T. G.; Bentley, Stephen D.; Gosbell, Iain B.; Jensen, Slade O.

2014-01-01

Objectives To obtain an expanded understanding of antibiotic resistance evolution in vivo, particularly in the context of vancomycin exposure. Methods The whole genomes of six consecutive methicillin-resistant Staphylococcus aureus blood culture isolates (ST239-MRSA-III) from a single patient exposed to various antimicrobials (over a 77 day period) were sequenced and analysed. Results Variant analysis revealed the existence of non-susceptible sub-populations derived from a common susceptible ancestor, with the predominant circulating clone(s) selected for by type and duration of antimicrobial exposure. Conclusions This study highlights the dynamic nature of bacterial evolution and that non-susceptible sub-populations can emerge from clouds of variation upon antimicrobial exposure. Diagnostically, this has direct implications for sample selection when using whole-genome sequencing as a tool to guide clinical therapy. In the context of bacteraemia, deep sequencing of bacterial DNA directly from patient blood samples would avoid culture ‘bias’ and identify mutations associated with circulating non-susceptible sub-populations, some of which may confer cross-resistance to alternate therapies. PMID:24047554

CT Image Sequence Restoration Based on Sparse and Low-Rank Decomposition

PubMed Central

Gou, Shuiping; Wang, Yueyue; Wang, Zhilong; Peng, Yong; Zhang, Xiaopeng; Jiao, Licheng; Wu, Jianshe

2013-01-01

Blurry organ boundaries and soft tissue structures present a major challenge in biomedical image restoration. In this paper, we propose a low-rank decomposition-based method for computed tomography (CT) image sequence restoration, where the CT image sequence is decomposed into a sparse component and a low-rank component. A new point spread function of Weiner filter is employed to efficiently remove blur in the sparse component; a wiener filtering with the Gaussian PSF is used to recover the average image of the low-rank component. And then we get the recovered CT image sequence by combining the recovery low-rank image with all recovery sparse image sequence. Our method achieves restoration results with higher contrast, sharper organ boundaries and richer soft tissue structure information, compared with existing CT image restoration methods. The robustness of our method was assessed with numerical experiments using three different low-rank models: Robust Principle Component Analysis (RPCA), Linearized Alternating Direction Method with Adaptive Penalty (LADMAP) and Go Decomposition (GoDec). Experimental results demonstrated that the RPCA model was the most suitable for the small noise CT images whereas the GoDec model was the best for the large noisy CT images. PMID:24023764

Assessing Diversity of DNA Structure-Related Sequence Features in Prokaryotic Genomes

PubMed Central

Huang, Yongjie; Mrázek, Jan

2014-01-01

Prokaryotic genomes are diverse in terms of their nucleotide and oligonucleotide composition as well as presence of various sequence features that can affect physical properties of the DNA molecule. We present a survey of local sequence patterns which have a potential to promote non-canonical DNA conformations (i.e. different from standard B-DNA double helix) and interpret the results in terms of relationships with organisms' habitats, phylogenetic classifications, and other characteristics. Our present work differs from earlier similar surveys not only by investigating a wider range of sequence patterns in a large number of genomes but also by using a more realistic null model to assess significant deviations. Our results show that simple sequence repeats and Z-DNA-promoting patterns are generally suppressed in prokaryotic genomes, whereas palindromes and inverted repeats are over-represented. Representation of patterns that promote Z-DNA and intrinsic DNA curvature increases with increasing optimal growth temperature (OGT), and decreases with increasing oxygen requirement. Additionally, representations of close direct repeats, palindromes and inverted repeats exhibit clear negative trends with increasing OGT. The observed relationships with environmental characteristics, particularly OGT, suggest possible evolutionary scenarios of structural adaptation of DNA to particular environmental niches. PMID:24408877

Portero versus portador: Spanish interpretation of genomic terminology during whole exome sequencing results disclosure.

PubMed

Gutierrez, Amanda M; Robinson, Jill O; Statham, Emily E; Scollon, Sarah; Bergstrom, Katie L; Slashinski, Melody J; Parsons, Donald W; Plon, Sharon E; McGuire, Amy L; Street, Richard L

2017-11-01

Describe modifications to technical genomic terminology made by interpreters during disclosure of whole exome sequencing (WES) results. Using discourse analysis, we identified and categorized interpretations of genomic terminology in 42 disclosure sessions where Spanish-speaking parents received their child's WES results either from a clinician using a medical interpreter, or directly from a bilingual physician. Overall, 76% of genomic terms were interpreted accordantly, 11% were misinterpreted and 13% were omitted. Misinterpretations made by interpreters and bilingual physicians included using literal and nonmedical terminology to interpret genomic concepts. Modifications to genomic terminology made during interpretation highlight the need to standardize bilingual genomic lexicons. We recommend Spanish terms that can be used to refer to genomic concepts.

Detection of Cytosine methylation in ancient DNA from five native american populations using bisulfite sequencing.

PubMed

Smith, Rick W A; Monroe, Cara; Bolnick, Deborah A

2015-01-01

While cytosine methylation has been widely studied in extant populations, relatively few studies have analyzed methylation in ancient DNA. Most existing studies of epigenetic marks in ancient DNA have inferred patterns of methylation in highly degraded samples using post-mortem damage to cytosines as a proxy for cytosine methylation levels. However, this approach limits the inference of methylation compared with direct bisulfite sequencing, the current gold standard for analyzing cytosine methylation at single nucleotide resolution. In this study, we used direct bisulfite sequencing to assess cytosine methylation in ancient DNA from the skeletal remains of 30 Native Americans ranging in age from approximately 230 to 4500 years before present. Unmethylated cytosines were converted to uracils by treatment with sodium bisulfite, bisulfite products of a CpG-rich retrotransposon were pyrosequenced, and C-to-T ratios were quantified for a single CpG position. We found that cytosine methylation is readily recoverable from most samples, given adequate preservation of endogenous nuclear DNA. In addition, our results indicate that the precision of cytosine methylation estimates is inversely correlated with aDNA preservation, such that samples of low DNA concentration show higher variability in measures of percent methylation than samples of high DNA concentration. In particular, samples in this study with a DNA concentration above 0.015 ng/μL generated the most consistent measures of cytosine methylation. This study presents evidence of cytosine methylation in a large collection of ancient human remains, and indicates that it is possible to analyze epigenetic patterns in ancient populations using direct bisulfite sequencing approaches.

Planning Ahead: Object-Directed Sequential Actions Decoded from Human Frontoparietal and Occipitotemporal Networks

PubMed Central

Gallivan, Jason P.; Johnsrude, Ingrid S.; Randall Flanagan, J.

2016-01-01

Object-manipulation tasks (e.g., drinking from a cup) typically involve sequencing together a series of distinct motor acts (e.g., reaching toward, grasping, lifting, and transporting the cup) in order to accomplish some overarching goal (e.g., quenching thirst). Although several studies in humans have investigated the neural mechanisms supporting the planning of visually guided movements directed toward objects (such as reaching or pointing), only a handful have examined how manipulatory sequences of actions—those that occur after an object has been grasped—are planned and represented in the brain. Here, using event-related functional MRI and pattern decoding methods, we investigated the neural basis of real-object manipulation using a delayed-movement task in which participants first prepared and then executed different object-directed action sequences that varied either in their complexity or final spatial goals. Consistent with previous reports of preparatory brain activity in non-human primates, we found that activity patterns in several frontoparietal areas reliably predicted entire action sequences in advance of movement. Notably, we found that similar sequence-related information could also be decoded from pre-movement signals in object- and body-selective occipitotemporal cortex (OTC). These findings suggest that both frontoparietal and occipitotemporal circuits are engaged in transforming object-related information into complex, goal-directed movements. PMID:25576538

Intra-Genomic Internal Transcribed Spacer Region Sequence Heterogeneity and Molecular Diagnosis in Clinical Microbiology.

PubMed

Zhao, Ying; Tsang, Chi-Ching; Xiao, Meng; Cheng, Jingwei; Xu, Yingchun; Lau, Susanna K P; Woo, Patrick C Y

2015-10-22

Internal transcribed spacer region (ITS) sequencing is the most extensively used technology for accurate molecular identification of fungal pathogens in clinical microbiology laboratories. Intra-genomic ITS sequence heterogeneity, which makes fungal identification based on direct sequencing of PCR products difficult, has rarely been reported in pathogenic fungi. During the process of performing ITS sequencing on 71 yeast strains isolated from various clinical specimens, direct sequencing of the PCR products showed ambiguous sequences in six of them. After cloning the PCR products into plasmids for sequencing, interpretable sequencing electropherograms could be obtained. For each of the six isolates, 10-49 clones were selected for sequencing and two to seven intra-genomic ITS copies were detected. The identities of these six isolates were confirmed to be Candida glabrata (n=2), Pichia (Candida) norvegensis (n=2), Candida tropicalis (n=1) and Saccharomyces cerevisiae (n=1). Multiple sequence alignment revealed that one to four intra-genomic ITS polymorphic sites were present in the six isolates, and all these polymorphic sites were located in the ITS1 and/or ITS2 regions. We report and describe the first evidence of intra-genomic ITS sequence heterogeneity in four different pathogenic yeasts, which occurred exclusively in the ITS1 and ITS2 spacer regions for the six isolates in this study.

Intra-Genomic Internal Transcribed Spacer Region Sequence Heterogeneity and Molecular Diagnosis in Clinical Microbiology

PubMed Central

Zhao, Ying; Tsang, Chi-Ching; Xiao, Meng; Cheng, Jingwei; Xu, Yingchun; Lau, Susanna K. P.; Woo, Patrick C. Y.

2015-01-01

Internal transcribed spacer region (ITS) sequencing is the most extensively used technology for accurate molecular identification of fungal pathogens in clinical microbiology laboratories. Intra-genomic ITS sequence heterogeneity, which makes fungal identification based on direct sequencing of PCR products difficult, has rarely been reported in pathogenic fungi. During the process of performing ITS sequencing on 71 yeast strains isolated from various clinical specimens, direct sequencing of the PCR products showed ambiguous sequences in six of them. After cloning the PCR products into plasmids for sequencing, interpretable sequencing electropherograms could be obtained. For each of the six isolates, 10–49 clones were selected for sequencing and two to seven intra-genomic ITS copies were detected. The identities of these six isolates were confirmed to be Candida glabrata (n = 2), Pichia (Candida) norvegensis (n = 2), Candida tropicalis (n = 1) and Saccharomyces cerevisiae (n = 1). Multiple sequence alignment revealed that one to four intra-genomic ITS polymorphic sites were present in the six isolates, and all these polymorphic sites were located in the ITS1 and/or ITS2 regions. We report and describe the first evidence of intra-genomic ITS sequence heterogeneity in four different pathogenic yeasts, which occurred exclusively in the ITS1 and ITS2 spacer regions for the six isolates in this study. PMID:26506340

Role of the Adenovirus DNA-Binding Protein in In Vitro Adeno-Associated Virus DNA Replication

PubMed Central

Ward, Peter; Dean, Frank B.; O’Donnell, Michael E.; Berns, Kenneth I.

1998-01-01

A basic question in adeno-associated virus (AAV) biology has been whether adenovirus (Ad) infection provided any function which directly promoted replication of AAV DNA. Previously in vitro assays for AAV DNA replication, using linear duplex AAV DNA as the template, uninfected or Ad-infected HeLa cell extracts, and exogenous AAV Rep protein, demonstrated that Ad infection provides a direct helper effect for AAV DNA replication. It was shown that the nature of this helper effect was to increase the processivity of AAV DNA replication. Left unanswered was the question of whether this effect was the result of cellular factors whose activity was enhanced by Ad infection or was the result of direct participation of Ad proteins in AAV DNA replication. In this report, we show that in the in vitro assay, enhancement of processivity occurs with the addition of either the Ad DNA-binding protein (Ad-DBP) or the human single-stranded DNA-binding protein (replication protein A [RPA]). Clearly Ad-DBP is present after Ad infection but not before, whereas the cellular level of RPA is not apparently affected by Ad infection. However, we have not measured possible modifications of RPA which might occur after Ad infection and affect AAV DNA replication. When the substrate for replication was an AAV genome inserted into a plasmid vector, RPA was not an effective substitute for Ad-DBP. Extracts supplemented with Ad-DBP preferentially replicated AAV sequences rather than adjacent vector sequences; in contrast, extracts supplemented with RPA preferentially replicated vector sequences. PMID:9420241

N-ras Mutation Detection by Pyrosequencing in Adult Patients with Acute Myeloid Leukemia at a Single Institution

PubMed Central

Jeong, Ji Hun; Park, Soon Ho; Park, Mi Jung; Kim, Moon Jin; Kim, Kyung Hee; Park, Pil Whan; Seo, Yiel Hea; Lee, Jae Hoon; Park, Jinny; Hong, Junshik

2013-01-01

Background N-ras mutations are one of the most commonly detected abnormalities of myeloid origin. N-ras mutations result in a constitutively active N-ras protein that induces uncontrolled cell proliferation and inhibits apoptosis. We analyzed N-ras mutations in adult patients with AML at a particular institution and compared pyrosequencing analysis with a direct sequencing method for the detection of N-ras mutations. Methods We analyzed 90 bone marrow samples from 83 AML patients. We detected N-ras mutations in codons 12, 13, and 61 using the pyrosequencing method and subsequently confirmed all data by direct sequencing. Using these methods, we screened the N-ras mutation quantitatively and determined the incidence and characteristic of N-ras mutation. Results The incidence of N-ras mutation was 7.2% in adult AML patients. The patients with N-ras mutations showed significant higher hemoglobin levels (P=0.022) and an increased incidence of FLT3 mutations (P=0.003). We observed 3 cases with N-ras mutations in codon 12 (3.6%), 2 cases in codon 13 (2.4%), and 1 case in codon 61 (1.2%). All the mutations disappeared during chemotherapy. Conclusions There is a low incidence (7.2%) of N-ras mutations in AML patients compared with other populations. Similar data is obtained by both pyrosequencing and direct sequencing. This study showed the correlation between the N-ras mutation and the therapeutic response. However, pyrosequencing provides quantitative data and is useful for monitoring therapeutic responses. PMID:23667841

Seismic stratigraphy and structure of the Chukchi Borderland: implications for the opening of the Canada Basin

NASA Astrophysics Data System (ADS)

Ilhan, I.; Coakley, B.

2015-12-01

Interpretation of seismic reflection data from the western Chukchi Borderland has illuminated the structure and stratigraphy of the area. Basement rotated fault blocks are offset by two border fault systems (BFS1 and BFS2) and by secondary faults, striking curvilinear in the NW-SE direction, dipping to the NE. The BFS1 dissects the Chukchi Plateau into two first-order rotated blocks bounding two major sedimentary depocentres, the North Chukchi Basin and the Chukchi Plateau Central Basin. The BFS2, which has a larger offset than BFS1, forms the western boundary of the Northwind Basin. Much of the stratigraphy is controlled by sediment supply. The basins were starved early in their history, resulting in a limited syn-rift section. Substantial sediment accumulation in the Borderland appears to post-date large scale progradation of the depostional shelf edge across the Chukchi Shelf. Basin infill stratigraphies are subdivided into pre-rift, syn-rift, early-, middle-, late post-rift, and glacio-marine sequences (SB1-SB5). SB1 shows truncation of the remnants of the pre-rift strata below and onlap of the syn-rift sequence(s) above; the SB2 marks the termination of the rifting stage and is bounded by bi-directional onlap surface of the early post-rift strata above; the base of SB3 is an onlap surface marks the arrival of the prograding shelf margin sequence(s); the SB4 shows evidence of erosion at the base of the prograding late post-rift sequence(s); and the SB5 is an downloap surface marking the first arrival of the glacio-marine sediments eroded from the Chukchi Shelf. Two ages of the major sequence boundaries, the SB3 and SB4, can be directly tied to Popcorn and Crackerjack Chukchi Shelf well data, and the older ones, the end of rifting and the top of the pre-rift, are inferred based on stratigraphic observations. The stratigraphic relationship suggests that the Chukchi Borderland stratigraphy can be correlated in part to the Chukchi Shelf stratigraphy. The first and second-order rotated fault blocks and depositional history suggest that the Chukchi Borderland has been coupled to the Chukchi Shelf at least since the extension of the Borderland. Therefore we infer only small horizontal offsets between the Chukchi Borderland and the Chukchi Shelf, which have largely a shared geologic history.

Mechanistic considerations on the wavelength-dependent variations of UVR genotoxicity and mutagenesis in skin: the discrimination of UVA-signature from UV-signature mutation.

PubMed

Ikehata, Hironobu

2018-05-31

Ultraviolet radiation (UVR) predominantly induces UV-signature mutations, C → T and CC → TT base substitutions at dipyrimidine sites, in the cellular and skin genome. I observed in our in vivo mutation studies of mouse skin that these UVR-specific mutations show a wavelength-dependent variation in their sequence-context preference. The C → T mutation occurs most frequently in the 5'-TCG-3' sequence regardless of the UVR wavelength, but is recovered more preferentially there as the wavelength increases, resulting in prominent occurrences exclusively in the TCG sequence in the UVA wavelength range, which I will designate as a "UVA signature" in this review. The preference of the UVB-induced C → T mutation for the sequence contexts shows a mixed pattern of UVC- and UVA-induced mutations, and a similar pattern is also observed for natural sunlight, in which UVB is the most genotoxic component. In addition, the CC → TT mutation hardly occurs at UVA1 wavelengths, although it is detected rarely but constantly in the UVC and UVB ranges. This wavelength-dependent variation in the sequence-context preference of the UVR-specific mutations could be explained by two different photochemical mechanisms of cyclobutane pyrimidine dimer (CPD) formation. The UV-signature mutations observed in the UVC and UVB ranges are known to be caused mainly by CPDs produced through the conventional singlet/triplet excitation of pyrimidine bases after the direct absorption of the UVC/UVB photon energy in those bases. On the other hand, a novel photochemical mechanism through the direct absorption of the UVR energy to double-stranded DNA, which is called "collective excitation", has been proposed for the UVA-induced CPD formation. The UVA photons directly absorbed by DNA produce CPDs with a sequence context preference different from that observed for CPDs caused by the UVC/UVB-mediated singlet/triplet excitation, causing CPD formation preferentially at thymine-containing dipyrimidine sites and probably also preferably at methyl CpG-associated dipyrimidine sites, which include the TCG sequence. In this review, I present a mechanistic consideration on the wavelength-dependent variation of the sequence context preference of the UVR-specific mutations and rationalize the proposition of the UVA-signature mutation, in addition to the UV-signature mutation.

Hybrid spread spectrum radio system

DOEpatents

Smith, Stephen F [London, TN; Dress, William B [Camas, WA

2010-02-09

Systems and methods are described for hybrid spread spectrum radio systems. A method, includes receiving a hybrid spread spectrum signal including: fast frequency hopping demodulating and direct sequence demodulating a direct sequence spread spectrum signal, wherein multiple frequency hops occur within a single data-bit time and each bit is represented by chip transmissions at multiple frequencies.

Design of multi-phase dynamic chemical networks

NASA Astrophysics Data System (ADS)

Chen, Chenrui; Tan, Junjun; Hsieh, Ming-Chien; Pan, Ting; Goodwin, Jay T.; Mehta, Anil K.; Grover, Martha A.; Lynn, David G.

2017-08-01

Template-directed polymerization reactions enable the accurate storage and processing of nature's biopolymer information. This mutualistic relationship of nucleic acids and proteins, a network known as life's central dogma, is now marvellously complex, and the progressive steps necessary for creating the initial sequence and chain-length-specific polymer templates are lost to time. Here we design and construct dynamic polymerization networks that exploit metastable prion cross-β phases. Mixed-phase environments have been used for constructing synthetic polymers, but these dynamic phases emerge naturally from the growing peptide oligomers and create environments suitable both to nucleate assembly and select for ordered templates. The resulting templates direct the amplification of a phase containing only chain-length-specific peptide-like oligomers. Such multi-phase biopolymer dynamics reveal pathways for the emergence, self-selection and amplification of chain-length- and possibly sequence-specific biopolymers.

Hadoop-BAM: directly manipulating next generation sequencing data in the cloud.

PubMed

Niemenmaa, Matti; Kallio, Aleksi; Schumacher, André; Klemelä, Petri; Korpelainen, Eija; Heljanko, Keijo

2012-03-15

Hadoop-BAM is a novel library for the scalable manipulation of aligned next-generation sequencing data in the Hadoop distributed computing framework. It acts as an integration layer between analysis applications and BAM files that are processed using Hadoop. Hadoop-BAM solves the issues related to BAM data access by presenting a convenient API for implementing map and reduce functions that can directly operate on BAM records. It builds on top of the Picard SAM JDK, so tools that rely on the Picard API are expected to be easily convertible to support large-scale distributed processing. In this article we demonstrate the use of Hadoop-BAM by building a coverage summarizing tool for the Chipster genome browser. Our results show that Hadoop offers good scalability, and one should avoid moving data in and out of Hadoop between analysis steps.

A soluble acid invertase is directed to the vacuole by a signal anchor mechanism.

PubMed

Rae, Anne L; Casu, Rosanne E; Perroux, Jai M; Jackson, Mark A; Grof, Christopher P L

2011-06-15

Enzyme activities in the vacuole have an important impact on the net concentration of sucrose. In sugarcane (Saccharum hybrid), immunolabelling demonstrated that a soluble acid invertase (β-fructofuranosidase; EC 3.2.1.26) is present in the vacuole of storage parenchyma cells during sucrose accumulation. Examination of sequences from sugarcane, barley and rice showed that the N-terminus of the invertase sequence contains a signal anchor and a tyrosine motif, characteristic of single-pass membrane proteins destined for lysosomal compartments. The N-terminal peptide from the barley invertase was shown to be capable of directing the green fluorescent protein to the vacuole in sugarcane cells. The results suggest that soluble acid invertase is sorted to the vacuole in a membrane-bound form. Copyright © 2010 Elsevier GmbH. All rights reserved.

Extrinsic finger and thumb muscles command a virtual hand to allow individual finger and grasp control.

PubMed

Birdwell, J Alexander; Hargrove, Levi J; Weir, Richard F ff; Kuiken, Todd A

2015-01-01

Fine-wire intramuscular electrodes were used to obtain electromyogram (EMG) signals from six extrinsic hand muscles associated with the thumb, index, and middle fingers. Subjects' EMG activity was used to control a virtual three-degree-of-freedom (DOF) hand as they conformed the hand to a sequence of hand postures testing two controllers: direct EMG control and pattern recognition control. Subjects tested two conditions using each controller: starting the hand from a predefined neutral posture before each new posture and starting the hand from the previous posture in the sequence. Subjects demonstrated their abilities to simultaneously, yet individually, move all three DOFs during the direct EMG control trials; however, results showed subjects did not often utilize this feature. Performance metrics such as failure rate and completion time showed no significant difference between the two controllers.

Transpressional Structure in Chiayi Area, Taiwan: Insight from the 2017 ML5.1 Zhongpu Earthquake Sequence

NASA Astrophysics Data System (ADS)

Feng, K. F.; Huang, H. H.

2017-12-01

The Chiayi area is located at the deformation front of active fold-and-thrust belt of Taiwan, where the fault system is composed primarily of a series of north-south-trending east-dipping thrusts and also an east-west-trending strike-slip fault (Meishan Fault, MSF) with right-lateral faulting. On 24th May 2017, a ML 5.1 earthquake occurred at Zhongpu, Chiayi (namely Zhongpu earthquake), however, shows a left-lateral strike-slip faulting distinct from the known structure in the area. The distribution of the reported aftershocks is difficult to distinguish the actual fault plane. To determine the fault plane of this abnormal earthquake and investigate its structural relationships to the regional tectonics, we relocate the earthquake sequence and estimate the rupture directivity of the mainshock by using the 3-D double difference hypocenter relocation method (Lin, 2013) and the 3-D directivity moment tensor inversion method (DMT, Huang et al., 2017, submitted). The DMT results show that the rupture directivity of the Zhongpu earthquake is west- and down-ward along the east-west fault plane, which also agrees with east-west-distributed aftershocks after relocation. As a result, the Zhongpu earthquake reveals an undiscovered east-west-trending structure which is sub-parallel with the MSF but with opposite faulting direction, exhibiting a complex transpressional tectonic regime in the Chiayi area.

Sequence-structure mapping errors in the PDB: OB-fold domains

PubMed Central

Venclovas, Česlovas; Ginalski, Krzysztof; Kang, Chulhee

2004-01-01

The Protein Data Bank (PDB) is the single most important repository of structural data for proteins and other biologically relevant molecules. Therefore, it is critically important to keep the PDB data, as much as possible, error-free. In this study, we have analyzed PDB crystal structures possessing oligonucleotide/oligosaccharide binding (OB)-fold, one of the highly populated folds, for the presence of sequence-structure mapping errors. Using energy-based structure quality assessment coupled with sequence analyses, we have found that there are at least five OB-structures in the PDB that have regions where sequences have been incorrectly mapped onto the structure. We have demonstrated that the combination of these computation techniques is effective not only in detecting sequence-structure mapping errors, but also in providing guidance to correct them. Namely, we have used results of computational analysis to direct a revision of X-ray data for one of the PDB entries containing a fairly inconspicuous sequence-structure mapping error. The revised structure has been deposited with the PDB. We suggest use of computational energy assessment and sequence analysis techniques to facilitate structure determination when homologs having known structure are available to use as a reference. Such computational analysis may be useful in either guiding the sequence-structure assignment process or verifying the sequence mapping within poorly defined regions. PMID:15133161

Directionality analysis on functional magnetic resonance imaging during motor task using Granger causality.

PubMed

Anwar, A R; Muthalib, M; Perrey, S; Galka, A; Granert, O; Wolff, S; Deuschl, G; Raethjen, J; Heute, U; Muthuraman, M

2012-01-01

Directionality analysis of signals originating from different parts of brain during motor tasks has gained a lot of interest. Since brain activity can be recorded over time, methods of time series analysis can be applied to medical time series as well. Granger Causality is a method to find a causal relationship between time series. Such causality can be referred to as a directional connection and is not necessarily bidirectional. The aim of this study is to differentiate between different motor tasks on the basis of activation maps and also to understand the nature of connections present between different parts of the brain. In this paper, three different motor tasks (finger tapping, simple finger sequencing, and complex finger sequencing) are analyzed. Time series for each task were extracted from functional magnetic resonance imaging (fMRI) data, which have a very good spatial resolution and can look into the sub-cortical regions of the brain. Activation maps based on fMRI images show that, in case of complex finger sequencing, most parts of the brain are active, unlike finger tapping during which only limited regions show activity. Directionality analysis on time series extracted from contralateral motor cortex (CMC), supplementary motor area (SMA), and cerebellum (CER) show bidirectional connections between these parts of the brain. In case of simple finger sequencing and complex finger sequencing, the strongest connections originate from SMA and CMC, while connections originating from CER in either direction are the weakest ones in magnitude during all paradigms.

Mutation Analysis in Classical Phenylketonuria Patients Followed by Detecting Haplotypes Linked to Some PAH Mutations.

PubMed

Dehghanian, Fatemeh; Silawi, Mohammad; Tabei, Seyed M B

2017-02-01

Deficiency of phenylalanine hydroxylase (PAH) enzyme and elevation of phenylalanine in body fluids cause phenylketonuria (PKU). The gold standard for confirming PKU and PAH deficiency is detecting causal mutations by direct sequencing of the coding exons and splicing involved sequences of the PAH gene. Furthermore, haplotype analysis could be considered as an auxiliary approach for detecting PKU causative mutations before direct sequencing of the PAH gene by making comparisons between prior detected mutation linked-haplotypes and new PKU case haplotypes with undetermined mutations. In this study, 13 unrelated classical PKU patients took part in the study detecting causative mutations. Mutations were identified by polymerase chain reaction (PCR) and direct sequencing in all patients. After that, haplotype analysis was performed by studying VNTR and PAHSTR markers (linked genetic markers of the PAH gene) through application of PCR and capillary electrophoresis (CE). Mutation analysis was performed successfully and the detected mutations were as follows: c.782G>A, c.754C>T, c.842C>G, c.113-115delTCT, c.688G>A, and c.696A>G. Additionally, PAHSTR/VNTR haplotypes were detected to discover haplotypes linked to each mutation. Mutation detection is the best approach for confirming PAH enzyme deficiency in PKU patients. Due to the relatively large size of the PAH gene and high cost of the direct sequencing in developing countries, haplotype analysis could be used before DNA sequencing and mutation detection for a faster and cheaper way via identifying probable mutated exons.

Evaluation of next generation mtGenome sequencing using the Ion Torrent Personal Genome Machine (PGM)☆

PubMed Central

Parson, Walther; Strobl, Christina; Huber, Gabriela; Zimmermann, Bettina; Gomes, Sibylle M.; Souto, Luis; Fendt, Liane; Delport, Rhena; Langit, Reina; Wootton, Sharon; Lagacé, Robert; Irwin, Jodi

2013-01-01

Insights into the human mitochondrial phylogeny have been primarily achieved by sequencing full mitochondrial genomes (mtGenomes). In forensic genetics (partial) mtGenome information can be used to assign haplotypes to their phylogenetic backgrounds, which may, in turn, have characteristic geographic distributions that would offer useful information in a forensic case. In addition and perhaps even more relevant in the forensic context, haplogroup-specific patterns of mutations form the basis for quality control of mtDNA sequences. The current method for establishing (partial) mtDNA haplotypes is Sanger-type sequencing (STS), which is laborious, time-consuming, and expensive. With the emergence of Next Generation Sequencing (NGS) technologies, the body of available mtDNA data can potentially be extended much more quickly and cost-efficiently. Customized chemistries, laboratory workflows and data analysis packages could support the community and increase the utility of mtDNA analysis in forensics. We have evaluated the performance of mtGenome sequencing using the Personal Genome Machine (PGM) and compared the resulting haplotypes directly with conventional Sanger-type sequencing. A total of 64 mtGenomes (>1 million bases) were established that yielded high concordance with the corresponding STS haplotypes (<0.02% differences). About two-thirds of the differences were observed in or around homopolymeric sequence stretches. In addition, the sequence alignment algorithm employed to align NGS reads played a significant role in the analysis of the data and the resulting mtDNA haplotypes. Further development of alignment software would be desirable to facilitate the application of NGS in mtDNA forensic genetics. PMID:23948325

Sequence periodicity in nucleosomal DNA and intrinsic curvature.

PubMed

Nair, T Murlidharan

2010-05-17

Most eukaryotic DNA contained in the nucleus is packaged by wrapping DNA around histone octamers. Histones are ubiquitous and bind most regions of chromosomal DNA. In order to achieve smooth wrapping of the DNA around the histone octamer, the DNA duplex should be able to deform and should possess intrinsic curvature. The deformability of DNA is a result of the non-parallelness of base pair stacks. The stacking interaction between base pairs is sequence dependent. The higher the stacking energy the more rigid the DNA helix, thus it is natural to expect that sequences that are involved in wrapping around the histone octamer should be unstacked and possess intrinsic curvature. Intrinsic curvature has been shown to be dictated by the periodic recurrence of certain dinucleotides. Several genome-wide studies directed towards mapping of nucleosome positions have revealed periodicity associated with certain stretches of sequences. In the current study, these sequences have been analyzed with a view to understand their sequence-dependent structures. Higher order DNA structures and the distribution of molecular bend loci associated with 146 base nucleosome core DNA sequence from C. elegans and chicken have been analyzed using the theoretical model for DNA curvature. The curvature dispersion calculated by cyclically permuting the sequences revealed that the molecular bend loci were delocalized throughout the nucleosome core region and had varying degrees of intrinsic curvature. The higher order structures associated with nucleosomes of C.elegans and chicken calculated from the sequences revealed heterogeneity with respect to the deviation of the DNA axis. The results points to the possibility of context dependent curvature of varying degrees to be associated with nucleosomal DNA.

Digit-color synaesthesia only enhances memory for colors in a specific context: A new method of duration thresholds to measure serial recall.

PubMed

Teichmann, A Lina; Nieuwenstein, Mark R; Rich, Anina N

2017-08-01

For digit-color synaesthetes, digits elicit vivid experiences of color that are highly consistent for each individual. The conscious experience of synaesthesia is typically unidirectional: Digits evoke colors but not vice versa. There is an ongoing debate about whether synaesthetes have a memory advantage over non-synaesthetes. One key question in this debate is whether synaesthetes have a general superiority or whether any benefit is specific to a certain type of material. Here, we focus on immediate serial recall and ask digit-color synaesthetes and controls to memorize digit and color sequences. We developed a sensitive staircase method manipulating presentation duration to measure participants' serial recall of both overlearned and novel sequences. Our results show that synaesthetes can activate digit information to enhance serial memory for color sequences. When color sequences corresponded to ascending or descending digit sequences, synaesthetes encoded these sequences at a faster rate than their non-synaesthetes counterparts and faster than non-structured color sequences. However, encoding color sequences is approximately 200 ms slower than encoding digit sequences directly, independent of group and condition, which shows that the translation process is time consuming. These results suggest memory advantages in synaesthesia require a modified dual-coding account, in which secondary (synaesthetically linked) information is useful only if it is more memorable than the primary information to be recalled. Our study further shows that duration thresholds are a sensitive method to measure subtle differences in serial recall performance. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

Transcription Factor Information System (TFIS): A Tool for Detection of Transcription Factor Binding Sites.

PubMed

Narad, Priyanka; Kumar, Abhishek; Chakraborty, Amlan; Patni, Pranav; Sengupta, Abhishek; Wadhwa, Gulshan; Upadhyaya, K C

2017-09-01

Transcription factors are trans-acting proteins that interact with specific nucleotide sequences known as transcription factor binding site (TFBS), and these interactions are implicated in regulation of the gene expression. Regulation of transcriptional activation of a gene often involves multiple interactions of transcription factors with various sequence elements. Identification of these sequence elements is the first step in understanding the underlying molecular mechanism(s) that regulate the gene expression. For in silico identification of these sequence elements, we have developed an online computational tool named transcription factor information system (TFIS) for detecting TFBS for the first time using a collection of JAVA programs and is mainly based on TFBS detection using position weight matrix (PWM). The database used for obtaining position frequency matrices (PFM) is JASPAR and HOCOMOCO, which is an open-access database of transcription factor binding profiles. Pseudo-counts are used while converting PFM to PWM, and TFBS detection is carried out on the basis of percent score taken as threshold value. TFIS is equipped with advanced features such as direct sequence retrieving from NCBI database using gene identification number and accession number, detecting binding site for common TF in a batch of gene sequences, and TFBS detection after generating PWM from known raw binding sequences in addition to general detection methods. TFIS can detect the presence of potential TFBSs in both the directions at the same time. This feature increases its efficiency. And the results for this dual detection are presented in different colors specific to the orientation of the binding site. Results obtained by the TFIS are more detailed and specific to the detected TFs as integration of more informative links from various related web servers are added in the result pages like Gene Ontology, PAZAR database and Transcription Factor Encyclopedia in addition to NCBI and UniProt. Common TFs like SP1, AP1 and NF-KB of the Amyloid beta precursor gene is easily detected using TFIS along with multiple binding sites. In another scenario of embryonic developmental process, TFs of the FOX family (FOXL1 and FOXC1) were also identified. TFIS is platform-independent which is publicly available along with its support and documentation at http://tfistool.appspot.com and http://www.bioinfoplus.com/tfis/ . TFIS is licensed under the GNU General Public License, version 3 (GPL-3.0).

Detection of a new bat gammaherpesvirus in the Philippines.

PubMed

Watanabe, Shumpei; Ueda, Naoya; Iha, Koichiro; Masangkay, Joseph S; Fujii, Hikaru; Alviola, Phillip; Mizutani, Tetsuya; Maeda, Ken; Yamane, Daisuke; Walid, Azab; Kato, Kentaro; Kyuwa, Shigeru; Tohya, Yukinobu; Yoshikawa, Yasuhiro; Akashi, Hiroomi

2009-08-01

A new bat herpesvirus was detected in the spleen of an insectivorous bat (Hipposideros diadema, family Hipposideridae) collected on Panay Island, the Philippines. PCR analyses were performed using COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOPs) targeting the herpesvirus DNA polymerase (DPOL) gene. Although we obtained PCR products with CODEHOPs, direct sequencing using the primers was not possible because of high degree of degeneracy. Direct sequencing technology developed in our rapid determination system of viral RNA sequences (RDV) was applied in this study, and a partial DPOL nucleotide sequence was determined. In addition, a partial gB gene nucleotide sequence was also determined using the same strategy. We connected the partial gB and DPOL sequences with long-distance PCR, and a 3741-bp nucleotide fragment, including the 3' part of the gB gene and the 5' part of the DPOL gene, was finally determined. Phylogenetic analysis showed that the sequence was novel and most similar to those of the subfamily Gammaherpesvirinae.

Effects of sequence on DNA wrapping around histones

NASA Astrophysics Data System (ADS)

Ortiz, Vanessa

2011-03-01

A central question in biophysics is whether the sequence of a DNA strand affects its mechanical properties. In epigenetics, these are thought to influence nucleosome positioning and gene expression. Theoretical and experimental attempts to answer this question have been hindered by an inability to directly resolve DNA structure and dynamics at the base-pair level. In our previous studies we used a detailed model of DNA to measure the effects of sequence on the stability of naked DNA under bending. Sequence was shown to influence DNA's ability to form kinks, which arise when certain motifs slide past others to form non-native contacts. Here, we have now included histone-DNA interactions to see if the results obtained for naked DNA are transferable to the problem of nucleosome positioning. Different DNA sequences interacting with the histone protein complex are studied, and their equilibrium and mechanical properties are compared among themselves and with the naked case. NLM training grant to the Computation and Informatics in Biology and Medicine Training Program (NLM T15LM007359).

On the Detection and Characterization of Polluted White Dwarfs

NASA Astrophysics Data System (ADS)

Steele, Amy; Debes, John H.; Deming, Drake

2017-06-01

There is evidence of circumstellar material around main sequence, giant, and white dwarf stars. What happens to this material after the main sequence? With this work, we focus on the characterization of the material around WD 1145+017. The goals are to monitor the white dwarf—which has a transiting, disintegrating planetesimal and determine the composition of the evaporated material for that same white dwarf by looking at high-resolution spectra. We also present preliminary results of follow-up photometric observations of known polluted WDs. If rocky bodies survive red giant branch evolution, then the material raining down on a WD atmosphere is a direct probe of main sequence cosmochemistry. If rocky bodies do not survive the evolution, then this informs the degree of post-main-sequence processing. These case studies will provide the community with further insight about debris disk modeling, the degree of post-main-sequence processing of circumstellar material, and the composition of a disintegrating planetesimal.

Engineering of a DNA Polymerase for Direct m6 A Sequencing.

PubMed

Aschenbrenner, Joos; Werner, Stephan; Marchand, Virginie; Adam, Martina; Motorin, Yuri; Helm, Mark; Marx, Andreas

2018-01-08

Methods for the detection of RNA modifications are of fundamental importance for advancing epitranscriptomics. N 6 -methyladenosine (m 6 A) is the most abundant RNA modification in mammalian mRNA and is involved in the regulation of gene expression. Current detection techniques are laborious and rely on antibody-based enrichment of m 6 A-containing RNA prior to sequencing, since m 6 A modifications are generally "erased" during reverse transcription (RT). To overcome the drawbacks associated with indirect detection, we aimed to generate novel DNA polymerase variants for direct m 6 A sequencing. Therefore, we developed a screen to evolve an RT-active KlenTaq DNA polymerase variant that sets a mark for N 6 -methylation. We identified a mutant that exhibits increased misincorporation opposite m 6 A compared to unmodified A. Application of the generated DNA polymerase in next-generation sequencing allowed the identification of m 6 A sites directly from the sequencing data of untreated RNA samples. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Pyrosequencing analysis for detection of a BRAFV600E mutation in an FNAB specimen of thyroid nodules.

PubMed

Kim, Suk Kyeong; Kim, Dong-Lim; Han, Hye Seung; Kim, Wan Seop; Kim, Seung Ja; Moon, Won Jin; Oh, Seo Young; Hwang, Tae Sook

2008-06-01

Fine-needle aspiration biopsy (FNAB) is the primary means of distinguishing benign from malignant and of guiding therapeutic intervention in thyroid nodules. However, 10% to 30% of cases with indeterminate cytology in FNAB need other diagnostic tools to refine diagnosis. We compared the pyrosequencing method with the conventional direct DNA sequencing analysis and investigated the usefulness of preoperative BRAF mutation analysis as an adjunct diagnostic tool with routine FNAB. A total of 103 surgically confirmed patients' FNA slides were recruited and DNA was extracted after atypical cells were scraped from the slides. BRAF mutation was analyzed by pyrosequencing and direct DNA sequencing. Sixty-three (77.8%) of 81 histopathologically diagnosed malignant nodules revealed positive BRAF mutation on pyrosequencing analysis. In detail, 63 (84.0%) of 75 papillary thyroid carcinoma (PTC) samples showed positive BRAF mutation, whereas 3 follicular thyroid carcinomas, 1 anaplastic carcinoma, 1 medullary thyroid carcinoma, and 1 metastatic lung carcinoma did not show BRAF mutation. None of 22 benign nodules had BRAF mutation in both pyrosequencing and direct DNA sequencing. Out of 27 thyroid nodules classified as 'indeterminate' on cytologic examination preoperatively, 21 (77.8%) cases turned out to be malignant: 18 PTCs (including 2 follicular variant types) and 3 follicular thyroid carcinomas. Among these, 13 (61.9%) classic PTCs had BRAF mutation. None of 6 benign nodules, including 3 follicular adenomas and 3 nodular hyperplasias, had BRAF mutation. Among 63 PTCs with positive BRAF mutation detected by pyrosequencing analysis, 3 cases did not show BRAF mutation by direct DNA sequencing. Although it was not statistically significant, pyrosequencing was superior to direct DNA sequencing in detecting the BRAF mutation of thyroid nodules (P=0.25). Detecting BRAF mutation by pyrosequencing is more sensitive, faster, and less expensive than direct DNA sequencing and is proposed as an adjunct diagnostic tool in evaluating thyroid nodules of indeterminate cytology.

Draft genome sequence and annotation of Lactobacillus acetotolerans BM-LA14527, a beer-spoilage bacteria.

PubMed

Liu, Junyan; Li, Lin; Peters, Brian M; Li, Bing; Deng, Yang; Xu, Zhenbo; Shirtliff, Mark E

2016-09-01

Lactobacillus acetotolerans is a hard-to-culture beer-spoilage bacterium capable of entering into the viable putative nonculturable (VPNC) state. As part of an initial strategy to investigate the phenotypic behavior of L. acetotolerans, draft genome sequencing was performed. Results demonstrated a total of 1824 predicted annotated genes, with several potential VPNC- and beer-spoilage-associated genes identified. Importantly, this is the first genome sequence of L. acetotolerans as beer-spoilage bacteria and it may aid in further analysis of L. acetotolerans and other beer-spoilage bacteria, with direct implications for food safety control in the beer brewing industry. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Predicting DNA binding proteins using support vector machine with hybrid fractal features.

PubMed

Niu, Xiao-Hui; Hu, Xue-Hai; Shi, Feng; Xia, Jing-Bo

2014-02-21

DNA-binding proteins play a vitally important role in many biological processes. Prediction of DNA-binding proteins from amino acid sequence is a significant but not fairly resolved scientific problem. Chaos game representation (CGR) investigates the patterns hidden in protein sequences, and visually reveals previously unknown structure. Fractal dimensions (FD) are good tools to measure sizes of complex, highly irregular geometric objects. In order to extract the intrinsic correlation with DNA-binding property from protein sequences, CGR algorithm, fractal dimension and amino acid composition are applied to formulate the numerical features of protein samples in this paper. Seven groups of features are extracted, which can be computed directly from the primary sequence, and each group is evaluated by the 10-fold cross-validation test and Jackknife test. Comparing the results of numerical experiments, the group of amino acid composition and fractal dimension (21-dimension vector) gets the best result, the average accuracy is 81.82% and average Matthew's correlation coefficient (MCC) is 0.6017. This resulting predictor is also compared with existing method DNA-Prot and shows better performances. © 2013 The Authors. Published by Elsevier Ltd All rights reserved.

Wind data mining by Kohonen Neural Networks.

PubMed

Fayos, José; Fayos, Carolina

2007-02-14

Time series of Circulation Weather Type (CWT), including daily averaged wind direction and vorticity, are self-classified by similarity using Kohonen Neural Networks (KNN). It is shown that KNN is able to map by similarity all 7300 five-day CWT sequences during the period of 1975-94, in London, United Kingdom. It gives, as a first result, the most probable wind sequences preceding each one of the 27 CWT Lamb classes in that period. Inversely, as a second result, the observed diffuse correlation between both five-day CWT sequences and the CWT of the 6(th) day, in the long 20-year period, can be generalized to predict the last from the previous CWT sequence in a different test period, like 1995, as both time series are similar. Although the average prediction error is comparable to that obtained by forecasting standard methods, the KNN approach gives complementary results, as they depend only on an objective classification of observed CWT data, without any model assumption. The 27 CWT of the Lamb Catalogue were coded with binary three-dimensional vectors, pointing to faces, edges and vertex of a "wind-cube," so that similar CWT vectors were close.

Exome sequencing is an efficient tool for variant late-infantile neuronal ceroid lipofuscinosis molecular diagnosis.

PubMed

Patiño, Liliana Catherine; Battu, Rajani; Ortega-Recalde, Oscar; Nallathambi, Jeyabalan; Anandula, Venkata Ramana; Renukaradhya, Umashankar; Laissue, Paul

2014-01-01

The neuronal ceroid-lipofuscinoses (NCL) is a group of neurodegenerative disorders characterized by epilepsy, visual failure, progressive mental and motor deterioration, myoclonus, dementia and reduced life expectancy. Classically, NCL-affected individuals have been classified into six categories, which have been mainly defined regarding the clinical onset of symptoms. However, some patients cannot be easily included in a specific group because of significant variation in the age of onset and disease progression. Molecular genetics has emerged in recent years as a useful tool for enhancing NCL subtype classification. Fourteen NCL genetic forms (CLN1 to CLN14) have been described to date. The variant late-infantile form of the disease has been linked to CLN5, CLN6, CLN7 (MFSD8) and CLN8 mutations. Despite advances in the diagnosis of neurodegenerative disorders mutations in these genes may cause similar phenotypes, which rends difficult accurate candidate gene selection for direct sequencing. Three siblings who were affected by variant late-infantile NCL are reported in the present study. We used whole-exome sequencing, direct sequencing and in silico approaches to identify the molecular basis of the disease. We identified the novel c.1219T>C (p.Trp407Arg) and c.1361T>C (p.Met454Thr) MFSD8 pathogenic mutations. Our results highlighted next generation sequencing as a novel and powerful methodological approach for the rapid determination of the molecular diagnosis of NCL. They also provide information regarding the phenotypic and molecular spectrum of CLN7 disease.

A model of directional selection applied to the evolution of drug resistance in HIV-1.

PubMed

Seoighe, Cathal; Ketwaroo, Farahnaz; Pillay, Visva; Scheffler, Konrad; Wood, Natasha; Duffet, Rodger; Zvelebil, Marketa; Martinson, Neil; McIntyre, James; Morris, Lynn; Hide, Winston

2007-04-01

Understanding how pathogens acquire resistance to drugs is important for the design of treatment strategies, particularly for rapidly evolving viruses such as HIV-1. Drug treatment can exert strong selective pressures and sites within targeted genes that confer resistance frequently evolve far more rapidly than the neutral rate. Rapid evolution at sites that confer resistance to drugs can be used to help elucidate the mechanisms of evolution of drug resistance and to discover or corroborate novel resistance mutations. We have implemented standard maximum likelihood methods that are used to detect diversifying selection and adapted them for use with serially sampled reverse transcriptase (RT) coding sequences isolated from a group of 300 HIV-1 subtype C-infected women before and after single-dose nevirapine (sdNVP) to prevent mother-to-child transmission. We have also extended the standard models of codon evolution for application to the detection of directional selection. Through simulation, we show that the directional selection model can provide a substantial improvement in sensitivity over models of diversifying selection. Five of the sites within the RT gene that are known to harbor mutations that confer resistance to nevirapine (NVP) strongly supported the directional selection model. There was no evidence that other mutations that are known to confer NVP resistance were selected in this cohort. The directional selection model, applied to serially sampled sequences, also had more power than the diversifying selection model to detect selection resulting from factors other than drug resistance. Because inference of selection from serial samples is unlikely to be adversely affected by recombination, the methods we describe may have general applicability to the analysis of positive selection affecting recombining coding sequences when serially sampled data are available.

Improving performance of DS-CDMA systems using chaotic complex Bernoulli spreading codes

NASA Astrophysics Data System (ADS)

Farzan Sabahi, Mohammad; Dehghanfard, Ali

2014-12-01

The most important goal of spreading spectrum communication system is to protect communication signals against interference and exploitation of information by unintended listeners. In fact, low probability of detection and low probability of intercept are two important parameters to increase the performance of the system. In Direct Sequence Code Division Multiple Access (DS-CDMA) systems, these properties are achieved by multiplying the data information in spreading sequences. Chaotic sequences, with their particular properties, have numerous applications in constructing spreading codes. Using one-dimensional Bernoulli chaotic sequence as spreading code is proposed in literature previously. The main feature of this sequence is its negative auto-correlation at lag of 1, which with proper design, leads to increase in efficiency of the communication system based on these codes. On the other hand, employing the complex chaotic sequences as spreading sequence also has been discussed in several papers. In this paper, use of two-dimensional Bernoulli chaotic sequences is proposed as spreading codes. The performance of a multi-user synchronous and asynchronous DS-CDMA system will be evaluated by applying these sequences under Additive White Gaussian Noise (AWGN) and fading channel. Simulation results indicate improvement of the performance in comparison with conventional spreading codes like Gold codes as well as similar complex chaotic spreading sequences. Similar to one-dimensional Bernoulli chaotic sequences, the proposed sequences also have negative auto-correlation. Besides, construction of complex sequences with lower average cross-correlation is possible with the proposed method.

PepLine: a software pipeline for high-throughput direct mapping of tandem mass spectrometry data on genomic sequences.

PubMed

Ferro, Myriam; Tardif, Marianne; Reguer, Erwan; Cahuzac, Romain; Bruley, Christophe; Vermat, Thierry; Nugues, Estelle; Vigouroux, Marielle; Vandenbrouck, Yves; Garin, Jérôme; Viari, Alain

2008-05-01

PepLine is a fully automated software which maps MS/MS fragmentation spectra of trypsic peptides to genomic DNA sequences. The approach is based on Peptide Sequence Tags (PSTs) obtained from partial interpretation of QTOF MS/MS spectra (first module). PSTs are then mapped on the six-frame translations of genomic sequences (second module) giving hits. Hits are then clustered to detect potential coding regions (third module). Our work aimed at optimizing the algorithms of each component to allow the whole pipeline to proceed in a fully automated manner using raw nucleic acid sequences (i.e., genomes that have not been "reduced" to a database of ORFs or putative exons sequences). The whole pipeline was tested on controlled MS/MS spectra sets from standard proteins and from Arabidopsis thaliana envelope chloroplast samples. Our results demonstrate that PepLine competed with protein database searching softwares and was fast enough to potentially tackle large data sets and/or high size genomes. We also illustrate the potential of this approach for the detection of the intron/exon structure of genes.

Highly conserved intragenic HSV-2 sequences: Results from next-generation sequencing of HSV-2 UL and US regions from genital swabs collected from 3 continents.

PubMed

Johnston, Christine; Magaret, Amalia; Roychoudhury, Pavitra; Greninger, Alexander L; Cheng, Anqi; Diem, Kurt; Fitzgibbon, Matthew P; Huang, Meei-Li; Selke, Stacy; Lingappa, Jairam R; Celum, Connie; Jerome, Keith R; Wald, Anna; Koelle, David M

2017-10-01

Understanding the variability in circulating herpes simplex virus type 2 (HSV-2) genomic sequences is critical to the development of HSV-2 vaccines. Genital lesion swabs containing ≥ 10 7 log 10 copies HSV DNA collected from Africa, the USA, and South America underwent next-generation sequencing, followed by K-mer based filtering and de novo genomic assembly. Sites of heterogeneity within coding regions in unique long and unique short (U L _U S ) regions were identified. Phylogenetic trees were created using maximum likelihood reconstruction. Among 46 samples from 38 persons, 1468 intragenic base-pair substitutions were identified. The maximum nucleotide distance between strains for concatenated U L_ U S segments was 0.4%. Phylogeny did not reveal geographic clustering. The most variable proteins had non-synonymous mutations in < 3% of amino acids. Unenriched HSV-2 DNA can undergo next-generation sequencing to identify intragenic variability. The use of clinical swabs for sequencing expands the information that can be gathered directly from these specimens. Copyright © 2017 Elsevier Inc. All rights reserved.

Association of Amine-Receptor DNA Sequence Variants with Associative Learning in the Honeybee.

PubMed

Lagisz, Malgorzata; Mercer, Alison R; de Mouzon, Charlotte; Santos, Luana L S; Nakagawa, Shinichi

2016-03-01

Octopamine- and dopamine-based neuromodulatory systems play a critical role in learning and learning-related behaviour in insects. To further our understanding of these systems and resulting phenotypes, we quantified DNA sequence variations at six loci coding octopamine-and dopamine-receptors and their association with aversive and appetitive learning traits in a population of honeybees. We identified 79 polymorphic sequence markers (mostly SNPs and a few insertions/deletions) located within or close to six candidate genes. Intriguingly, we found that levels of sequence variation in the protein-coding regions studied were low, indicating that sequence variation in the coding regions of receptor genes critical to learning and memory is strongly selected against. Non-coding and upstream regions of the same genes, however, were less conserved and sequence variations in these regions were weakly associated with between-individual differences in learning-related traits. While these associations do not directly imply a specific molecular mechanism, they suggest that the cross-talk between dopamine and octopamine signalling pathways may influence olfactory learning and memory in the honeybee.

Dynamically hot galaxies. I - Structural properties

NASA Technical Reports Server (NTRS)

Bender, Ralf; Burstein, David; Faber, S. M.

1992-01-01

Results are reported from an analysis of the structural properties of dynamically hot galaxies which combines central velocity dispersion, effective surface brightness, and effective radius into a new 3-space (k), in which the axes are parameters that are physically meaningful. Hot galaxies are found to divide into groups in k-space that closely parallel conventional morphological classifications, namely, luminous ellipticals, compacts, bulges, bright dwarfs, and dwarf spheroidals. A major sequence is defined by luminous ellipticals, bulges, and most compacts, which together constitute a smooth continuum in k-space. Several properties vary smoothly with mass along this continuum, including bulge-to-disk ratio, radio properties, rotation, degree of velocity anisotropy, and 'unrelaxed'. A second major sequence is comprised of dwarf ellipticals and dwarf spheroidals. It is suggested that mass loss is a major factor in hot dwarf galaxies, but the dwarf sequence cannot be simply a mass-loss sequence, as it has the wrong direction in k-space.

Denoising time-resolved microscopy image sequences with singular value thresholding.

PubMed

Furnival, Tom; Leary, Rowan K; Midgley, Paul A

2017-07-01

Time-resolved imaging in microscopy is important for the direct observation of a range of dynamic processes in both the physical and life sciences. However, the image sequences are often corrupted by noise, either as a result of high frame rates or a need to limit the radiation dose received by the sample. Here we exploit both spatial and temporal correlations using low-rank matrix recovery methods to denoise microscopy image sequences. We also make use of an unbiased risk estimator to address the issue of how much thresholding to apply in a robust and automated manner. The performance of the technique is demonstrated using simulated image sequences, as well as experimental scanning transmission electron microscopy data, where surface adatom motion and nanoparticle structural dynamics are recovered at rates of up to 32 frames per second. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Quantification of fetal heart rate regularity using symbolic dynamics

NASA Astrophysics Data System (ADS)

van Leeuwen, P.; Cysarz, D.; Lange, S.; Geue, D.; Groenemeyer, D.

2007-03-01

Fetal heart rate complexity was examined on the basis of RR interval time series obtained in the second and third trimester of pregnancy. In each fetal RR interval time series, short term beat-to-beat heart rate changes were coded in 8bit binary sequences. Redundancies of the 28 different binary patterns were reduced by two different procedures. The complexity of these sequences was quantified using the approximate entropy (ApEn), resulting in discrete ApEn values which were used for classifying the sequences into 17 pattern sets. Also, the sequences were grouped into 20 pattern classes with respect to identity after rotation or inversion of the binary value. There was a specific, nonuniform distribution of the sequences in the pattern sets and this differed from the distribution found in surrogate data. In the course of gestation, the number of sequences increased in seven pattern sets, decreased in four and remained unchanged in six. Sequences that occurred less often over time, both regular and irregular, were characterized by patterns reflecting frequent beat-to-beat reversals in heart rate. They were also predominant in the surrogate data, suggesting that these patterns are associated with stochastic heart beat trains. Sequences that occurred more frequently over time were relatively rare in the surrogate data. Some of these sequences had a high degree of regularity and corresponded to prolonged heart rate accelerations or decelerations which may be associated with directed fetal activity or movement or baroreflex activity. Application of the pattern classes revealed that those sequences with a high degree of irregularity correspond to heart rate patterns resulting from complex physiological activity such as fetal breathing movements. The results suggest that the development of the autonomic nervous system and the emergence of fetal behavioral states lead to increases in not only irregular but also regular heart rate patterns. Using symbolic dynamics to examine the cardiovascular system may thus lead to new insight with respect to fetal development.

enoLOGOS: a versatile web tool for energy normalized sequence logos

PubMed Central

Workman, Christopher T.; Yin, Yutong; Corcoran, David L.; Ideker, Trey; Stormo, Gary D.; Benos, Panayiotis V.

2005-01-01

enoLOGOS is a web-based tool that generates sequence logos from various input sources. Sequence logos have become a popular way to graphically represent DNA and amino acid sequence patterns from a set of aligned sequences. Each position of the alignment is represented by a column of stacked symbols with its total height reflecting the information content in this position. Currently, the available web servers are able to create logo images from a set of aligned sequences, but none of them generates weighted sequence logos directly from energy measurements or other sources. With the advent of high-throughput technologies for estimating the contact energy of different DNA sequences, tools that can create logos directly from binding affinity data are useful to researchers. enoLOGOS generates sequence logos from a variety of input data, including energy measurements, probability matrices, alignment matrices, count matrices and aligned sequences. Furthermore, enoLOGOS can represent the mutual information of different positions of the consensus sequence, a unique feature of this tool. Another web interface for our software, C2H2-enoLOGOS, generates logos for the DNA-binding preferences of the C2H2 zinc-finger transcription factor family members. enoLOGOS and C2H2-enoLOGOS are accessible over the web at . PMID:15980495

Detection of Mixed Infection from Bacterial Whole Genome Sequence Data Allows Assessment of Its Role in Clostridium difficile Transmission

PubMed Central

Eyre, David W.; Cule, Madeleine L.; Griffiths, David; Crook, Derrick W.; Peto, Tim E. A.

2013-01-01

Bacterial whole genome sequencing offers the prospect of rapid and high precision investigation of infectious disease outbreaks. Close genetic relationships between microorganisms isolated from different infected cases suggest transmission is a strong possibility, whereas transmission between cases with genetically distinct bacterial isolates can be excluded. However, undetected mixed infections—infection with ≥2 unrelated strains of the same species where only one is sequenced—potentially impairs exclusion of transmission with certainty, and may therefore limit the utility of this technique. We investigated the problem by developing a computationally efficient method for detecting mixed infection without the need for resource-intensive independent sequencing of multiple bacterial colonies. Given the relatively low density of single nucleotide polymorphisms within bacterial sequence data, direct reconstruction of mixed infection haplotypes from current short-read sequence data is not consistently possible. We therefore use a two-step maximum likelihood-based approach, assuming each sample contains up to two infecting strains. We jointly estimate the proportion of the infection arising from the dominant and minor strains, and the sequence divergence between these strains. In cases where mixed infection is confirmed, the dominant and minor haplotypes are then matched to a database of previously sequenced local isolates. We demonstrate the performance of our algorithm with in silico and in vitro mixed infection experiments, and apply it to transmission of an important healthcare-associated pathogen, Clostridium difficile. Using hospital ward movement data in a previously described stochastic transmission model, 15 pairs of cases enriched for likely transmission events associated with mixed infection were selected. Our method identified four previously undetected mixed infections, and a previously undetected transmission event, but no direct transmission between the pairs of cases under investigation. These results demonstrate that mixed infections can be detected without additional sequencing effort, and this will be important in assessing the extent of cryptic transmission in our hospitals. PMID:23658511

[Vertical variability of Pinus sylvestris var. mongolica tree ring delta13C and its relationship with tree ring width in northern Daxing' an Mountains of Northeast China].

PubMed

Shang, Zhi-Yuan; Wang, Jian; Zhang, Wen; Li, Yan-Yan; Cui, Ming-Xing; Chen, Zhen-Ju; Zhao, Xing-Yun

2013-01-01

A measurement was made on the vertical direction tree ring stable carbon isotope ratio (delta13C) and tree ring width of Pinus sylvestris var. mongolica in northern Daxing' an Mountains of Northeast China, with the relationship between the vertical direction variations of the tree ring delta13C and tree ring width analyzed. In the whole ring of xylem, earlywood (EW) and bark endodermis, the delta13C all exhibited an increasing trend from the top to the base at first, with the maximum at the bottom of tree crown, and then, decreased rapidly to the minimum downward. The EW and late-wood (LW) had an increasing ratio of average tree ring width from the base to the top. The average annual sequence of the delta13C in vertical direction had an obvious reverse correspondence with the average annual sequence of tree ring width, and had a trend comparatively in line with the average annual sequence of the tree ring width ratio of EW to LW above tree crown. The variance analysis showed that there existed significant differences in the sequences of tree ring delta13C and ring width in vertical direction, and the magnitude of vertical delta13C variability was basically the same as that of the inter-annual delta13C variability. The year-to-year variation trend of the vertical delta13C sequence was approximately identical. For each sample, the delta13C sequence at the same heights was negatively correlated with the ring width sequence, but the statistical significance differed with tree height.

GC-rich coding sequences reduce transposon-like, small RNA-mediated transgene silencing.

PubMed

Sidorenko, Lyudmila V; Lee, Tzuu-Fen; Woosley, Aaron; Moskal, William A; Bevan, Scott A; Merlo, P Ann Owens; Walsh, Terence A; Wang, Xiujuan; Weaver, Staci; Glancy, Todd P; Wang, PoHao; Yang, Xiaozeng; Sriram, Shreedharan; Meyers, Blake C

2017-11-01

The molecular basis of transgene susceptibility to silencing is poorly characterized in plants; thus, we evaluated several transgene design parameters as means to reduce heritable transgene silencing. Analyses of Arabidopsis plants with transgenes encoding a microalgal polyunsaturated fatty acid (PUFA) synthase revealed that small RNA (sRNA)-mediated silencing, combined with the use of repetitive regulatory elements, led to aggressive transposon-like silencing of canola-biased PUFA synthase transgenes. Diversifying regulatory sequences and using native microalgal coding sequences (CDSs) with higher GC content improved transgene expression and resulted in a remarkable trans-generational stability via reduced accumulation of sRNAs and DNA methylation. Further experiments in maize with transgenes individually expressing three crystal (Cry) proteins from Bacillus thuringiensis (Bt) tested the impact of CDS recoding using different codon bias tables. Transgenes with higher GC content exhibited increased transcript and protein accumulation. These results demonstrate that the sequence composition of transgene CDSs can directly impact silencing, providing design strategies for increasing transgene expression levels and reducing risks of heritable loss of transgene expression.

Utilization of RNA polymerase I promoter and terminator sequences to develop a DNA transfection system for the study of hepatitis C virus internal ribosomal entry site-dependent translation.

PubMed

Oem, Jae-Ku; Xiang, Zhonghua; Zhou, Yan; Babiuk, Lorne A; Liu, Qiang

2007-09-01

Hepatitis C virus (HCV) causes severe liver diseases in a large population worldwide. HCV protein translation is controlled by an internal ribosomal entry site (IRES) within the 5'-untranslated region (UTR). HCV IRES-dependent translation is critical for HCV-associated pathogenesis. To develop a plasmid DNA transfection system by using RNA polymerase I promoter and terminator sequences for studying HCV IRES-dependent translation. A gene cassette containing HCV 5'-UTR, Renilla luciferase reporter gene, and HCV 3'-UTR was inserted between RNA polymerase I promoter and terminator sequences. HCV IRES-directed translation was determined by luciferase assay after transfection. Transfection of the RNA polymerase I-HCV IRES plasmid into human hepatoma Huh-7 and HepG2 cells resulted in luciferase gene expression. Deletion of the IIIf domain in HCV IRES dramatically reduced luciferase activity. Our results indicated that the plasmid vector system-based on RNA polymerase I promoter and terminator sequences represents an effective approach for the study of HCV IRES-dependent translation.

Observing complex action sequences: The role of the fronto-parietal mirror neuron system.

PubMed

Molnar-Szakacs, Istvan; Kaplan, Jonas; Greenfield, Patricia M; Iacoboni, Marco

2006-11-15

A fronto-parietal mirror neuron network in the human brain supports the ability to represent and understand observed actions allowing us to successfully interact with others and our environment. Using functional magnetic resonance imaging (fMRI), we wanted to investigate the response of this network in adults during observation of hierarchically organized action sequences of varying complexity that emerge at different developmental stages. We hypothesized that fronto-parietal systems may play a role in coding the hierarchical structure of object-directed actions. The observation of all action sequences recruited a common bilateral network including the fronto-parietal mirror neuron system and occipito-temporal visual motion areas. Activity in mirror neuron areas varied according to the motoric complexity of the observed actions, but not according to the developmental sequence of action structures, possibly due to the fact that our subjects were all adults. These results suggest that the mirror neuron system provides a fairly accurate simulation process of observed actions, mimicking internally the level of motoric complexity. We also discuss the results in terms of the links between mirror neurons, language development and evolution.

Evolutionary and biophysical relationships among the papillomavirus E2 proteins.

PubMed

Blakaj, Dukagjin M; Fernandez-Fuentes, Narcis; Chen, Zigui; Hegde, Rashmi; Fiser, Andras; Burk, Robert D; Brenowitz, Michael

2009-01-01

Infection by human papillomavirus (HPV) may result in clinical conditions ranging from benign warts to invasive cancer. The HPV E2 protein represses oncoprotein transcription and is required for viral replication. HPV E2 binds to palindromic DNA sequences of highly conserved four base pair sequences flanking an identical length variable 'spacer'. E2 proteins directly contact the conserved but not the spacer DNA. Variation in naturally occurring spacer sequences results in differential protein affinity that is dependent on their sensitivity to the spacer DNA's unique conformational and/or dynamic properties. This article explores the biophysical character of this core viral protein with the goal of identifying characteristics that associated with risk of virally caused malignancy. The amino acid sequence, 3d structure and electrostatic features of the E2 protein DNA binding domain are highly conserved; specific interactions with DNA binding sites have also been conserved. In contrast, the E2 protein's transactivation domain does not have extensive surfaces of highly conserved residues. Rather, regions of high conservation are localized to small surface patches. Implications to cancer biology are discussed.

Can We Improve Structured Sequence Processing? Exploring the Direct and Indirect Effects of Computerized Training Using a Mediational Model

PubMed Central

Smith, Gretchen N. L.; Conway, Christopher M.; Bauernschmidt, Althea; Pisoni, David B.

2015-01-01

Recent research suggests that language acquisition may rely on domain-general learning abilities, such as structured sequence processing, which is the ability to extract, encode, and represent structured patterns in a temporal sequence. If structured sequence processing supports language, then it may be possible to improve language function by enhancing this foundational learning ability. The goal of the present study was to use a novel computerized training task as a means to better understand the relationship between structured sequence processing and language function. Participants first were assessed on pre-training tasks to provide baseline behavioral measures of structured sequence processing and language abilities. Participants were then quasi-randomly assigned to either a treatment group involving adaptive structured visuospatial sequence training, a treatment group involving adaptive non-structured visuospatial sequence training, or a control group. Following four days of sequence training, all participants were assessed with the same pre-training measures. Overall comparison of the post-training means revealed no group differences. However, in order to examine the potential relations between sequence training, structured sequence processing, and language ability, we used a mediation analysis that showed two competing effects. In the indirect effect, adaptive sequence training with structural regularities had a positive impact on structured sequence processing performance, which in turn had a positive impact on language processing. This finding not only identifies a potential novel intervention to treat language impairments but also may be the first demonstration that structured sequence processing can be improved and that this, in turn, has an impact on language processing. However, in the direct effect, adaptive sequence training with structural regularities had a direct negative impact on language processing. This unexpected finding suggests that adaptive training with structural regularities might potentially interfere with language processing. Taken together, these findings underscore the importance of pursuing designs that promote a better understanding of the mechanisms underlying training-related changes, so that regimens can be developed that help reduce these types of negative effects while simultaneously maximizing the benefits to outcome measures of interest. PMID:25946222

Can we improve structured sequence processing? Exploring the direct and indirect effects of computerized training using a mediational model.

PubMed

Smith, Gretchen N L; Conway, Christopher M; Bauernschmidt, Althea; Pisoni, David B

2015-01-01

Recent research suggests that language acquisition may rely on domain-general learning abilities, such as structured sequence processing, which is the ability to extract, encode, and represent structured patterns in a temporal sequence. If structured sequence processing supports language, then it may be possible to improve language function by enhancing this foundational learning ability. The goal of the present study was to use a novel computerized training task as a means to better understand the relationship between structured sequence processing and language function. Participants first were assessed on pre-training tasks to provide baseline behavioral measures of structured sequence processing and language abilities. Participants were then quasi-randomly assigned to either a treatment group involving adaptive structured visuospatial sequence training, a treatment group involving adaptive non-structured visuospatial sequence training, or a control group. Following four days of sequence training, all participants were assessed with the same pre-training measures. Overall comparison of the post-training means revealed no group differences. However, in order to examine the potential relations between sequence training, structured sequence processing, and language ability, we used a mediation analysis that showed two competing effects. In the indirect effect, adaptive sequence training with structural regularities had a positive impact on structured sequence processing performance, which in turn had a positive impact on language processing. This finding not only identifies a potential novel intervention to treat language impairments but also may be the first demonstration that structured sequence processing can be improved and that this, in turn, has an impact on language processing. However, in the direct effect, adaptive sequence training with structural regularities had a direct negative impact on language processing. This unexpected finding suggests that adaptive training with structural regularities might potentially interfere with language processing. Taken together, these findings underscore the importance of pursuing designs that promote a better understanding of the mechanisms underlying training-related changes, so that regimens can be developed that help reduce these types of negative effects while simultaneously maximizing the benefits to outcome measures of interest.

Complete mitochondrial genome sequences of Brassica rapa (Chinese cabbage and mizuna), and intraspecific differentiation of cytoplasm in B. rapa and Brassica juncea.

PubMed

Hatono, Saki; Nishimura, Kaori; Murakami, Yoko; Tsujimura, Mai; Yamagishi, Hiroshi

2017-09-01

The complete sequence of the mitochondrial genome was determined for two cultivars of Brassica rapa . After determining the sequence of a Chinese cabbage variety, 'Oushou hakusai', the sequence of a mizuna variety, 'Chusei shiroguki sensuji kyomizuna', was mapped against the sequence of Chinese cabbage. The precise sequences where the two varieties demonstrated variation were ascertained by direct sequencing. It was found that the mitochondrial genomes of the two varieties are identical over 219,775 bp, with a single nucleotide polymorphism (SNP) between the genomes. Because B. rapa is the maternal species of an amphidiploid crop species, Brassica juncea , the distribution of the SNP was observed both in B. rapa and B. juncea . While the mizuna type SNP was restricted mainly to cultivars of mizuna (japonica group) in B. rapa , the mizuna type was widely distributed in B. juncea . The finding that the two Brassica species have these SNP types in common suggests that the nucleotide substitution occurred in wild B. rapa before both mitotypes were domesticated. It was further inferred that the interspecific hybridization between B. rapa and B. nigra took place twice and resulted in the two mitotypes of cultivated B. juncea .

The genome of Diuraphis noxia, a global pest of small grains

USDA-ARS?s Scientific Manuscript database

The Russian wheat aphid (Diuraphis noxia) is the world's most destructive grain aphid, producing unique phytotoxic damage symptoms that result directly from salivary proteins injected into the host plant while feeding. We sequenced and assembled the genome of D. noxia biotype 2, the most widely des...

Brushless direct-current motors

NASA Technical Reports Server (NTRS)

Bahm, E. J.

1970-01-01

Survey results are presented on the use of unconventional motor windings and switching sequences to optimize performance of brushless dc motors. A motor was built, each coil terminal having a separate, accessible lead. With the shaft and all electronics excluded, length and outside diameter measured 1.25 and 0.75 in., respectively.

Tandem Mass Spectrum Sequencing: An Alternative to Database Search Engines in Shotgun Proteomics.

PubMed

Muth, Thilo; Rapp, Erdmann; Berven, Frode S; Barsnes, Harald; Vaudel, Marc

2016-01-01

Protein identification via database searches has become the gold standard in mass spectrometry based shotgun proteomics. However, as the quality of tandem mass spectra improves, direct mass spectrum sequencing gains interest as a database-independent alternative. In this chapter, the general principle of this so-called de novo sequencing is introduced along with pitfalls and challenges of the technique. The main tools available are presented with a focus on user friendly open source software which can be directly applied in everyday proteomic workflows.

Direct inhibition of excision/synthesis DNA repair activities by cadmium: analysis on dedicated biochips.

PubMed

Candéias, S; Pons, B; Viau, M; Caillat, S; Sauvaigo, S

2010-12-10

The well established toxicity of cadmium and cadmium compounds results from their additive effects on several key cellular processes, including DNA repair. Mammalian cells have evolved several biochemical pathways to repair DNA lesions and maintain genomic integrity. By interfering with the homeostasis of redox metals and antioxidant systems, cadmium promotes the development of an intracellular environment that results in oxidative DNA damage which can be mutagenic if unrepaired. Small base lesions are recognised by specialized glycosylases and excised from the DNA molecule. The resulting abasic sites are incised, and the correct sequences restored by DNA polymerases using the opposite strands as template. Bulky lesions are recognised by a different set of proteins and excised from DNA as part of an oligonucleotide. As in base repair, the resulting gaps are filled by DNA polymerases using the opposite strands as template. Thus, these two repair pathways consist in excision of the lesion followed by DNA synthesis. In this study, we analysed in vitro the direct effects of cadmium exposure on the functionality of base and nucleotide DNA repair pathways. To this end, we used recently described dedicated microarrays that allow the parallel monitoring in cell extracts of the repair activities directed against several model base and/or nucleotide lesions. Both base and nucleotide excision/repair pathways are inhibited by CdCl₂, with different sensitivities. The inhibitory effects of cadmium affect mainly the recognition and excision stages of these processes. Furthermore, our data indicate that the repair activities directed against different damaged bases also exhibit distinct sensitivities, and the direct comparison of cadmium effects on the excision of uracile in different sequences even allows us to propose a hierarchy of cadmium sensibility within the glycosylases removing U from DNA. These results indicate that, in our experimental conditions, cadmium is a very potent DNA repair poison. Copyright © 2010 Elsevier B.V. All rights reserved.

Psychiatric symptoms mediate the effects of neurological soft signs on functional outcomes in patients with chronic schizophrenia: A longitudinal path-analytic study.

PubMed

Fong, Ted C T; Ho, Rainbow T H; Wan, Adrian H Y; Au-Yeung, Friendly S W

2017-03-01

Neurological soft signs (NSS) in motor coordination and sequencing occur in schizophrenia patients and are an intrinsic sign of the underlying neural dysfunctions. The present longitudinal study explored the relationships among NSS, psychiatric symptoms, and functional outcomes in 151 Chinese patients with chronic schizophrenia across a 6-month period. The participants completed neurological assessments at baseline (Time 1), psychiatric interviews at Time 1 and 3-month follow-up (Time 2), and self-report measures on daily functioning at 6-month follow-up (Time 3). Two possible (combined and cascading) path models were examined on predicting the functional outcomes. Direct and indirect effects of Time 1 NSS on Time 3 functional outcomes via Time 2 psychiatric symptoms were evaluated using path analysis under bootstrapping. Motor coordination and sequencing NSS did not have significant direct effects on functional outcomes. Motor coordination NSS exerted significant and negative indirect effects on functional outcomes via psychiatric symptoms. These results contribute to a better understanding of the determinants of functional outcomes by showing significant indirect pathways from motor coordination NSS to functional outcomes via psychiatric symptoms. That motor sequencing NSS did not affect functional outcomes either directly or indirectly may be explained by their trait marking features. Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.

Distortion correction for diffusion-weighted MRI tractography and fMRI in the temporal lobes.

PubMed

Embleton, Karl V; Haroon, Hamied A; Morris, David M; Ralph, Matthew A Lambon; Parker, Geoff J M

2010-10-01

Single shot echo-planar imaging (EPI) sequences are currently the most commonly used sequences for diffusion-weighted imaging (DWI) and functional magnetic resonance imaging (fMRI) as they allow relatively high signal to noise with rapid acquisition time. A major drawback of EPI is the substantial geometric distortion and signal loss that can occur due to magnetic field inhomogeneities close to air-tissue boundaries. If DWI-based tractography and fMRI are to be applied to these regions, then the distortions must be accurately corrected to achieve meaningful results. We describe robust acquisition and processing methods for correcting such distortions in spin echo (SE) EPI using a variant of the reversed direction k space traversal method with a number of novel additions. We demonstrate that dual direction k space traversal with maintained diffusion-encoding gradient strength and direction results in correction of the great majority of eddy current-associated distortions in DWI, in addition to those created by variations in magnetic susceptibility. We also provide examples to demonstrate that the presence of severe distortions cannot be ignored if meaningful tractography results are desired. The distortion correction routine was applied to SE-EPI fMRI acquisitions and allowed detection of activation in the temporal lobe that had been previously found using PET but not conventional fMRI. © 2010 Wiley-Liss, Inc.

Facile Site-Directed Mutagenesis of Large Constructs Using Gibson Isothermal DNA Assembly.

PubMed

Yonemoto, Isaac T; Weyman, Philip D

2017-01-01

Site-directed mutagenesis is a commonly used molecular biology technique to manipulate biological sequences, and is especially useful for studying sequence determinants of enzyme function or designing proteins with improved activity. We describe a strategy using Gibson Isothermal DNA Assembly to perform site-directed mutagenesis on large (>~20 kbp) constructs that are outside the effective range of standard techniques such as QuikChange II (Agilent Technologies), but more reliable than traditional cloning using restriction enzymes and ligation.

The involvement of immunoglobulin E isotype switch in scleroderma skin tissue.

PubMed

Ohtsuka, Tsutomu; Yamazaki, Soji

2005-08-01

The involvement of mast cell, which is activated by immunoglobulin E (IgE), has been reported in the formation of systemic sclerosis (SSc) abnormality. IgE is generated with isotype switch. During isotype switch, switch circles resulting from direct mu to epsilon, or from sequential mu to gamma via epsilon switching will be created. We studied whether switching occurs in SSc. We used nested polymerase chain reaction to analyze the S fragments from switch circles. Fifty-two patients with SSc, and 62 healthy women were studied. Neither of 62 normal skin tissues showed direct switch, nor sequential switch. Neither of seven normal whole blood cells showed direct switch, nor sequential switch. In 52SSc skin tissues, three (5.8%) showed direct switch, and two (3.8%) showed sequential switch. As a result, five (9.6%) of SSc skin tissue showed immunogobulin E class switch. These results were confirmed by DNA sequencing. These results demonstrated that isotype switch to the epsilon locus achieved by direct and/or sequential switch are involved in SSc skin.

Complete plastid genome sequence of Daucus carota: Implications for biotechnology and phylogeny of angiosperms

PubMed Central

Ruhlman, Tracey; Lee, Seung-Bum; Jansen, Robert K; Hostetler, Jessica B; Tallon, Luke J; Town, Christopher D; Daniell, Henry

2006-01-01

Background Carrot (Daucus carota) is a major food crop in the US and worldwide. Its capacity for storage and its lifecycle as a biennial make it an attractive species for the introduction of foreign genes, especially for oral delivery of vaccines and other therapeutic proteins. Until recently efforts to express recombinant proteins in carrot have had limited success in terms of protein accumulation in the edible tap roots. Plastid genetic engineering offers the potential to overcome this limitation, as demonstrated by the accumulation of BADH in chromoplasts of carrot taproots to confer exceedingly high levels of salt resistance. The complete plastid genome of carrot provides essential information required for genetic engineering. Additionally, the sequence data add to the rapidly growing database of plastid genomes for assessing phylogenetic relationships among angiosperms. Results The complete carrot plastid genome is 155,911 bp in length, with 115 unique genes and 21 duplicated genes within the IR. There are four ribosomal RNAs, 30 distinct tRNA genes and 18 intron-containing genes. Repeat analysis reveals 12 direct and 2 inverted repeats ≥ 30 bp with a sequence identity ≥ 90%. Phylogenetic analysis of nucleotide sequences for 61 protein-coding genes using both maximum parsimony (MP) and maximum likelihood (ML) were performed for 29 angiosperms. Phylogenies from both methods provide strong support for the monophyly of several major angiosperm clades, including monocots, eudicots, rosids, asterids, eurosids II, euasterids I, and euasterids II. Conclusion The carrot plastid genome contains a number of dispersed direct and inverted repeats scattered throughout coding and non-coding regions. This is the first sequenced plastid genome of the family Apiaceae and only the second published genome sequence of the species-rich euasterid II clade. Both MP and ML trees provide very strong support (100% bootstrap) for the sister relationship of Daucus with Panax in the euasterid II clade. These results provide the best taxon sampling of complete chloroplast genomes and the strongest support yet for the sister relationship of Caryophyllales to the asterids. The availability of the complete plastid genome sequence should facilitate improved transformation efficiency and foreign gene expression in carrot through utilization of endogenous flanking sequences and regulatory elements. PMID:16945140

Unique Conformation in a Natural Interruption Sequence of Type XIX Collagen Revealed by Its High-Resolution Crystal Structure.

PubMed

Xu, Tingting; Zhou, Cong-Zhao; Xiao, Jianxi; Liu, Jinsong

2018-02-20

Naturally occurring interruptions in nonfibrillar collagen play key roles in molecular flexibility, collagen degradation, and ligand binding. The structural feature of the interruption sequences and the molecular basis for their functions have not been well studied. Here, we focused on a G5G type natural interruption sequence G-POALO-G from human type XIX collagen, a homotrimer collagen, as this sequence possesses distinct properties compared with those of a pathological similar Gly mutation sequence in collagen mimic peptides. We determined the crystal structures of the host-guest peptide (GPO) 3 -GPOALO-(GPO) 4 to 1.03 Å resolution in two crystal forms. In these structures, the interruption zone brings localized disruptions to the triple helix and introduces a light 6-8° bend with the same directional preference to the whole molecule, which may correspond structurally to the first physiological kink site in type XIX collagen. Furthermore, at the G5G interruption site, the presence of Ala and Leu residues, both with free N-H groups, allows the formation of more direct and water-mediated interchain hydrogen bonds than in the related Gly → Ala structure. These could partly explain the difference in thermal stability between the different interruptions. In addition, our structures provide a detailed view of the dynamic property of such an interrupted zone with respect to hydrogen bonding topology, torsion angles, and helical parameters. Our results, for the first time, also identified the binding of zinc to the end of the triple helix. These findings will shed light on how the interruption sequence influences the conformation of the collagen molecule and provide a structural basis for further functional studies.

Improved Annotation of 3′ Untranslated Regions and Complex Loci by Combination of Strand-Specific Direct RNA Sequencing, RNA-Seq and ESTs

PubMed Central

Song, Junfang; Duc, Céline; Storey, Kate G.; McLean, W. H. Irwin; Brown, Sara J.; Simpson, Gordon G.; Barton, Geoffrey J.

2014-01-01

The reference annotations made for a genome sequence provide the framework for all subsequent analyses of the genome. Correct and complete annotation in addition to the underlying genomic sequence is particularly important when interpreting the results of RNA-seq experiments where short sequence reads are mapped against the genome and assigned to genes according to the annotation. Inconsistencies in annotations between the reference and the experimental system can lead to incorrect interpretation of the effect on RNA expression of an experimental treatment or mutation in the system under study. Until recently, the genome-wide annotation of 3′ untranslated regions received less attention than coding regions and the delineation of intron/exon boundaries. In this paper, data produced for samples in Human, Chicken and A. thaliana by the novel single-molecule, strand-specific, Direct RNA Sequencing technology from Helicos Biosciences which locates 3′ polyadenylation sites to within +/− 2 nt, were combined with archival EST and RNA-Seq data. Nine examples are illustrated where this combination of data allowed: (1) gene and 3′ UTR re-annotation (including extension of one 3′ UTR by 5.9 kb); (2) disentangling of gene expression in complex regions; (3) clearer interpretation of small RNA expression and (4) identification of novel genes. While the specific examples displayed here may become obsolete as genome sequences and their annotations are refined, the principles laid out in this paper will be of general use both to those annotating genomes and those seeking to interpret existing publically available annotations in the context of their own experimental data. PMID:24722185

Characterization of expressed sequence tag-derived simple sequence repeat markers for Aspergillus flavus: emphasis on variability of isolates from the southern United States.

PubMed

Wang, Xinwang; Wadl, Phillip A; Wood-Jones, Alicia; Windham, Gary; Trigiano, Robert N; Scruggs, Mary; Pilgrim, Candace; Baird, Richard

2012-12-01

Simple sequence repeat (SSR) markers were developed from Aspergillus flavus expressed sequence tag (EST) database to conduct an analysis of genetic relationships of Aspergillus isolates from numerous host species and geographical regions, but primarily from the United States. Twenty-nine primers were designed from 362 tri-nucleotide EST-SSR sequences. Eighteen polymorphic loci were used to genotype 96 Aspergillus species isolates. The number of alleles detected per locus ranged from 2 to 24 with a mean of 8.2 alleles. Haploid diversity ranged from 0.28 to 0.91. Genetic distance matrix was used to perform principal coordinates analysis (PCA) and to generate dendrograms using unweighted pair group method with arithmetic mean (UPGMA). Two principal coordinates explained more than 75 % of the total variation among the isolates. One clade was identified for A. flavus isolates (n = 87) with the other Aspergillus species (n = 7) using PCA, but five distinct clusters were present when the others taxa were excluded from the analysis. Six groups were noted when the EST-SSR data were compared using UPGMA. However, the latter PCA or UPGMA comparison resulted in no direct associations with host species, geographical region or aflatoxin production. Furthermore, there was no direct correlation to visible morphological features such as sclerotial types. The isolates from Mississippi Delta region, which contained the largest percentage of isolates, did not show any unusual clustering except for isolates K32, K55, and 199. Further studies of these three isolates are warranted to evaluate their pathogenicity, aflatoxin production potential, additional gene sequences (e.g., RPB2), and morphological comparisons.

Single-molecule, full-length transcript sequencing provides insight into the extreme metabolism of the ruby-throated hummingbird Archilochus colubris

PubMed Central

Workman, Rachael E; Myrka, Alexander M; Wong, G William; Tseng, Elizabeth

2018-01-01

Abstract Background Hummingbirds oxidize ingested nectar sugars directly to fuel foraging but cannot sustain this fuel use during fasting periods, such as during the night or during long-distance migratory flights. Instead, fasting hummingbirds switch to oxidizing stored lipids that are derived from ingested sugars. The hummingbird liver plays a key role in moderating energy homeostasis and this remarkable capacity for fuel switching. Additionally, liver is the principle location of de novo lipogenesis, which can occur at exceptionally high rates, such as during premigratory fattening. Yet understanding how this tissue and whole organism moderates energy turnover is hampered by a lack of information regarding how relevant enzymes differ in sequence, expression, and regulation. Findings We generated a de novo transcriptome of the hummingbird liver using PacBio full-length cDNA sequencing (Iso-Seq), yielding 8.6Gb of sequencing data, or 2.6M reads from 4 different size fractions. We analyzed data using the SMRTAnalysis v3.1 Iso-Seq pipeline, then clustered isoforms into gene families to generate de novo gene contigs using Cogent. We performed orthology analysis to identify closely related sequences between our transcriptome and other avian and human gene sets. Finally, we closely examined homology of critical lipid metabolism genes between our transcriptome data and avian and human genomes. Conclusions We confirmed high levels of sequence divergence within hummingbird lipogenic enzymes, suggesting a high probability of adaptive divergent function in the hepatic lipogenic pathways. Our results leverage cutting-edge technology and a novel bioinformatics pipeline to provide a first direct look at the transcriptome of this incredible organism. PMID:29618047

Sequence Learning Is Preserved in Individuals with Cerebellar Degeneration when the Movements Are Directly Cued

ERIC Educational Resources Information Center

Spencer, Rebecca M. C.; Ivry, Richard B.

2009-01-01

Cerebellar pathology is associated with impairments on a range of motor learning tasks including sequence learning. However, various lines of evidence are at odds with the idea that the cerebellum plays a central role in the associative processes underlying sequence learning. Behavioral studies indicate that sequence learning, at least with short…

Microsatellite analysis in the genome of Acanthaceae: An in silico approach

PubMed Central

Kaliswamy, Priyadharsini; Vellingiri, Srividhya; Nathan, Bharathi; Selvaraj, Saravanakumar

2015-01-01

Background: Acanthaceae is one of the advanced and specialized families with conventionally used medicinal plants. Simple sequence repeats (SSRs) play a major role as molecular markers for genome analysis and plant breeding. The microsatellites existing in the complete genome sequences would help to attain a direct role in the genome organization, recombination, gene regulation, quantitative genetic variation, and evolution of genes. Objective: The current study reports the frequency of microsatellites and appropriate markers for the Acanthaceae family genome sequences. Materials and Methods: The whole nucleotide sequences of Acanthaceae species were obtained from National Center for Biotechnology Information database and screened for the presence of SSRs. SSR Locator tool was used to predict the microsatellites and inbuilt Primer3 module was used for primer designing. Results: Totally 110 repeats from 108 sequences of Acanthaceae family plant genomes were identified, and the occurrence of dinucleotide repeats was found to be abundant in the genome sequences. The essential amino acid isoleucine was found rich in all the sequences. We also designed the SSR-based primers/markers for 59 sequences of this family that contains microsatellite repeats in their genome. Conclusion: The identified microsatellites and primers might be useful for breeding and genetic studies of plants that belong to Acanthaceae family in the future. PMID:25709226

Schema vs. primitive perceptual grouping: the relative weighting of sequential vs. spatial cues during an auditory grouping task in frogs.

PubMed

Farris, Hamilton E; Ryan, Michael J

2017-03-01

Perceptually, grouping sounds based on their sources is critical for communication. This is especially true in túngara frog breeding aggregations, where multiple males produce overlapping calls that consist of an FM 'whine' followed by harmonic bursts called 'chucks'. Phonotactic females use at least two cues to group whines and chucks: whine-chuck spatial separation and sequence. Spatial separation is a primitive cue, whereas sequence is schema-based, as chuck production is morphologically constrained to follow whines, meaning that males cannot produce the components simultaneously. When one cue is available, females perceptually group whines and chucks using relative comparisons: components with the smallest spatial separation or those closest to the natural sequence are more likely grouped. By simultaneously varying the temporal sequence and spatial separation of a single whine and two chucks, this study measured between-cue perceptual weighting during a specific grouping task. Results show that whine-chuck spatial separation is a stronger grouping cue than temporal sequence, as grouping is more likely for stimuli with smaller spatial separation and non-natural sequence than those with larger spatial separation and natural sequence. Compared to the schema-based whine-chuck sequence, we propose that spatial cues have less variance, potentially explaining their preferred use when grouping during directional behavioral responses.

High-Frame-Rate Doppler Ultrasound Using a Repeated Transmit Sequence

PubMed Central

Podkowa, Anthony S.; Oelze, Michael L.; Ketterling, Jeffrey A.

2018-01-01

The maximum detectable velocity of high-frame-rate color flow Doppler ultrasound is limited by the imaging frame rate when using coherent compounding techniques. Traditionally, high quality ultrasonic images are produced at a high frame rate via coherent compounding of steered plane wave reconstructions. However, this compounding operation results in an effective downsampling of the slow-time signal, thereby artificially reducing the frame rate. To alleviate this effect, a new transmit sequence is introduced where each transmit angle is repeated in succession. This transmit sequence allows for direct comparison between low resolution, pre-compounded frames at a short time interval in ways that are resistent to sidelobe motion. Use of this transmit sequence increases the maximum detectable velocity by a scale factor of the transmit sequence length. The performance of this new transmit sequence was evaluated using a rotating cylindrical phantom and compared with traditional methods using a 15-MHz linear array transducer. Axial velocity estimates were recorded for a range of ±300 mm/s and compared to the known ground truth. Using these new techniques, the root mean square error was reduced from over 400 mm/s to below 50 mm/s in the high-velocity regime compared to traditional techniques. The standard deviation of the velocity estimate in the same velocity range was reduced from 250 mm/s to 30 mm/s. This result demonstrates the viability of the repeated transmit sequence methods in detecting and quantifying high-velocity flow. PMID:29910966

SU-F-J-112: Clinical Feasibility Test of An RF Pulse-Based MRI Method for the Quantitative Fat-Water Segmentation

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

Yee, S; Wloch, J; Pirkola, M

Purpose: Quantitative fat-water segmentation is important not only because of the clinical utility of fat-suppressed MRI images in better detecting lesions of clinical significance (in the midst of bright fat signal) but also because of the possible physical need, in which CT-like images based on the materials’ photon attenuation properties may have to be generated from MR images; particularly, as in the case of MR-only radiation oncology environment to obtain radiation dose calculation or as in the case of hybrid PET/MR modality to obtain attenuation correction map for the quantitative PET reconstruction. The majority of such fat-water quantitative segmentations havemore » been performed by utilizing the Dixon’s method and its variations, which have to enforce the proper settings (often predefined) of echo time (TE) in the pulse sequences. Therefore, such methods have been unable to be directly combined with those ultrashort TE (UTE) sequences that, taking the advantage of very low TE values (∼ 10’s microsecond), might be beneficial to directly detect bones. Recently, an RF pulse-based method (http://dx.doi.org/10.1016/j.mri.2015.11.006), termed as PROD pulse method, was introduced as a method of quantitative fat-water segmentation that does not have to depend on predefined TE settings. Here, the clinical feasibility of this method is verified in brain tumor patients by combining the PROD pulse with several sequences. Methods: In a clinical 3T MRI, the PROD pulse was combined with turbo spin echo (e.g. TR=1500, TE=16 or 60, ETL=15) or turbo field echo (e.g. TR=5.6, TE=2.8, ETL=12) sequences without specifying TE values. Results: The fat-water segmentation was possible without having to set specific TE values. Conclusion: The PROD pulse method is clinically feasible. Although not yet combined with UTE sequences in our laboratory, the method is potentially compatible with UTE sequences, and thus, might be useful to directly segment fat, water, bone and air.« less

CAMELOT: A machine learning approach for coarse-grained simulations of aggregation of block-copolymeric protein sequences

PubMed Central

Ruff, Kiersten M.; Harmon, Tyler S.; Pappu, Rohit V.

2015-01-01

We report the development and deployment of a coarse-graining method that is well suited for computer simulations of aggregation and phase separation of protein sequences with block-copolymeric architectures. Our algorithm, named CAMELOT for Coarse-grained simulations Aided by MachinE Learning Optimization and Training, leverages information from converged all atom simulations that is used to determine a suitable resolution and parameterize the coarse-grained model. To parameterize a system-specific coarse-grained model, we use a combination of Boltzmann inversion, non-linear regression, and a Gaussian process Bayesian optimization approach. The accuracy of the coarse-grained model is demonstrated through direct comparisons to results from all atom simulations. We demonstrate the utility of our coarse-graining approach using the block-copolymeric sequence from the exon 1 encoded sequence of the huntingtin protein. This sequence comprises of 17 residues from the N-terminal end of huntingtin (N17) followed by a polyglutamine (polyQ) tract. Simulations based on the CAMELOT approach are used to show that the adsorption and unfolding of the wild type N17 and its sequence variants on the surface of polyQ tracts engender a patchy colloid like architecture that promotes the formation of linear aggregates. These results provide a plausible explanation for experimental observations, which show that N17 accelerates the formation of linear aggregates in block-copolymeric N17-polyQ sequences. The CAMELOT approach is versatile and is generalizable for simulating the aggregation and phase behavior of a range of block-copolymeric protein sequences. PMID:26723608

Direct comparisons of Illumina vs. Roche 454 sequencing technologies on the same microbial community DNA sample.

PubMed

Luo, Chengwei; Tsementzi, Despina; Kyrpides, Nikos; Read, Timothy; Konstantinidis, Konstantinos T

2012-01-01

Next-generation sequencing (NGS) is commonly used in metagenomic studies of complex microbial communities but whether or not different NGS platforms recover the same diversity from a sample and their assembled sequences are of comparable quality remain unclear. We compared the two most frequently used platforms, the Roche 454 FLX Titanium and the Illumina Genome Analyzer (GA) II, on the same DNA sample obtained from a complex freshwater planktonic community. Despite the substantial differences in read length and sequencing protocols, the platforms provided a comparable view of the community sampled. For instance, derived assemblies overlapped in ~90% of their total sequences and in situ abundances of genes and genotypes (estimated based on sequence coverage) correlated highly between the two platforms (R(2)>0.9). Evaluation of base-call error, frameshift frequency, and contig length suggested that Illumina offered equivalent, if not better, assemblies than Roche 454. The results from metagenomic samples were further validated against DNA samples of eighteen isolate genomes, which showed a range of genome sizes and G+C% content. We also provide quantitative estimates of the errors in gene and contig sequences assembled from datasets characterized by different levels of complexity and G+C% content. For instance, we noted that homopolymer-associated, single-base errors affected ~1% of the protein sequences recovered in Illumina contigs of 10× coverage and 50% G+C; this frequency increased to ~3% when non-homopolymer errors were also considered. Collectively, our results should serve as a useful practical guide for choosing proper sampling strategies and data possessing protocols for future metagenomic studies.

Genetic Requirements for the Single-Strand Annealing Pathway of Double-Strand Break Repair in Saccharomyces Cerevisiae

PubMed Central

Ivanov, E. L.; Sugawara, N.; Fishman-Lobell, J.; Haber, J. E.

1996-01-01

HO endonuclease-induced double-strand breaks (DSBs) within a direct duplication of Escherichia coli lacZ genes are repaired either by gene conversion or by single-strand annealing (SSA), with >80% being SSA. Previously it was demonstrated that the RAD52 gene is required for DSB-induced SSA. In the present study, the effects of other genes belonging to the RAD52 epistasis group were analyzed. We show that RAD51, RAD54, RAD55, and RAD57 genes are not required for SSA irrespective of whether recombination occurred in plasmid or chromosomal DNA. In both plasmid and chromosomal constructs with homologous sequences in direct orientation, the proportion of SSA events over gene conversion was significantly elevated in the mutant strains. However, gene conversion was not affected when the two lacZ sequences were in inverted orientation. These results suggest that there is a competition between SSA and gene conversion processes that favors SSA in the absence of RAD51, RAD54, RAD55 and RAD57. Mutations in RAD50 and XRS2 genes do not prevent the completion, but markedly retard the kinetics, of DSB repair by both mechanisms in the lacZ direct repeat plasmid, a result resembling the effects of these genes during mating-type (MAT) switching. PMID:8849880

Investigating Delamination Migration in Composite Tape Laminates

NASA Technical Reports Server (NTRS)

Ratcliffe, James G.; DeCarvalho, Nelson V.

2014-01-01

A modification to a recently developed test specimen designed to investigate migration of a delamination between neighboring ply interfaces in tape laminates is presented. The specimen is a cross-ply laminated beam consisting of 40 plies with a polytetrafluoroethylene insert spanning part way along its length. The insert is located between a lower 0-degree ply (specimen length direction) and a stack of four 90-degree plies (specimen width direction). The modification involved a stacking sequence that promotes stable delamination growth prior to migration, and included a relocation of the insert from the specimen midplane to the interface between plies 14 and 15. Specimens were clamped at both ends onto a rigid baseplate and loaded on their upper surface via a piano hinge assembly, resulting in a predominantly flexural loading condition. Tests were conducted with the load-application point positioned at various locations along a specimen's span. This position affected the sequence of damage events during a test.

Combining stress transfer and source directivity: the case of the 2012 Emilia seismic sequence

PubMed Central

Convertito, Vincenzo; Catalli, Flaminia; Emolo, Antonio

2013-01-01

The Emilia seismic sequence (Northern Italy) started on May 2012 and caused 17 casualties, severe damage to dwellings and forced the closure of several factories. The total number of events recorded in one month was about 2100, with local magnitude ranging between 1.0 and 5.9. We investigate potential mechanisms (static and dynamic triggering) that may describe the evolution of the sequence. We consider rupture directivity in the dynamic strain field and observe that, for each main earthquake, its aftershocks and the subsequent large event occurred in an area characterized by higher dynamic strains and corresponding to the dominant rupture direction. We find that static stress redistribution alone is not capable of explaining the locations of subsequent events. We conclude that dynamic triggering played a significant role in driving the sequence. This triggering was also associated with a variation in permeability and a pore pressure increase in an area characterized by a massive presence of fluids. PMID:24177982

Bending and stretching finite element analysis of anisotropic viscoelastic composite plates

NASA Technical Reports Server (NTRS)

Hilton, Harry H.; Yi, Sung

1990-01-01

Finite element algorithms have been developed to analyze linear anisotropic viscoelastic plates, with or without holes, subjected to mechanical (bending, tension), temperature, and hygrothermal loadings. The analysis is based on Laplace transforms rather than direct time integrations in order to improve the accuracy of the results and save on extensive computational time and storage. The time dependent displacement fields in the transverse direction for the cross ply and angle ply laminates are calculated and the stacking sequence effects of the laminates are discussed in detail. Creep responses for the plates with or without a circular hole are also studied. The numerical results compare favorably with analytical solutions, i.e. within 1.8 percent for bending and 10(exp -3) 3 percent for tension. The tension results of the present method are compared with those using the direct time integration scheme.

Helper-dependent adenoviral vectors for liver-directed gene therapy

PubMed Central

Brunetti-Pierri, Nicola; Ng, Philip

2011-01-01

Helper-dependent adenoviral (HDAd) vectors devoid of all viral-coding sequences are promising non-integrating vectors for liver-directed gene therapy because they have a large cloning capacity, can efficiently transduce a wide variety of cell types from various species independent of the cell cycle and can result in long-term transgene expression without chronic toxicity. The main obstacle preventing clinical applications of HDAd for liver-directed gene therapy is the host innate inflammatory response against the vector capsid proteins that occurs shortly after intravascular vector administration resulting in acute toxicity, the severity of which is dependent on vector dose. Intense efforts have been focused on elucidating the factors involved in this acute response and various strategies have been investigated to improve the therapeutic index of HDAd vectors. These strategies have yielded encouraging results with the potential for clinical translation. PMID:21470977

Moleculo Long-Read Sequencing Facilitates Assembly and Genomic Binning from Complex Soil Metagenomes

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

White, Richard Allen; Bottos, Eric M.; Roy Chowdhury, Taniya

ABSTRACT Soil metagenomics has been touted as the “grand challenge” for metagenomics, as the high microbial diversity and spatial heterogeneity of soils make them unamenable to current assembly platforms. Here, we aimed to improve soil metagenomic sequence assembly by applying the Moleculo synthetic long-read sequencing technology. In total, we obtained 267 Gbp of raw sequence data from a native prairie soil; these data included 109.7 Gbp of short-read data (~100 bp) from the Joint Genome Institute (JGI), an additional 87.7 Gbp of rapid-mode read data (~250 bp), plus 69.6 Gbp (>1.5 kbp) from Moleculo sequencing. The Moleculo data alone yielded over 5,600more » reads of >10 kbp in length, and over 95% of the unassembled reads mapped to contigs of >1.5 kbp. Hybrid assembly of all data resulted in more than 10,000 contigs over 10 kbp in length. We mapped three replicate metatranscriptomes derived from the same parent soil to the Moleculo subassembly and found that 95% of the predicted genes, based on their assignments to Enzyme Commission (EC) numbers, were expressed. The Moleculo subassembly also enabled binning of >100 microbial genome bins. We obtained via direct binning the first complete genome, that of “CandidatusPseudomonas sp. strain JKJ-1” from a native soil metagenome. By mapping metatranscriptome sequence reads back to the bins, we found that several bins corresponding to low-relative-abundanceAcidobacteriawere highly transcriptionally active, whereas bins corresponding to high-relative-abundanceVerrucomicrobiawere not. These results demonstrate that Moleculo sequencing provides a significant advance for resolving complex soil microbial communities. IMPORTANCESoil microorganisms carry out key processes for life on our planet, including cycling of carbon and other nutrients and supporting growth of plants. However, there is poor molecular-level understanding of their functional roles in ecosystem stability and responses to environmental perturbations. This knowledge gap is largely due to the difficulty in culturing the majority of soil microbes. Thus, use of culture-independent approaches, such as metagenomics, promises the direct assessment of the functional potential of soil microbiomes. Soil is, however, a challenge for metagenomic assembly due to its high microbial diversity and variable evenness, resulting in low coverage and uneven sampling of microbial genomes. Despite increasingly large soil metagenome data volumes (>200 Gbp), the majority of the data do not assemble. Here, we used the cutting-edge approach of synthetic long-read sequencing technology (Moleculo) to assemble soil metagenome sequence data into long contigs and used the assemblies for binning of genomes. Author Video: Anauthor video summaryof this article is available.« less

Moleculo Long-Read Sequencing Facilitates Assembly and Genomic Binning from Complex Soil Metagenomes

PubMed Central

White, Richard Allen; Bottos, Eric M.; Roy Chowdhury, Taniya; Zucker, Jeremy D.; Brislawn, Colin J.; Nicora, Carrie D.; Fansler, Sarah J.; Glaesemann, Kurt R.; Glass, Kevin

2016-01-01

ABSTRACT Soil metagenomics has been touted as the “grand challenge” for metagenomics, as the high microbial diversity and spatial heterogeneity of soils make them unamenable to current assembly platforms. Here, we aimed to improve soil metagenomic sequence assembly by applying the Moleculo synthetic long-read sequencing technology. In total, we obtained 267 Gbp of raw sequence data from a native prairie soil; these data included 109.7 Gbp of short-read data (~100 bp) from the Joint Genome Institute (JGI), an additional 87.7 Gbp of rapid-mode read data (~250 bp), plus 69.6 Gbp (>1.5 kbp) from Moleculo sequencing. The Moleculo data alone yielded over 5,600 reads of >10 kbp in length, and over 95% of the unassembled reads mapped to contigs of >1.5 kbp. Hybrid assembly of all data resulted in more than 10,000 contigs over 10 kbp in length. We mapped three replicate metatranscriptomes derived from the same parent soil to the Moleculo subassembly and found that 95% of the predicted genes, based on their assignments to Enzyme Commission (EC) numbers, were expressed. The Moleculo subassembly also enabled binning of >100 microbial genome bins. We obtained via direct binning the first complete genome, that of “Candidatus Pseudomonas sp. strain JKJ-1” from a native soil metagenome. By mapping metatranscriptome sequence reads back to the bins, we found that several bins corresponding to low-relative-abundance Acidobacteria were highly transcriptionally active, whereas bins corresponding to high-relative-abundance Verrucomicrobia were not. These results demonstrate that Moleculo sequencing provides a significant advance for resolving complex soil microbial communities. IMPORTANCE Soil microorganisms carry out key processes for life on our planet, including cycling of carbon and other nutrients and supporting growth of plants. However, there is poor molecular-level understanding of their functional roles in ecosystem stability and responses to environmental perturbations. This knowledge gap is largely due to the difficulty in culturing the majority of soil microbes. Thus, use of culture-independent approaches, such as metagenomics, promises the direct assessment of the functional potential of soil microbiomes. Soil is, however, a challenge for metagenomic assembly due to its high microbial diversity and variable evenness, resulting in low coverage and uneven sampling of microbial genomes. Despite increasingly large soil metagenome data volumes (>200 Gbp), the majority of the data do not assemble. Here, we used the cutting-edge approach of synthetic long-read sequencing technology (Moleculo) to assemble soil metagenome sequence data into long contigs and used the assemblies for binning of genomes. Author Video: An author video summary of this article is available. PMID:27822530

New mutations in the NHS gene in Nance-Horan Syndrome families from the Netherlands.

PubMed

Florijn, Ralph J; Loves, Willem; Maillette de Buy Wenniger-Prick, Liesbeth J J M; Mannens, Marcel M A M; Tijmes, Nel; Brooks, Simon P; Hardcastle, Alison J; Bergen, Arthur A B

2006-09-01

Mutations in the NHS gene cause Nance-Horan Syndrome (NHS), a rare X-chromosomal recessive disorder with variable features, including congenital cataract, microphthalmia, a peculiar form of the ear and dental anomalies. We investigated the NHS gene in four additional families with NHS from the Netherlands, by dHPLC and direct sequencing. We identified an unique mutation in each family. Three out of these four mutations were not reported before. We report here the first splice site sequence alteration mutation and three protein truncating mutations. Our results suggest that X-linked cataract and NHS are allelic disorders.

GRIL-Seq, a method for identifying direct targets of bacterial small regulatory RNA by in vivo proximity ligation

PubMed Central

Han, Kook; Tjaden, Brian; Lory, Stephen

2017-01-01

The first step in the post-transcriptional regulatory function of most bacterial small non-coding RNAs (sRNAs) is base-pairing with partially complementary sequences of targeted transcripts. We present a simple method for identifying sRNA targets in vivo and defining processing sites of the regulated transcripts. The technique (referred to as GRIL-Seq) is based on preferential ligation of sRNAs to ends of base-paired targets in bacteria co-expressing T4 RNA ligase, followed by sequencing to identify the chimeras. In addition to the RNA chaperone Hfq, the GRIL-Seq method depends on the activity of the pyrophosphorylase RppH. Using PrrF1, an iron-regulated sRNA in Pseudomonas aeruginosa, we demonstrate that direct regulatory targets of this sRNA can be readily identified. Therefore, GRIL-Seq represents a powerful tool not only for identifying direct targets of sRNAs in a variety of environments, but can also result in uncovering novel roles for sRNAs and their targets in complex regulatory networks. PMID:28005055

Estimation of a Killer Whale (Orcinus orca) Population’s Diet Using Sequencing Analysis of DNA from Feces

PubMed Central

Ford, Michael J.; Hempelmann, Jennifer; Hanson, M. Bradley; Ayres, Katherine L.; Baird, Robin W.; Emmons, Candice K.; Lundin, Jessica I.; Schorr, Gregory S.; Wasser, Samuel K.; Park, Linda K.

2016-01-01

Estimating diet composition is important for understanding interactions between predators and prey and thus illuminating ecosystem function. The diet of many species, however, is difficult to observe directly. Genetic analysis of fecal material collected in the field is therefore a useful tool for gaining insight into wild animal diets. In this study, we used high-throughput DNA sequencing to quantitatively estimate the diet composition of an endangered population of wild killer whales (Orcinus orca) in their summer range in the Salish Sea. We combined 175 fecal samples collected between May and September from five years between 2006 and 2011 into 13 sample groups. Two known DNA composition control groups were also created. Each group was sequenced at a ~330bp segment of the 16s gene in the mitochondrial genome using an Illumina MiSeq sequencing system. After several quality controls steps, 4,987,107 individual sequences were aligned to a custom sequence database containing 19 potential fish prey species and the most likely species of each fecal-derived sequence was determined. Based on these alignments, salmonids made up >98.6% of the total sequences and thus of the inferred diet. Of the six salmonid species, Chinook salmon made up 79.5% of the sequences, followed by coho salmon (15%). Over all years, a clear pattern emerged with Chinook salmon dominating the estimated diet early in the summer, and coho salmon contributing an average of >40% of the diet in late summer. Sockeye salmon appeared to be occasionally important, at >18% in some sample groups. Non-salmonids were rarely observed. Our results are consistent with earlier results based on surface prey remains, and confirm the importance of Chinook salmon in this population’s summer diet. PMID:26735849

Estimation of a Killer Whale (Orcinus orca) Population's Diet Using Sequencing Analysis of DNA from Feces.

PubMed

Ford, Michael J; Hempelmann, Jennifer; Hanson, M Bradley; Ayres, Katherine L; Baird, Robin W; Emmons, Candice K; Lundin, Jessica I; Schorr, Gregory S; Wasser, Samuel K; Park, Linda K

2016-01-01

Estimating diet composition is important for understanding interactions between predators and prey and thus illuminating ecosystem function. The diet of many species, however, is difficult to observe directly. Genetic analysis of fecal material collected in the field is therefore a useful tool for gaining insight into wild animal diets. In this study, we used high-throughput DNA sequencing to quantitatively estimate the diet composition of an endangered population of wild killer whales (Orcinus orca) in their summer range in the Salish Sea. We combined 175 fecal samples collected between May and September from five years between 2006 and 2011 into 13 sample groups. Two known DNA composition control groups were also created. Each group was sequenced at a ~330bp segment of the 16s gene in the mitochondrial genome using an Illumina MiSeq sequencing system. After several quality controls steps, 4,987,107 individual sequences were aligned to a custom sequence database containing 19 potential fish prey species and the most likely species of each fecal-derived sequence was determined. Based on these alignments, salmonids made up >98.6% of the total sequences and thus of the inferred diet. Of the six salmonid species, Chinook salmon made up 79.5% of the sequences, followed by coho salmon (15%). Over all years, a clear pattern emerged with Chinook salmon dominating the estimated diet early in the summer, and coho salmon contributing an average of >40% of the diet in late summer. Sockeye salmon appeared to be occasionally important, at >18% in some sample groups. Non-salmonids were rarely observed. Our results are consistent with earlier results based on surface prey remains, and confirm the importance of Chinook salmon in this population's summer diet.

Detection of a divergent variant of grapevine virus F by next-generation sequencing.

PubMed

Molenaar, Nicholas; Burger, Johan T; Maree, Hans J

2015-08-01

The complete genome sequence of a South African isolate of grapevine virus F (GVF) is presented. It was first detected by metagenomic next-generation sequencing of field samples and validated through direct Sanger sequencing. The genome sequence of GVF isolate V5 consists of 7539 nucleotides and contains a poly(A) tail. It has a typical vitivirus genome arrangement that comprises five open reading frames (ORFs), which share only 88.96 % nucleotide sequence identity with the existing complete GVF genome sequence (JX105428).

Structure and specificity of the RNA-guided endonuclease Cas9 during DNA interrogation, target binding and cleavage

PubMed Central

Josephs, Eric A.; Kocak, D. Dewran; Fitzgibbon, Christopher J.; McMenemy, Joshua; Gersbach, Charles A.; Marszalek, Piotr E.

2015-01-01

CRISPR-associated endonuclease Cas9 cuts DNA at variable target sites designated by a Cas9-bound RNA molecule. Cas9's ability to be directed by single ‘guide RNA’ molecules to target nearly any sequence has been recently exploited for a number of emerging biological and medical applications. Therefore, understanding the nature of Cas9's off-target activity is of paramount importance for its practical use. Using atomic force microscopy (AFM), we directly resolve individual Cas9 and nuclease-inactive dCas9 proteins as they bind along engineered DNA substrates. High-resolution imaging allows us to determine their relative propensities to bind with different guide RNA variants to targeted or off-target sequences. Mapping the structural properties of Cas9 and dCas9 to their respective binding sites reveals a progressive conformational transformation at DNA sites with increasing sequence similarity to its target. With kinetic Monte Carlo (KMC) simulations, these results provide evidence of a ‘conformational gating’ mechanism driven by the interactions between the guide RNA and the 14th–17th nucleotide region of the targeted DNA, the stabilities of which we find correlate significantly with reported off-target cleavage rates. KMC simulations also reveal potential methodologies to engineer guide RNA sequences with improved specificity by considering the invasion of guide RNAs into targeted DNA duplex. PMID:26384421

Targeted gene panel sequencing in children with very early onset inflammatory bowel disease--evaluation and prospective analysis.

PubMed

Kammermeier, Jochen; Drury, Suzanne; James, Chela T; Dziubak, Robert; Ocaka, Louise; Elawad, Mamoun; Beales, Philip; Lench, Nicholas; Uhlig, Holm H; Bacchelli, Chiara; Shah, Neil

2014-11-01

Multiple monogenetic conditions with partially overlapping phenotypes can present with inflammatory bowel disease (IBD)-like intestinal inflammation. With novel genotype-specific therapies emerging, establishing a molecular diagnosis is becoming increasingly important. We have introduced targeted next-generation sequencing (NGS) technology as a prospective screening tool in children with very early onset IBD (VEOIBD). We evaluated the coverage of 40 VEOIBD genes in two separate cohorts undergoing targeted gene panel sequencing (TGPS) (n=25) and whole exome sequencing (WES) (n=20). TGPS revealed causative mutations in four genes (IL10RA, EPCAM, TTC37 and SKIV2L) discovered unexpected phenotypes and directly influenced clinical decision making by supporting as well as avoiding haematopoietic stem cell transplantation. TGPS resulted in significantly higher median coverage when compared with WES, fewer coverage deficiencies and improved variant detection across established VEOIBD genes. Excluding or confirming known VEOIBD genotypes should be considered early in the disease course in all cases of therapy-refractory VEOIBD, as it can have a direct impact on patient management. To combine both described NGS technologies would compensate for the limitations of WES for disease-specific application while offering the opportunity for novel gene discovery in the research setting. 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 expression of APC-1B but not APC-1A by the deletion of promoter 1B is responsible for familial adenomatous polyposis.

PubMed

Yamaguchi, Kiyoshi; Nagayama, Satoshi; Shimizu, Eigo; Komura, Mitsuhiro; Yamaguchi, Rui; Shibuya, Tetsuo; Arai, Masami; Hatakeyama, Seira; Ikenoue, Tsuneo; Ueno, Masashi; Miyano, Satoru; Imoto, Seiya; Furukawa, Yoichi

2016-05-24

Germline mutations in the tumor suppressor gene APC are associated with familial adenomatous polyposis (FAP). Here we applied whole-genome sequencing (WGS) to the DNA of a sporadic FAP patient in which we did not find any pathological APC mutations by direct sequencing. WGS identified a promoter deletion of approximately 10 kb encompassing promoter 1B and exon1B of APC. Additional allele-specific expression analysis by deep cDNA sequencing revealed that the deletion reduced the expression of the mutated APC allele to as low as 11.2% in the total APC transcripts, suggesting that the residual mutant transcripts were driven by other promoter(s). Furthermore, cap analysis of gene expression (CAGE) demonstrated that the deleted promoter 1B region is responsible for the great majority of APC transcription in many tissues except the brain. The deletion decreased the transcripts of APC-1B to 39-45% in the patient compared to the healthy controls, but it did not decrease those of APC-1A. Different deletions including promoter 1B have been reported in FAP patients. Taken together, our results strengthen the evidence that analysis of structural variations in promoter 1B should be considered for the FAP patients whose pathological mutations are not identified by conventional direct sequencing.

Torque measurements reveal sequence-specific cooperative transitions in supercoiled DNA

PubMed Central

Oberstrass, Florian C.; Fernandes, Louis E.; Bryant, Zev

2012-01-01

B-DNA becomes unstable under superhelical stress and is able to adopt a wide range of alternative conformations including strand-separated DNA and Z-DNA. Localized sequence-dependent structural transitions are important for the regulation of biological processes such as DNA replication and transcription. To directly probe the effect of sequence on structural transitions driven by torque, we have measured the torsional response of a panel of DNA sequences using single molecule assays that employ nanosphere rotational probes to achieve high torque resolution. The responses of Z-forming d(pGpC)n sequences match our predictions based on a theoretical treatment of cooperative transitions in helical polymers. “Bubble” templates containing 50–100 bp mismatch regions show cooperative structural transitions similar to B-DNA, although less torque is required to disrupt strand–strand interactions. Our mechanical measurements, including direct characterization of the torsional rigidity of strand-separated DNA, establish a framework for quantitative predictions of the complex torsional response of arbitrary sequences in their biological context. PMID:22474350

Probe-Directed Degradation (PDD) for Flexible Removal of Unwanted cDNA Sequences from RNA-Seq Libraries.

PubMed

Archer, Stuart K; Shirokikh, Nikolay E; Preiss, Thomas

2015-04-01

Most applications for RNA-seq require the depletion of abundant transcripts to gain greater coverage of the underlying transcriptome. The sequences to be targeted for depletion depend on application and species and in many cases may not be supported by commercial depletion kits. This unit describes a method for generating RNA-seq libraries that incorporates probe-directed degradation (PDD), which can deplete any unwanted sequence set, with the low-bias split-adapter method of library generation (although many other library generation methods are in principle compatible). The overall strategy is suitable for applications requiring customized sequence depletion or where faithful representation of fragment ends and lack of sequence bias is paramount. We provide guidelines to rapidly design specific probes against the target sequence, and a detailed protocol for library generation using the split-adapter method including several strategies for streamlining the technique and reducing adapter dimer content. Copyright © 2015 John Wiley & Sons, Inc.

Estimation of Airline Benefits from Avionics Upgrade under Preferential Merge Re-sequence Scheduling

NASA Technical Reports Server (NTRS)

Kotegawa, Tatsuya; Cayabyab, Charlene Anne; Almog, Noam

2013-01-01

Modernization of the airline fleet avionics is essential to fully enable future technologies and procedures for increasing national airspace system capacity. However in the current national airspace system, system-wide benefits gained by avionics upgrade are not fully directed to aircraft/airlines that upgrade, resulting in slow fleet modernization rate. Preferential merge re-sequence scheduling is a best-equipped-best-served concept designed to incentivize avionics upgrade among airlines by allowing aircraft with new avionics (high-equipped) to be re-sequenced ahead of aircraft without the upgrades (low-equipped) at enroute merge waypoints. The goal of this study is to investigate the potential benefits gained or lost by airlines under a high or low-equipped fleet scenario if preferential merge resequence scheduling is implemented.

Next-Generation Sequencing of Coccidioides immitis Isolated during Cluster Investigation

PubMed Central

Engelthaler, David M.; Chiller, Tom; Schupp, James A.; Colvin, Joshua; Beckstrom-Sternberg, Stephen M.; Driebe, Elizabeth M.; Moses, Tracy; Tembe, Waibhav; Sinari, Shripad; Beckstrom-Sternberg, James S.; Christoforides, Alexis; Pearson, John V.; Carpten, John; Keim, Paul; Peterson, Ashley; Terashita, Dawn

2011-01-01

Next-generation sequencing enables use of whole-genome sequence typing (WGST) as a viable and discriminatory tool for genotyping and molecular epidemiologic analysis. We used WGST to confirm the linkage of a cluster of Coccidioides immitis isolates from 3 patients who received organ transplants from a single donor who later had positive test results for coccidioidomycosis. Isolates from the 3 patients were nearly genetically identical (a total of 3 single-nucleotide polymorphisms identified among them), thereby demonstrating direct descent of the 3 isolates from an original isolate. We used WGST to demonstrate the genotypic relatedness of C. immitis isolates that were also epidemiologically linked. Thus, WGST offers unique benefits to public health for investigation of clusters considered to be linked to a single source. PMID:21291593

Working memory in children assessed with serial chaining and Simon tasks.

PubMed

Parrish, Audrey E; Perdue, Bonnie M; Kelly, Andrew J; Beran, Michael J

2018-06-06

In the serial chaining task, participants are required to produce a sequence of responses to stimuli in the correct order, and sometimes must determine the sequence at trial outset if stimuli are masked after the first response is made. Similarly, the Simon memory span task presents a participant with a sequence of colors, and the participant must recreate the sequence after the full series is shown. In efforts to directly link the comparative literature on sequential planning behavior and working memory span with the developmental literature, we presented preschool children with the serial chaining task using masked Arabic numerals (N = 44) and the Simon memory span task (N = 65). Older children outperformed younger children in each task, sequencing a longer string of numbers in the serial chaining task and remembering a greater number of items in the Simon task. Controlling for the role of age, there was a significant positive relationship between task scores. These results highlight the emergence of working memory skills that might underlie planning capacities in children using a task developed for nonhuman animals, and the results indicate that improvement in general executive functions could be measured using either or both of these tasks among human children and nonhuman species. Copyright © 2018 Elsevier B.V. All rights reserved.

A computer aided thermodynamic approach for predicting the formation of Z-DNA in naturally occurring sequences

NASA Technical Reports Server (NTRS)

Ho, P. S.; Ellison, M. J.; Quigley, G. J.; Rich, A.

1986-01-01

The ease with which a particular DNA segment adopts the left-handed Z-conformation depends largely on the sequence and on the degree of negative supercoiling to which it is subjected. We describe a computer program (Z-hunt) that is designed to search long sequences of naturally occurring DNA and retrieve those nucleotide combinations of up to 24 bp in length which show a strong propensity for Z-DNA formation. Incorporated into Z-hunt is a statistical mechanical model based on empirically determined energetic parameters for the B to Z transition accumulated to date. The Z-forming potential of a sequence is assessed by ranking its behavior as a function of negative superhelicity relative to the behavior of similar sized randomly generated nucleotide sequences assembled from over 80,000 combinations. The program makes it possible to compare directly the Z-forming potential of sequences with different base compositions and different sequence lengths. Using Z-hunt, we have analyzed the DNA sequences of the bacteriophage phi X174, plasmid pBR322, the animal virus SV40 and the replicative form of the eukaryotic adenovirus-2. The results are compared with those previously obtained by others from experiments designed to locate Z-DNA forming regions in these sequences using probes which show specificity for the left-handed DNA conformation.

A comprehensive evaluation of assembly scaffolding tools

PubMed Central

2014-01-01

Background Genome assembly is typically a two-stage process: contig assembly followed by the use of paired sequencing reads to join contigs into scaffolds. Scaffolds are usually the focus of reported assembly statistics; longer scaffolds greatly facilitate the use of genome sequences in downstream analyses, and it is appealing to present larger numbers as metrics of assembly performance. However, scaffolds are highly prone to errors, especially when generated using short reads, which can directly result in inflated assembly statistics. Results Here we provide the first independent evaluation of scaffolding tools for second-generation sequencing data. We find large variations in the quality of results depending on the tool and dataset used. Even extremely simple test cases of perfect input, constructed to elucidate the behaviour of each algorithm, produced some surprising results. We further dissect the performance of the scaffolders using real and simulated sequencing data derived from the genomes of Staphylococcus aureus, Rhodobacter sphaeroides, Plasmodium falciparum and Homo sapiens. The results from simulated data are of high quality, with several of the tools producing perfect output. However, at least 10% of joins remains unidentified when using real data. Conclusions The scaffolders vary in their usability, speed and number of correct and missed joins made between contigs. Results from real data highlight opportunities for further improvements of the tools. Overall, SGA, SOPRA and SSPACE generally outperform the other tools on our datasets. However, the quality of the results is highly dependent on the read mapper and genome complexity. PMID:24581555

Biosensors for DNA sequence detection

NASA Technical Reports Server (NTRS)

Vercoutere, Wenonah; Akeson, Mark

2002-01-01

DNA biosensors are being developed as alternatives to conventional DNA microarrays. These devices couple signal transduction directly to sequence recognition. Some of the most sensitive and functional technologies use fibre optics or electrochemical sensors in combination with DNA hybridization. In a shift from sequence recognition by hybridization, two emerging single-molecule techniques read sequence composition using zero-mode waveguides or electrical impedance in nanoscale pores.

Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples

PubMed Central

Quick, Josh; Grubaugh, Nathan D; Pullan, Steven T; Claro, Ingra M; Smith, Andrew D; Gangavarapu, Karthik; Oliveira, Glenn; Robles-Sikisaka, Refugio; Rogers, Thomas F; Beutler, Nathan A; Burton, Dennis R; Lewis-Ximenez, Lia Laura; de Jesus, Jaqueline Goes; Giovanetti, Marta; Hill, Sarah; Black, Allison; Bedford, Trevor; Carroll, Miles W; Nunes, Marcio; Alcantara, Luiz Carlos; Sabino, Ester C; Baylis, Sally A; Faria, Nuno; Loose, Matthew; Simpson, Jared T; Pybus, Oliver G; Andersen, Kristian G; Loman, Nicholas J

2018-01-01

Genome sequencing has become a powerful tool for studying emerging infectious diseases; however, genome sequencing directly from clinical samples without isolation remains challenging for viruses such as Zika, where metagenomic sequencing methods may generate insufficient numbers of viral reads. Here we present a protocol for generating coding-sequence complete genomes comprising an online primer design tool, a novel multiplex PCR enrichment protocol, optimised library preparation methods for the portable MinION sequencer (Oxford Nanopore Technologies) and the Illumina range of instruments, and a bioinformatics pipeline for generating consensus sequences. The MinION protocol does not require an internet connection for analysis, making it suitable for field applications with limited connectivity. Our method relies on multiplex PCR for targeted enrichment of viral genomes from samples containing as few as 50 genome copies per reaction. Viral consensus sequences can be achieved starting with clinical samples in 1-2 days following a simple laboratory workflow. This method has been successfully used by several groups studying Zika virus evolution and is facilitating an understanding of the spread of the virus in the Americas. PMID:28538739

Direct Detection and Sequencing of Damaged DNA Bases

PubMed Central

2011-01-01

Products of various forms of DNA damage have been implicated in a variety of important biological processes, such as aging, neurodegenerative diseases, and cancer. Therefore, there exists great interest to develop methods for interrogating damaged DNA in the context of sequencing. Here, we demonstrate that single-molecule, real-time (SMRT®) DNA sequencing can directly detect damaged DNA bases in the DNA template - as a by-product of the sequencing method - through an analysis of the DNA polymerase kinetics that are altered by the presence of a modified base. We demonstrate the sequencing of several DNA templates containing products of DNA damage, including 8-oxoguanine, 8-oxoadenine, O6-methylguanine, 1-methyladenine, O4-methylthymine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, or thymine dimers, and show that these base modifications can be readily detected with single-modification resolution and DNA strand specificity. We characterize the distinct kinetic signatures generated by these DNA base modifications. PMID:22185597

Direct detection and sequencing of damaged DNA bases.

PubMed

Clark, Tyson A; Spittle, Kristi E; Turner, Stephen W; Korlach, Jonas

2011-12-20

Products of various forms of DNA damage have been implicated in a variety of important biological processes, such as aging, neurodegenerative diseases, and cancer. Therefore, there exists great interest to develop methods for interrogating damaged DNA in the context of sequencing. Here, we demonstrate that single-molecule, real-time (SMRT®) DNA sequencing can directly detect damaged DNA bases in the DNA template - as a by-product of the sequencing method - through an analysis of the DNA polymerase kinetics that are altered by the presence of a modified base. We demonstrate the sequencing of several DNA templates containing products of DNA damage, including 8-oxoguanine, 8-oxoadenine, O6-methylguanine, 1-methyladenine, O4-methylthymine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, or thymine dimers, and show that these base modifications can be readily detected with single-modification resolution and DNA strand specificity. We characterize the distinct kinetic signatures generated by these DNA base modifications.

The Steens Mountain (Oregon) geomagnetic polarity transition: 1. Directional history, duration of episodes, and rock magnetism

USGS Publications Warehouse

Mankinen, Edward A.; Prevot, M.; Gromme, C. Sherman; Coe, Robert S.

1985-01-01

The thick sequence of Miocene lava flows exposed on Steens Mountain in southeastern Oregon is well known for containing a detailed record of a reversed‐to‐normal geomagnetic polarity transition. Paleomagnetic samples were obtained from the sequence for a combined study of the directional and intensity variations recorded; the paleointensity study is reported in a companion paper. This effort has resulted in the first detailed history of total geomagnetic field behavior during a reversal of polarity. A comparison of the directional variation history of the reversed and normal polarity intervals on either side of the transition with the Holocene record has allowed an estimate of the duration of these periods to be made. These time estimates were then used to calculate accumulation rates for the volcanic sequence and thereby provide a means for estimating time periods within the transition itself. The polarity transition was found to consist of two phases, each with quite different characteristics. At the onset of the first phase, a one‐third decrease in magnetic field intensity may have preceded the first intermediate field directions by about 600 years. Changes in field direction were confined near the local north‐south vertical plane when the actual reversal in direction occurred and normal polarity directions may have been attained within 550±150 years. The end of the first phase of the transition was marked by a brief (possibly 100–300 years) period with normal polarity and a pretransitional intensity which suggests a quasi‐normal dipole field structure existed during this interval. The second phase of the transition was characterized by a return to very low field intensities with the changes in direction describing a long counterclockwise loop in contrast to the earlier narrowly constrained changes. This second phase lasted 2900±300 years, and both normal directions and intensities were recovered at the same time. Both directional and intensity data document very erratic geomagnetic field behavior during the polarity transition. Changes in magnetic field direction were variable and occurred either (1) in a regular, progressive manner, (2) with sudden, extremely rapid angular changes (58°±21°/year), or (3) with little or no movement for periods of the order of 600±200 years. Changes in magnetic intensity occurred in a like manner and were sometimes correlated with changes in direction, but during other periods both directional and intensity changes occurred independently. Directional changes following the polarity transition occurred in a seemingly normal manner, although intensity fluctuations attest to some instability of the newly reestablished dipole.

Differences in selection drive olfactory receptor genes in different directions in dogs and wolf.

PubMed

Chen, Rui; Irwin, David M; Zhang, Ya-Ping

2012-11-01

The olfactory receptor (OR) gene family is the largest gene family found in mammalian genomes. It is known to evolve through a birth-and-death process. Here, we characterized the sequences of 16 segregating OR pseudogenes in the samples of the wolf and the Chinese village dog (CVD) and compared them with the sequences from dogs of different breeds. Our results show that the segregating OR pseudogenes in breed dogs are under strong purifying selection, while evolving neutrally in the CVD, and show a more complicated pattern in the wolf. In the wolf, we found a trend to remove deleterious polymorphisms and accumulate nondeleterious polymorphisms. On the basis of protein structure of the ORs, we found that the distribution of different types of polymorphisms (synonymous, nonsynonymous, tolerated, and untolerated) varied greatly between the wolf and the breed dogs. In summary, our results suggest that different forms of selection have acted on the segregating OR pseudogenes in the CVD since domestication, breed dogs after breed formation, and ancestral wolf population, which has driven the evolution of these genes in different directions.

T85C polymorphisms of the dihydropyrimidine dehydrogenase gene detected in gastric cancer tissues by high-resolution melting curve analysis.

PubMed

Fang, Weijia; Xu, Nong; Jin, Dazhi; Chen, Yu; Chen, Xiaogang; Zheng, Yi; Shen, Hong; Yuan, Ying; Zheng, Shusen

2012-01-01

Dihydropyrimidine dehydrogenase is a key enzyme acting on the metabolic pathway of medications for gastric cancer. High-resolution melting curve technology, which was developed recently, can distinguish the wild-type dihydropyrimidine dehydrogenase gene from multiple polymorphisms by fluorescent quantitative polymerase chain reaction products in a direct and effective manner. T85C polymorphisms of dihydropyrimidine dehydrogenase in the peripheral blood of 112 Chinese gastric cancer patients were detected by real-time polymerase chain reaction combined with high-resolution melting curve technology. Primer design, along with the reaction system and conditions, was optimized based on the GenBank sequence. Seventy nine cases of wild-type (TT, [70.5%]), 29 cases of heterozygous (TC, [25.9%]), and 4 cases of homozygous mutant (CC, [3.6%]) were observed. The result was completely consistent with the results of the sequencing. Real-time polymerase chain reaction combined with high-resolution melting curve technology is a rapid, simple, reliable, direct-viewing, and convenient method for the detection and screening of polymorphisms.

BiDiBlast: comparative genomics pipeline for the PC.

PubMed

de Almeida, João M G C F

2010-06-01

Bi-directional BLAST is a simple approach to detect, annotate, and analyze candidate orthologous or paralogous sequences in a single go. This procedure is usually confined to the realm of customized Perl scripts, usually tuned for UNIX-like environments. Porting those scripts to other operating systems involves refactoring them, and also the installation of the Perl programming environment with the required libraries. To overcome these limitations, a data pipeline was implemented in Java. This application submits two batches of sequences to local versions of the NCBI BLAST tool, manages result lists, and refines both bi-directional and simple hits. GO Slim terms are attached to hits, several statistics are derived, and molecular evolution rates are estimated through PAML. The results are written to a set of delimited text tables intended for further analysis. The provided graphic user interface allows a friendly interaction with this application, which is documented and available to download at http://moodle.fct.unl.pt/course/view.php?id=2079 or https://sourceforge.net/projects/bidiblast/ under the GNU GPL license. Copyright 2010 Beijing Genomics Institute. Published by Elsevier Ltd. All rights reserved.

Unbiased Strain-Typing of Arbovirus Directly from Mosquitoes Using Nanopore Sequencing: A Field-forward Biosurveillance Protocol.

PubMed

Russell, Joseph A; Campos, Brittany; Stone, Jennifer; Blosser, Erik M; Burkett-Cadena, Nathan; Jacobs, Jonathan L

2018-04-03

The future of infectious disease surveillance and outbreak response is trending towards smaller hand-held solutions for point-of-need pathogen detection. Here, samples of Culex cedecei mosquitoes collected in Southern Florida, USA were tested for Venezuelan Equine Encephalitis Virus (VEEV), a previously-weaponized arthropod-borne RNA-virus capable of causing acute and fatal encephalitis in animal and human hosts. A single 20-mosquito pool tested positive for VEEV by quantitative reverse transcription polymerase chain reaction (RT-qPCR) on the Biomeme two3. The virus-positive sample was subjected to unbiased metatranscriptome sequencing on the Oxford Nanopore MinION and shown to contain Everglades Virus (EVEV), an alphavirus in the VEEV serocomplex. Our results demonstrate, for the first time, the use of unbiased sequence-based detection and subtyping of a high-consequence biothreat pathogen directly from an environmental sample using field-forward protocols. The development and validation of methods designed for field-based diagnostic metagenomics and pathogen discovery, such as those suitable for use in mobile "pocket laboratories", will address a growing demand for public health teams to carry out their mission where it is most urgent: at the point-of-need.

Precision of working memory for visual motion sequences and transparent motion surfaces

PubMed Central

Zokaei, Nahid; Gorgoraptis, Nikos; Bahrami, Bahador; Bays, Paul M; Husain, Masud

2012-01-01

Recent studies investigating working memory for location, colour and orientation support a dynamic resource model. We examined whether this might also apply to motion, using random dot kinematograms (RDKs) presented sequentially or simultaneously. Mean precision for motion direction declined as sequence length increased, with precision being lower for earlier RDKs. Two alternative models of working memory were compared specifically to distinguish between the contributions of different sources of error that corrupt memory (Zhang & Luck (2008) vs. Bays et al (2009)). The latter provided a significantly better fit for the data, revealing that decrease in memory precision for earlier items is explained by an increase in interference from other items in a sequence, rather than random guessing or a temporal decay of information. Misbinding feature attributes is an important source of error in working memory. Precision of memory for motion direction decreased when two RDKs were presented simultaneously as transparent surfaces, compared to sequential RDKs. However, precision was enhanced when one motion surface was prioritized, demonstrating that selective attention can improve recall precision. These results are consistent with a resource model that can be used as a general conceptual framework for understanding working memory across a range of visual features. PMID:22135378

Identification of distant drug off-targets by direct superposition of binding pocket surfaces.

PubMed

Schumann, Marcel; Armen, Roger S

2013-01-01

Correctly predicting off-targets for a given molecular structure, which would have the ability to bind a large range of ligands, is both particularly difficult and important if they share no significant sequence or fold similarity with the respective molecular target ("distant off-targets"). A novel approach for identification of off-targets by direct superposition of protein binding pocket surfaces is presented and applied to a set of well-studied and highly relevant drug targets, including representative kinases and nuclear hormone receptors. The entire Protein Data Bank is searched for similar binding pockets and convincing distant off-target candidates were identified that share no significant sequence or fold similarity with the respective target structure. These putative target off-target pairs are further supported by the existence of compounds that bind strongly to both with high topological similarity, and in some cases, literature examples of individual compounds that bind to both. Also, our results clearly show that it is possible for binding pockets to exhibit a striking surface similarity, while the respective off-target shares neither significant sequence nor significant fold similarity with the respective molecular target ("distant off-target").

Identification of Distant Drug Off-Targets by Direct Superposition of Binding Pocket Surfaces

PubMed Central

Schumann, Marcel; Armen, Roger S.

2013-01-01

Correctly predicting off-targets for a given molecular structure, which would have the ability to bind a large range of ligands, is both particularly difficult and important if they share no significant sequence or fold similarity with the respective molecular target (“distant off-targets”). A novel approach for identification of off-targets by direct superposition of protein binding pocket surfaces is presented and applied to a set of well-studied and highly relevant drug targets, including representative kinases and nuclear hormone receptors. The entire Protein Data Bank is searched for similar binding pockets and convincing distant off-target candidates were identified that share no significant sequence or fold similarity with the respective target structure. These putative target off-target pairs are further supported by the existence of compounds that bind strongly to both with high topological similarity, and in some cases, literature examples of individual compounds that bind to both. Also, our results clearly show that it is possible for binding pockets to exhibit a striking surface similarity, while the respective off-target shares neither significant sequence nor significant fold similarity with the respective molecular target (“distant off-target”). PMID:24391782

Conifer genomics and adaptation: at the crossroads of genetic diversity and genome function.

PubMed

Prunier, Julien; Verta, Jukka-Pekka; MacKay, John J

2016-01-01

Conifers have been understudied at the genomic level despite their worldwide ecological and economic importance but the situation is rapidly changing with the development of next generation sequencing (NGS) technologies. With NGS, genomics research has simultaneously gained in speed, magnitude and scope. In just a few years, genomes of 20-24 gigabases have been sequenced for several conifers, with several others expected in the near future. Biological insights have resulted from recent sequencing initiatives as well as genetic mapping, gene expression profiling and gene discovery research over nearly two decades. We review the knowledge arising from conifer genomics research emphasizing genome evolution and the genomic basis of adaptation, and outline emerging questions and knowledge gaps. We discuss future directions in three areas with potential inputs from NGS technologies: the evolutionary impacts of adaptation in conifers based on the adaptation-by-speciation model; the contributions of genetic variability of gene expression in adaptation; and the development of a broader understanding of genetic diversity and its impacts on genome function. These research directions promise to sustain research aimed at addressing the emerging challenges of adaptation that face conifer trees. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

A new rapid methodological strategy to assess BRCA mutational status.

PubMed

Vuttariello, Emilia; Borra, Marco; Calise, Celeste; Mauriello, Elvira; Greggi, Stefano; Vecchione, Aldo; Biffali, Elio; Chiappetta, Gennaro

2013-07-01

Hereditary cancers account for approximately 10 % of breast and ovarian cancers. Mutations of the BRCA1 and BRCA2 genes, encoding two proteins involved in DNA repair, underlie most cases of such hereditary cancers. Women with BRCA mutations develop breast cancer in 50-80 % of cases and ovarian cancer in 10-40 % of cases. Assessing BRCA mutational status is needed to direct the clinical management of women with predisposition to these hereditary cancers. However, BRCA screening constitutes a bottleneck in terms of costs and time to deliver results. We developed a PCR-based assay using 73 primer pairs covering the entire coding regions of BRCA1 and BRCA2. PCR primers, containing at the 5' end the universal M13 primer sequences, were pre-spotted in 96-well plates. Following PCR, direct sequencing was performed using M13 primers, allowing to standardize the conditions. PCR amplification and sequencing were successful for each amplicon. We tested and validated the assay on 10 known gDNAs from patients with Hereditary breast and ovarian cancer (HBOC). Our strategy is a promising time and cost-effective method to detect BRCA mutations in the clinical setting, which is essential to formulate a personalized therapy for patients with HBOC.

Automatic segmentation of low-visibility moving objects through energy analyis of the local 3D spectrum

NASA Astrophysics Data System (ADS)

Nestares, Oscar; Miravet, Carlos; Santamaria, Javier; Fonolla Navarro, Rafael

1999-05-01

Automatic object segmentation in highly noisy image sequences, composed by a translating object over a background having a different motion, is achieved through joint motion-texture analysis. Local motion and/or texture is characterized by the energy of the local spatio-temporal spectrum, as different textures undergoing different translational motions display distinctive features in their 3D (x,y,t) spectra. Measurements of local spectrum energy are obtained using a bank of directional 3rd order Gaussian derivative filters in a multiresolution pyramid in space- time (10 directions, 3 resolution levels). These 30 energy measurements form a feature vector describing texture-motion for every pixel in the sequence. To improve discrimination capability and reduce computational cost, we automatically select those 4 features (channels) that best discriminate object from background, under the assumptions that the object is smaller than the background and has a different velocity or texture. In this way we reject features irrelevant or dominated by noise, that could yield wrong segmentation results. This method has been successfully applied to sequences with extremely low visibility and for objects that are even invisible for the eye in absence of motion.

Exome Sequencing Is an Efficient Tool for Variant Late-Infantile Neuronal Ceroid Lipofuscinosis Molecular Diagnosis

PubMed Central

Ortega-Recalde, Oscar; Nallathambi, Jeyabalan; Anandula, Venkata Ramana; Renukaradhya, Umashankar; Laissue, Paul

2014-01-01

The neuronal ceroid-lipofuscinoses (NCL) is a group of neurodegenerative disorders characterized by epilepsy, visual failure, progressive mental and motor deterioration, myoclonus, dementia and reduced life expectancy. Classically, NCL-affected individuals have been classified into six categories, which have been mainly defined regarding the clinical onset of symptoms. However, some patients cannot be easily included in a specific group because of significant variation in the age of onset and disease progression. Molecular genetics has emerged in recent years as a useful tool for enhancing NCL subtype classification. Fourteen NCL genetic forms (CLN1 to CLN14) have been described to date. The variant late-infantile form of the disease has been linked to CLN5, CLN6, CLN7 (MFSD8) and CLN8 mutations. Despite advances in the diagnosis of neurodegenerative disorders mutations in these genes may cause similar phenotypes, which rends difficult accurate candidate gene selection for direct sequencing. Three siblings who were affected by variant late-infantile NCL are reported in the present study. We used whole-exome sequencing, direct sequencing and in silico approaches to identify the molecular basis of the disease. We identified the novel c.1219T>C (p.Trp407Arg) and c.1361T>C (p.Met454Thr) MFSD8 pathogenic mutations. Our results highlighted next generation sequencing as a novel and powerful methodological approach for the rapid determination of the molecular diagnosis of NCL. They also provide information regarding the phenotypic and molecular spectrum of CLN7 disease. PMID:25333361

Multiple sequence alignment using multi-objective based bacterial foraging optimization algorithm.

PubMed

Rani, R Ranjani; Ramyachitra, D

2016-12-01

Multiple sequence alignment (MSA) is a widespread approach in computational biology and bioinformatics. MSA deals with how the sequences of nucleotides and amino acids are sequenced with possible alignment and minimum number of gaps between them, which directs to the functional, evolutionary and structural relationships among the sequences. Still the computation of MSA is a challenging task to provide an efficient accuracy and statistically significant results of alignments. In this work, the Bacterial Foraging Optimization Algorithm was employed to align the biological sequences which resulted in a non-dominated optimal solution. It employs Multi-objective, such as: Maximization of Similarity, Non-gap percentage, Conserved blocks and Minimization of gap penalty. BAliBASE 3.0 benchmark database was utilized to examine the proposed algorithm against other methods In this paper, two algorithms have been proposed: Hybrid Genetic Algorithm with Artificial Bee Colony (GA-ABC) and Bacterial Foraging Optimization Algorithm. It was found that Hybrid Genetic Algorithm with Artificial Bee Colony performed better than the existing optimization algorithms. But still the conserved blocks were not obtained using GA-ABC. Then BFO was used for the alignment and the conserved blocks were obtained. The proposed Multi-Objective Bacterial Foraging Optimization Algorithm (MO-BFO) was compared with widely used MSA methods Clustal Omega, Kalign, MUSCLE, MAFFT, Genetic Algorithm (GA), Ant Colony Optimization (ACO), Artificial Bee Colony (ABC), Particle Swarm Optimization (PSO) and Hybrid Genetic Algorithm with Artificial Bee Colony (GA-ABC). The final results show that the proposed MO-BFO algorithm yields better alignment than most widely used methods. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

PubMed Central

Currin, Andrew; Swainston, Neil; Day, Philip J.

2015-01-01

The amino acid sequence of a protein affects both its structure and its function. Thus, the ability to modify the sequence, and hence the structure and activity, of individual proteins in a systematic way, opens up many opportunities, both scientifically and (as we focus on here) for exploitation in biocatalysis. Modern methods of synthetic biology, whereby increasingly large sequences of DNA can be synthesised de novo, allow an unprecedented ability to engineer proteins with novel functions. However, the number of possible proteins is far too large to test individually, so we need means for navigating the ‘search space’ of possible protein sequences efficiently and reliably in order to find desirable activities and other properties. Enzymologists distinguish binding (K d) and catalytic (k cat) steps. In a similar way, judicious strategies have blended design (for binding, specificity and active site modelling) with the more empirical methods of classical directed evolution (DE) for improving k cat (where natural evolution rarely seeks the highest values), especially with regard to residues distant from the active site and where the functional linkages underpinning enzyme dynamics are both unknown and hard to predict. Epistasis (where the ‘best’ amino acid at one site depends on that or those at others) is a notable feature of directed evolution. The aim of this review is to highlight some of the approaches that are being developed to allow us to use directed evolution to improve enzyme properties, often dramatically. We note that directed evolution differs in a number of ways from natural evolution, including in particular the available mechanisms and the likely selection pressures. Thus, we stress the opportunities afforded by techniques that enable one to map sequence to (structure and) activity in silico, as an effective means of modelling and exploring protein landscapes. Because known landscapes may be assessed and reasoned about as a whole, simultaneously, this offers opportunities for protein improvement not readily available to natural evolution on rapid timescales. Intelligent landscape navigation, informed by sequence-activity relationships and coupled to the emerging methods of synthetic biology, offers scope for the development of novel biocatalysts that are both highly active and robust. PMID:25503938

Santorini eruption radiocarbon dated to 1627-1600 B.C.

PubMed

Friedrich, Walter L; Kromer, Bernd; Friedrich, Michael; Heinemeier, Jan; Pfeiffer, Tom; Talamo, Sahra

2006-04-28

Precise and direct dating of the Minoan eruption of Santorini (Thera) in Greece, a global Bronze Age time marker, has been made possible by the unique find of an olive tree, buried alive in life position by the tephra (pumice and ashes) on Santorini. We applied so-called radiocarbon wiggle-matching to a carbon-14 sequence of tree-ring segments to constrain the eruption date to the range 1627-1600 B.C. with 95.4% probability. Our result is in the range of previous, less precise, and less direct results of several scientific dating methods, but it is a century earlier than the date derived from traditional Egyptian chronologies.

Multiple nucleotide preferences determine cleavage-site recognition by the HIV-1 and M-MuLV RNases H.

PubMed

Schultz, Sharon J; Zhang, Miaohua; Champoux, James J

2010-03-19

The RNase H activity of reverse transcriptase is required during retroviral replication and represents a potential target in antiviral drug therapies. Sequence features flanking a cleavage site influence the three types of retroviral RNase H activity: internal, DNA 3'-end-directed, and RNA 5'-end-directed. Using the reverse transcriptases of HIV-1 (human immunodeficiency virus type 1) and Moloney murine leukemia virus (M-MuLV), we evaluated how individual base preferences at a cleavage site direct retroviral RNase H specificity. Strong test cleavage sites (designated as between nucleotide positions -1 and +1) for the HIV-1 and M-MuLV enzymes were introduced into model hybrid substrates designed to assay internal or DNA 3'-end-directed cleavage, and base substitutions were tested at specific nucleotide positions. For internal cleavage, positions +1, -2, -4, -5, -10, and -14 for HIV-1 and positions +1, -2, -6, and -7 for M-MuLV significantly affected RNase H cleavage efficiency, while positions -7 and -12 for HIV-1 and positions -4, -9, and -11 for M-MuLV had more modest effects. DNA 3'-end-directed cleavage was influenced substantially by positions +1, -2, -4, and -5 for HIV-1 and positions +1, -2, -6, and -7 for M-MuLV. Cleavage-site distance from the recessed end did not affect sequence preferences for M-MuLV reverse transcriptase. Based on the identified sequence preferences, a cleavage site recognized by both HIV-1 and M-MuLV enzymes was introduced into a sequence that was otherwise resistant to RNase H. The isolated RNase H domain of M-MuLV reverse transcriptase retained sequence preferences at positions +1 and -2 despite prolific cleavage in the absence of the polymerase domain. The sequence preferences of retroviral RNase H likely reflect structural features in the substrate that favor cleavage and represent a novel specificity determinant to consider in drug design. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Integrated in silico and biological validation of the blocking effect of Cot-1 DNA on Microarray-CGH.

PubMed

Kang, Seung-Hui; Park, Chan Hee; Jeung, Hei Cheul; Kim, Ki-Yeol; Rha, Sun Young; Chung, Hyun Cheol

2007-06-01

In array-CGH, various factors may act as variables influencing the result of experiments. Among them, Cot-1 DNA, which has been used as a repetitive sequence-blocking agent, may become an artifact-inducing factor in BAC array-CGH. To identify the effect of Cot-1 DNA on Microarray-CGH experiments, Cot-1 DNA was labeled directly and Microarray-CGH experiments were performed. The results confirmed that probes which hybridized more completely with Cot-1 DNA had a higher sequence similarity to the Alu element. Further, in the sex-mismatched Microarray-CGH experiments, the variation and intensity in the fluorescent signal were reduced in the high intensity probe group in which probes were better hybridized with Cot-1 DNA. Otherwise, those of the low intensity probe group showed no alterations regardless of Cot-1 DNA. These results confirmed by in silico methods that Cot-1 DNA could block repetitive sequences in gDNA and probes. In addition, it was confirmed biologically that the blocking effect of Cot-1 DNA could be presented via its repetitive sequences, especially Alu elements. Thus, in contrast to BAC-array CGH, the use of Cot-1 DNA is advantageous in controlling experimental variation in Microarray-CGH.

Compressing DNA sequence databases with coil.

PubMed

White, W Timothy J; Hendy, Michael D

2008-05-20

Publicly available DNA sequence databases such as GenBank are large, and are growing at an exponential rate. The sheer volume of data being dealt with presents serious storage and data communications problems. Currently, sequence data is usually kept in large "flat files," which are then compressed using standard Lempel-Ziv (gzip) compression - an approach which rarely achieves good compression ratios. While much research has been done on compressing individual DNA sequences, surprisingly little has focused on the compression of entire databases of such sequences. In this study we introduce the sequence database compression software coil. We have designed and implemented a portable software package, coil, for compressing and decompressing DNA sequence databases based on the idea of edit-tree coding. coil is geared towards achieving high compression ratios at the expense of execution time and memory usage during compression - the compression time represents a "one-off investment" whose cost is quickly amortised if the resulting compressed file is transmitted many times. Decompression requires little memory and is extremely fast. We demonstrate a 5% improvement in compression ratio over state-of-the-art general-purpose compression tools for a large GenBank database file containing Expressed Sequence Tag (EST) data. Finally, coil can efficiently encode incremental additions to a sequence database. coil presents a compelling alternative to conventional compression of flat files for the storage and distribution of DNA sequence databases having a narrow distribution of sequence lengths, such as EST data. Increasing compression levels for databases having a wide distribution of sequence lengths is a direction for future work.

GeneChip{sup {trademark}} screening assay for cystic fibrosis mutations

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

Cronn, M.T.; Miyada, C.G.; Fucini, R.V.

1994-09-01

GeneChip{sup {trademark}} assays are based on high density, carefully designed arrays of short oligonucleotide probes (13-16 bases) built directly on derivatized silica substrates. DNA target sequence analysis is achieved by hybridizing fluorescently labeled amplification products to these arrays. Fluorescent hybridization signals located within the probe array are translated into target sequence information using the known probe sequence at each array feature. The mutation screening assay for cystic fibrosis includes sets of oligonucleotide probes designed to detect numerous different mutations that have been described in 14 exons and one intron of the CFTR gene. Each mutation site is addressed by amore » sub-array of at least 40 probe sequences, half designed to detect the wild type gene sequence and half designed to detect the reported mutant sequence. Hybridization with homozygous mutant, homozygous wild type or heterozygous targets results in distinctive hybridization patterns within a sub-array, permitting specific discrimination of each mutation. The GeneChip probe arrays are very small (approximately 1 cm{sup 2}). There miniature size coupled with their high information content make GeneChip probe arrays a useful and practical means for providing CF mutation analysis in a clinical setting.« less

Predictive Place-Cell Sequences for Goal-Finding Emerge from Goal Memory and the Cognitive Map: A Computational Model

PubMed Central

Gönner, Lorenz; Vitay, Julien; Hamker, Fred H.

2017-01-01

Hippocampal place-cell sequences observed during awake immobility often represent previous experience, suggesting a role in memory processes. However, recent reports of goals being overrepresented in sequential activity suggest a role in short-term planning, although a detailed understanding of the origins of hippocampal sequential activity and of its functional role is still lacking. In particular, it is unknown which mechanism could support efficient planning by generating place-cell sequences biased toward known goal locations, in an adaptive and constructive fashion. To address these questions, we propose a model of spatial learning and sequence generation as interdependent processes, integrating cortical contextual coding, synaptic plasticity and neuromodulatory mechanisms into a map-based approach. Following goal learning, sequential activity emerges from continuous attractor network dynamics biased by goal memory inputs. We apply Bayesian decoding on the resulting spike trains, allowing a direct comparison with experimental data. Simulations show that this model (1) explains the generation of never-experienced sequence trajectories in familiar environments, without requiring virtual self-motion signals, (2) accounts for the bias in place-cell sequences toward goal locations, (3) highlights their utility in flexible route planning, and (4) provides specific testable predictions. PMID:29075187

Registration of retinal sequences from new video-ophthalmoscopic camera.

PubMed

Kolar, Radim; Tornow, Ralf P; Odstrcilik, Jan; Liberdova, Ivana

2016-05-20

Analysis of fast temporal changes on retinas has become an important part of diagnostic video-ophthalmology. It enables investigation of the hemodynamic processes in retinal tissue, e.g. blood-vessel diameter changes as a result of blood-pressure variation, spontaneous venous pulsation influenced by intracranial-intraocular pressure difference, blood-volume changes as a result of changes in light reflection from retinal tissue, and blood flow using laser speckle contrast imaging. For such applications, image registration of the recorded sequence must be performed. Here we use a new non-mydriatic video-ophthalmoscope for simple and fast acquisition of low SNR retinal sequences. We introduce a novel, two-step approach for fast image registration. The phase correlation in the first stage removes large eye movements. Lucas-Kanade tracking in the second stage removes small eye movements. We propose robust adaptive selection of the tracking points, which is the most important part of tracking-based approaches. We also describe a method for quantitative evaluation of the registration results, based on vascular tree intensity profiles. The achieved registration error evaluated on 23 sequences (5840 frames) is 0.78 ± 0.67 pixels inside the optic disc and 1.39 ± 0.63 pixels outside the optic disc. We compared the results with the commonly used approaches based on Lucas-Kanade tracking and scale-invariant feature transform, which achieved worse results. The proposed method can efficiently correct particular frames of retinal sequences for shift and rotation. The registration results for each frame (shift in X and Y direction and eye rotation) can also be used for eye-movement evaluation during single-spot fixation tasks.

Constraints on the evolution of a doublesex target gene arising from doublesex’s pleiotropic deployment

PubMed Central

Luo, Shengzhan D.; Baker, Bruce S.

2015-01-01

“Regulatory evolution,” that is, changes in a gene’s expression pattern through changes at its regulatory sequence, rather than changes at the coding sequence of the gene or changes of the upstream transcription factors, has been increasingly recognized as a pervasive evolution mechanism. Many somatic sexually dimorphic features of Drosophila melanogaster are the results of gene expression regulated by the doublesex (dsx) gene, which encodes sex-specific transcription factors (DSXF in females and DSXM in males). Rapid changes in such sexually dimorphic features are likely a result of changes at the regulatory sequence of the target genes. We focused on the Flavin-containing monooxygenase-2 (Fmo-2) gene, a likely direct dsx target, to elucidate how sexually dimorphic expression and its evolution are brought about. We found that dsx is deployed to regulate the Fmo-2 transcription both in the midgut and in fat body cells of the spermatheca (a female-specific tissue), through a canonical DSX-binding site in the Fmo-2 regulatory sequence. In the melanogaster group, Fmo-2 transcription in the midgut has evolved rapidly, in contrast to the conserved spermathecal transcription. We identified two cis-regulatory modules (CRM-p and CRM-d) that direct sexually monomorphic or dimorphic Fmo-2 transcription, respectively, in the midguts of these species. Changes of Fmo-2 transcription in the midgut from sexually dimorphic to sexually monomorphic in some species are caused by the loss of CRM-d function, but not the loss of the canonical DSX-binding site. Thus, conferring transcriptional regulation on a CRM level allows the regulation to evolve rapidly in one tissue while evading evolutionary constraints posed by other tissues. PMID:25675536

Identification of single amino acid substitutions (SAAS) in neuraminidase from influenza a virus (H1N1) via mass spectrometry analysis coupled with de novo peptide sequencing.

PubMed

Peng, Qisheng; Wang, Zijian; Wu, Donglin; Li, Xiaoou; Liu, Xiaofeng; Sun, Wanchun; Liu, Ning

2016-08-01

Amino acid substitutions in the neuraminidase of the influenza virus are the main cause of the emergence of resistance to zanamivir or oseltamivir during seasonal influenza treatment; they are the result of non-synonymous mutations in the viral genome that can be successfully detected by polymer chain reaction (PCR)-based approaches. There is always an urgent need to detect variation in amino acid sequences directly at the protein level. Mass spectrometry coupled with de novo sequencing has been explored as an alternative and straightforward strategy for detecting amino acid substitutions, as well - this approach is the primary focus of the present study. Influenza virus (A/Puerto Rico/8/1934 H1N1) propagated in embryonated chicken eggs was purified by ultracentrifugation, followed by PNGase F treatment. The deglycosylated virion was lysed and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The gel band corresponding to neuraminidase was picked up and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. LC-MS/MS analyses, coupled with manual de novo sequencing, allowed the determination of three amino acid substitutions: R346K, S349 N, and S370I/L, in the neuraminidase from the influenza virus (A/Puerto Rico/8/1934 H1N1), which were located in three mutated peptides of the neuraminidase: YGNGVWIGK, TKNHSSR, and PNGWTETDI/LK, respectively. We found that the amino acid substitutions in the proteins of RNA viruses (including influenza A virus) resulting from non-synonymous gene mutations can indeed be directly analyzed via mass spectrometry, and that manual interpretation of the MS/MS data may be beneficial. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

Internal control regions for transcription of eukaryotic tRNA genes.

PubMed Central

Sharp, S; DeFranco, D; Dingermann, T; Farrell, P; Söll, D

1981-01-01

We have identified the region within a eukaryotic tRNA gene required for initiation of transcription. These results were obtained by systematically constructing deletions extending from the 5' or the 3' flanking regions into a cloned Drosophila tRNAArg gene by using nuclease BAL 31. The ability of the newly generated deletion clones to direct the in vitro synthesis of tRNA precursors was measured in transcription systems from Xenopus laevis oocytes, Drosophila Kc cells, and HeLa cells. Two control regions within the coding sequence were identified. The first was essential for transcription and was contained between nucleotides 8 and 25 of the mature tRNA sequence. Genes devoid of the second control region, which was contained between nucleotides 50 and 58 of the mature tRNA sequence, could be transcribed but with reduced efficiency. Thus, the promoter regions within a tRNA gene encode the tRNA sequences of the D stem and D loop, the invariant uridine at position 8, and the semi-invariant G-T-psi-C sequence. Images PMID:6947245

RNA-Seq Alignment to Individualized Genomes Improves Transcript Abundance Estimates in Multiparent Populations

PubMed Central

Munger, Steven C.; Raghupathy, Narayanan; Choi, Kwangbom; Simons, Allen K.; Gatti, Daniel M.; Hinerfeld, Douglas A.; Svenson, Karen L.; Keller, Mark P.; Attie, Alan D.; Hibbs, Matthew A.; Graber, Joel H.; Chesler, Elissa J.; Churchill, Gary A.

2014-01-01

Massively parallel RNA sequencing (RNA-seq) has yielded a wealth of new insights into transcriptional regulation. A first step in the analysis of RNA-seq data is the alignment of short sequence reads to a common reference genome or transcriptome. Genetic variants that distinguish individual genomes from the reference sequence can cause reads to be misaligned, resulting in biased estimates of transcript abundance. Fine-tuning of read alignment algorithms does not correct this problem. We have developed Seqnature software to construct individualized diploid genomes and transcriptomes for multiparent populations and have implemented a complete analysis pipeline that incorporates other existing software tools. We demonstrate in simulated and real data sets that alignment to individualized transcriptomes increases read mapping accuracy, improves estimation of transcript abundance, and enables the direct estimation of allele-specific expression. Moreover, when applied to expression QTL mapping we find that our individualized alignment strategy corrects false-positive linkage signals and unmasks hidden associations. We recommend the use of individualized diploid genomes over reference sequence alignment for all applications of high-throughput sequencing technology in genetically diverse populations. PMID:25236449

An Integrated Physical, Genetic and Cytogenetic Map of Brachypodium distachyon, a Model System for Grass Research

PubMed Central

Febrer, Melanie; Goicoechea, Jose Luis; Wright, Jonathan; McKenzie, Neil; Song, Xiang; Lin, Jinke; Collura, Kristi; Wissotski, Marina; Yu, Yeisoo; Ammiraju, Jetty S. S.; Wolny, Elzbieta; Idziak, Dominika; Betekhtin, Alexander; Kudrna, Dave; Hasterok, Robert; Wing, Rod A.; Bevan, Michael W.

2010-01-01

The pooid subfamily of grasses includes some of the most important crop, forage and turf species, such as wheat, barley and Lolium. Developing genomic resources, such as whole-genome physical maps, for analysing the large and complex genomes of these crops and for facilitating biological research in grasses is an important goal in plant biology. We describe a bacterial artificial chromosome (BAC)-based physical map of the wild pooid grass Brachypodium distachyon and integrate this with whole genome shotgun sequence (WGS) assemblies using BAC end sequences (BES). The resulting physical map contains 26 contigs spanning the 272 Mb genome. BES from the physical map were also used to integrate a genetic map. This provides an independent vaildation and confirmation of the published WGS assembly. Mapped BACs were used in Fluorescence In Situ Hybridisation (FISH) experiments to align the integrated physical map and sequence assemblies to chromosomes with high resolution. The physical, genetic and cytogenetic maps, integrated with whole genome shotgun sequence assemblies, enhance the accuracy and durability of this important genome sequence and will directly facilitate gene isolation. PMID:20976139

SDSS-IV MaNGA: Spatially Resolved Star Formation Main Sequence and LI(N)ER Sequence

NASA Astrophysics Data System (ADS)

Hsieh, B. C.; Lin, Lihwai; Lin, J. H.; Pan, H. A.; Hsu, C. H.; Sánchez, S. F.; Cano-Díaz, M.; Zhang, K.; Yan, R.; Barrera-Ballesteros, J. K.; Boquien, M.; Riffel, R.; Brownstein, J.; Cruz-González, I.; Hagen, A.; Ibarra, H.; Pan, K.; Bizyaev, D.; Oravetz, D.; Simmons, A.

2017-12-01

We present our study on the spatially resolved Hα and M * relation for 536 star-forming and 424 quiescent galaxies taken from the MaNGA survey. We show that the star formation rate surface density ({{{Σ }}}{SFR}), derived based on the Hα emissions, is strongly correlated with the M * surface density ({{{Σ }}}* ) on kiloparsec scales for star-forming galaxies and can be directly connected to the global star-forming sequence. This suggests that the global main sequence may be a consequence of a more fundamental relation on small scales. On the other hand, our result suggests that ∼20% of quiescent galaxies in our sample still have star formation activities in the outer region with lower specific star formation rate (SSFR) than typical star-forming galaxies. Meanwhile, we also find a tight correlation between {{{Σ }}}{{H}α } and {{{Σ }}}* for LI(N)ER regions, named the resolved “LI(N)ER” sequence, in quiescent galaxies, which is consistent with the scenario that LI(N)ER emissions are primarily powered by the hot, evolved stars as suggested in the literature.

Biological nitrogen fixation in the oxygen-minimum region of the eastern tropical North Pacific ocean.

PubMed

Jayakumar, Amal; Chang, Bonnie X; Widner, Brittany; Bernhardt, Peter; Mulholland, Margaret R; Ward, Bess B

2017-10-01

Biological nitrogen fixation (BNF) was investigated above and within the oxygen-depleted waters of the oxygen-minimum zone of the Eastern Tropical North Pacific Ocean. BNF rates were estimated using an isotope tracer method that overcame the uncertainty of the conventional bubble method by directly measuring the tracer enrichment during the incubations. Highest rates of BNF (~4 nM day -1 ) occurred in coastal surface waters and lowest detectable rates (~0.2 nM day -1 ) were found in the anoxic region of offshore stations. BNF was not detectable in most samples from oxygen-depleted waters. The composition of the N 2 -fixing assemblage was investigated by sequencing of nifH genes. The diazotrophic assemblage in surface waters contained mainly Proteobacterial sequences (Cluster I nifH), while both Proteobacterial sequences and sequences with high identities to those of anaerobic microbes characterized as Clusters III and IV type nifH sequences were found in the anoxic waters. Our results indicate modest input of N through BNF in oxygen-depleted zones mainly due to the activity of proteobacterial diazotrophs.

Nanowire-nanopore transistor sensor for DNA detection during translocation

NASA Astrophysics Data System (ADS)

Xie, Ping; Xiong, Qihua; Fang, Ying; Qing, Quan; Lieber, Charles

2011-03-01

Nanopore sequencing, as a promising low cost, high throughput sequencing technique, has been proposed more than a decade ago. Due to the incompatibility between small ionic current signal and fast translocation speed and the technical difficulties on large scale integration of nanopore for direct ionic current sequencing, alternative methods rely on integrated DNA sensors have been proposed, such as using capacitive coupling or tunnelling current etc. But none of them have been experimentally demonstrated yet. Here we show that for the first time an amplified sensor signal has been experimentally recorded from a nanowire-nanopore field effect transistor sensor during DNA translocation. Independent multi-channel recording was also demonstrated for the first time. Our results suggest that the signal is from highly localized potential change caused by DNA translocation in none-balanced buffer condition. Given this method may produce larger signal for smaller nanopores, we hope our experiment can be a starting point for a new generation of nanopore sequencing devices with larger signal, higher bandwidth and large-scale multiplexing capability and finally realize the ultimate goal of low cost high throughput sequencing.

Exploring the role of hydration and confinement in the aggregation of amyloidogenic peptides Aβ16-22 and Sup357-13 in AOT reverse micelles

NASA Astrophysics Data System (ADS)

Martinez, Anna Victoria; Małolepsza, Edyta; Rivera, Eva; Lu, Qing; Straub, John E.

2014-12-01

Knowledge of how intermolecular interactions of amyloid-forming proteins cause protein aggregation and how those interactions are affected by sequence and solution conditions is essential to our understanding of the onset of many degenerative diseases. Of particular interest is the aggregation of the amyloid-β (Aβ) peptide, linked to Alzheimer's disease, and the aggregation of the Sup35 yeast prion peptide, which resembles the mammalian prion protein linked to spongiform encephalopathies. To facilitate the study of these important peptides, experimentalists have identified small peptide congeners of the full-length proteins that exhibit amyloidogenic behavior, including the KLVFFAE sub-sequence, Aβ16-22, and the GNNQQNY subsequence, Sup357-13. In this study, molecular dynamics simulations were used to examine these peptide fragments encapsulated in reverse micelles (RMs) in order to identify the fundamental principles that govern how sequence and solution environment influence peptide aggregation. Aβ16-22 and Sup357-13 are observed to organize into anti-parallel and parallel β-sheet arrangements. Confinement in the sodium bis(2-ethylhexyl) sulfosuccinate (AOT) reverse micelles is shown to stabilize extended peptide conformations and enhance peptide aggregation. Substantial fluctuations in the reverse micelle shape are observed, in agreement with earlier studies. Shape fluctuations are found to facilitate peptide solvation through interactions between the peptide and AOT surfactant, including direct interaction between non-polar peptide residues and the aliphatic surfactant tails. Computed amide I IR spectra are compared with experimental spectra and found to reflect changes in the peptide structures induced by confinement in the RM environment. Furthermore, examination of the rotational anisotropy decay of water in the RM demonstrates that the water dynamics are sensitive to the presence of peptide as well as the peptide sequence. Overall, our results demonstrate that the RM is a complex confining environment where substantial direct interaction between the surfactant and peptides plays an important role in determining the resulting ensemble of peptide conformations. By extension the results suggest that similarly complex sequence-dependent interactions may determine conformational ensembles of amyloid-forming peptides in a cellular environment.

Methods for sequencing GC-rich and CCT repeat DNA templates

DOEpatents

Robinson, Donna L.

2007-02-20

The present invention is directed to a PCR-based method of cycle sequencing DNA and other polynucleotide sequences having high CG content and regions of high GC content, and includes for example DNA strands with a high Cytosine and/or Guanosine content and repeated motifs such as CCT repeats.

Genomic deletions of OFD1 account for 23% of oral-facial-digital type 1 syndrome after negative DNA sequencing.

PubMed

Thauvin-Robinet, Christel; Franco, Brunella; Saugier-Veber, Pascale; Aral, Bernard; Gigot, Nadège; Donzel, Anne; Van Maldergem, Lionel; Bieth, Eric; Layet, Valérie; Mathieu, Michèle; Teebi, Ahmad; Lespinasse, James; Callier, Patrick; Mugneret, Francine; Masurel-Paulet, Alice; Gautier, Elodie; Huet, Frédéric; Teyssier, Jean-Raymond; Tosi, Mario; Frébourg, Thierry; Faivre, Laurence

2009-02-01

Oral-facial-digital type I syndrome (OFDI) is characterised by an X-linked dominant mode of inheritance with lethality in males. Clinical features include facial dysmorphism with oral, dental and distal abnormalities, polycystic kidney disease and central nervous system malformations. Considerable allelic heterogeneity has been reported within the OFD1 gene, but DNA bi-directional sequencing of the exons and intron-exon boundaries of the OFD1 gene remains negative in more than 20% of cases. We hypothesized that genomic rearrangements could account for the majority of the remaining undiagnosed cases. Thus, we took advantage of two independent available series of patients with OFDI syndrome and negative DNA bi-directional sequencing of the exons and intron-exon boundaries of the OFD1 gene from two different European labs: 13/36 cases from the French lab; 13/95 from the Italian lab. All patients were screened by a semiquantitative fluorescent multiplex method (QFMPSF) and relative quantification by real-time PCR (qPCR). Six OFD1 genomic deletions (exon 5, exons 1-8, exons 1-14, exons 10-11, exons 13-23 and exon 17) were identified, accounting for 5% of OFDI patients and for 23% of patients with negative mutation screening by DNA sequencing. The association of DNA direct sequencing, QFMPSF and qPCR detects OFD1 alteration in up to 85% of patients with a phenotype suggestive of OFDI syndrome. Given the average percentage of large genomic rearrangements (5%), we suggest that dosage methods should be performed in addition to DNA direct sequencing analysis to exclude the involvement of the OFD1 transcript when there are genetic counselling issues. (c) 2008 Wiley-Liss, Inc.

Sliding over the Blocks in Enzyme-Free RNA Copying – One-Pot Primer Extension in Ice

PubMed Central

Löffler, Philipp M. G.; Groen, Joost; Dörr, Mark; Monnard, Pierre-Alain

2013-01-01

Template-directed polymerization of RNA in the absence of enzymes is the basis for an information transfer in the ‘RNA-world’ hypothesis and in novel nucleic acid based technology. Previous investigations established that only cytidine rich strands are efficient templates in bulk aqueous solutions while a few specific sequences completely block the extension of hybridized primers. We show that a eutectic water/ice system can support Pb2+/Mg2+-ion catalyzed extension of a primer across such sequences, i.e. AA, AU and AG, in a one-pot synthesis. Using mixtures of imidazole activated nucleotide 5′-monophosphates, the two first “blocking” residues could be passed during template-directed polymerization, i.e., formation of triply extended products containing a high fraction of faithful copies was demonstrated. Across the AG sequence, a mismatch sequence was formed in similar amounts to the correct product due to U·G wobble pairing. Thus, the template-directed extension occurs both across pyrimidine and purine rich sequences and insertions of pyrimidines did not inhibit the subsequent insertions. Products were mainly formed with 2′-5′-phosphodiester linkages, however, the abundance of 3′–5′-linkages was higher than previously reported for pyrimidine insertions. When enzyme-free, template-directed RNA polymerization is performed in a eutectic water ice environment, various intrinsic reaction limitations observed in bulk solution can then be overcome. PMID:24058695

A sequential analysis of classroom discourse in Italian primary schools: the many faces of the IRF pattern.

PubMed

Molinari, Luisa; Mameli, Consuelo; Gnisci, Augusto

2013-09-01

A sequential analysis of classroom discourse is needed to investigate the conditions under which the triadic initiation-response-feedback (IRF) pattern may host different teaching orientations. The purpose of the study is twofold: first, to describe the characteristics of classroom discourse and, second, to identify and explore the different interactive sequences that can be captured with a sequential statistical analysis. Twelve whole-class activities were video recorded in three Italian primary schools. We observed classroom interaction as it occurs naturally on an everyday basis. In total, we collected 587 min of video recordings. Subsequently, 828 triadic IRF patterns were extracted from this material and analysed with the programme Generalized Sequential Query (GSEQ). The results indicate that classroom discourse may unfold in different ways. In particular, we identified and described four types of sequences. Dialogic sequences were triggered by authentic questions, and continued through further relaunches. Monologic sequences were directed to fulfil the teachers' pre-determined didactic purposes. Co-constructive sequences fostered deduction, reasoning, and thinking. Scaffolding sequences helped and sustained children with difficulties. The application of sequential analyses allowed us to show that interactive sequences may account for a variety of meanings, thus making a significant contribution to the literature and research practice in classroom discourse. © 2012 The British Psychological Society.

Molecular sequences derived from Paleocene Fort Union Formation coals vs. associated produced waters: Implications for CBM regeneration

USGS Publications Warehouse

Klein, Donald A.; Flores, Romeo M.; Venot, Christophe; Gabbert, Kendra; Schmidt, Raleigh; Stricker, Gary D.; Pruden, Amy; Mandernack, Kevin

2008-01-01

Coalbed methane regeneration is of increasing interest, and is gaining global attention with respect to enhancement of gas recovery. The objective of this study is to determine if there are differences in methanogen nucleic acid sequences associated with low rank coals from the Powder River Basin, Wyoming, in comparison with sequences that can be recovered from coal bed-associated produced waters. Based on results obtained to date, the sequences from the coals appear to be associated with putatively deep-rooted thermophilic autotrophic methanogens, whereas the sequences from the waters are associated with thermophilic autotrophic and heterotrophic methanogens. The recovered sequences associated with coal thus appear to be both phylogenetically and functionally distinct from those that are more closely associated with the produced water. To be able to relate such recovered sequences to organisms that might be present and possibly active in these environments, it is suggested that direct observation, followed by isolation and single cell-based physiological/molecular analyses, be used to characterize methanogenic consortia possibly associated with coals and/or produced waters. It is also important to characterize the microenvironment where these microbes might be found, in both ecological and geological contexts, to be able to develop effective, ecologically relevant coalbed methane regeneration processes.

Study design requirements for RNA sequencing-based breast cancer diagnostics.

PubMed

Mer, Arvind Singh; Klevebring, Daniel; Grönberg, Henrik; Rantalainen, Mattias

2016-02-01

Sequencing-based molecular characterization of tumors provides information required for individualized cancer treatment. There are well-defined molecular subtypes of breast cancer that provide improved prognostication compared to routine biomarkers. However, molecular subtyping is not yet implemented in routine breast cancer care. Clinical translation is dependent on subtype prediction models providing high sensitivity and specificity. In this study we evaluate sample size and RNA-sequencing read requirements for breast cancer subtyping to facilitate rational design of translational studies. We applied subsampling to ascertain the effect of training sample size and the number of RNA sequencing reads on classification accuracy of molecular subtype and routine biomarker prediction models (unsupervised and supervised). Subtype classification accuracy improved with increasing sample size up to N = 750 (accuracy = 0.93), although with a modest improvement beyond N = 350 (accuracy = 0.92). Prediction of routine biomarkers achieved accuracy of 0.94 (ER) and 0.92 (Her2) at N = 200. Subtype classification improved with RNA-sequencing library size up to 5 million reads. Development of molecular subtyping models for cancer diagnostics requires well-designed studies. Sample size and the number of RNA sequencing reads directly influence accuracy of molecular subtyping. Results in this study provide key information for rational design of translational studies aiming to bring sequencing-based diagnostics to the clinic.

BiRen: predicting enhancers with a deep-learning-based model using the DNA sequence alone.

PubMed

Yang, Bite; Liu, Feng; Ren, Chao; Ouyang, Zhangyi; Xie, Ziwei; Bo, Xiaochen; Shu, Wenjie

2017-07-01

Enhancer elements are noncoding stretches of DNA that play key roles in controlling gene expression programmes. Despite major efforts to develop accurate enhancer prediction methods, identifying enhancer sequences continues to be a challenge in the annotation of mammalian genomes. One of the major issues is the lack of large, sufficiently comprehensive and experimentally validated enhancers for humans or other species. Thus, the development of computational methods based on limited experimentally validated enhancers and deciphering the transcriptional regulatory code encoded in the enhancer sequences is urgent. We present a deep-learning-based hybrid architecture, BiRen, which predicts enhancers using the DNA sequence alone. Our results demonstrate that BiRen can learn common enhancer patterns directly from the DNA sequence and exhibits superior accuracy, robustness and generalizability in enhancer prediction relative to other state-of-the-art enhancer predictors based on sequence characteristics. Our BiRen will enable researchers to acquire a deeper understanding of the regulatory code of enhancer sequences. Our BiRen method can be freely accessed at https://github.com/wenjiegroup/BiRen . shuwj@bmi.ac.cn or boxc@bmi.ac.cn. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com

European external quality control study on the competence of laboratories to recognize rare sequence variants resulting in unusual genotyping results.

PubMed

Márki-Zay, János; Klein, Christoph L; Gancberg, David; Schimmel, Heinz G; Dux, László

2009-04-01

Depending on the method used, rare sequence variants adjacent to the single nucleotide polymorphism (SNP) of interest may cause unusual or erroneous genotyping results. Because such rare variants are known for many genes commonly tested in diagnostic laboratories, we organized a proficiency study to assess their influence on the accuracy of reported laboratory results. Four external quality control materials were processed and sent to 283 laboratories through 3 EQA organizers for analysis of the prothrombin 20210G>A mutation. Two of these quality control materials contained sequence variants introduced by site-directed mutagenesis. One hundred eighty-nine laboratories participated in the study. When samples gave a usual result with the method applied, the error rate was 5.1%. Detailed analysis showed that more than 70% of the failures were reported from only 9 laboratories. Allele-specific amplification-based PCR had a much higher error rate than other methods (18.3% vs 2.9%). The variants 20209C>T and [20175T>G; 20179_20180delAC] resulted in unusual genotyping results in 67 and 85 laboratories, respectively. Eighty-three (54.6%) of these unusual results were not recognized, 32 (21.1%) were attributed to technical issues, and only 37 (24.3%) were recognized as another sequence variant. Our findings revealed that some of the participating laboratories were not able to recognize and correctly interpret unusual genotyping results caused by rare SNPs. Our study indicates that the majority of the failures could be avoided by improved training and careful selection and validation of the methods applied.

Effects of cloning and root-tip size on observations of fungal ITS sequences from Picea glauca roots

Treesearch

Daniel L. Lindner; Mark T. Banik

2009-01-01

To better understand the effects of cloning on observations of fungal ITS sequences from Picea glauca (white spruce) roots two techniques were compared: (i) direct sequencing of fungal ITS regions from individual root tips without cloning and (ii) cloning and sequencing of fungal ITS regions from individual root tips. Effect of root tip size was...

End Joining-Mediated Gene Expression in Mammalian Cells Using PCR-Amplified DNA Constructs that Contain Terminator in Front of Promoter.

PubMed

Nakamura, Mikiko; Suzuki, Ayako; Akada, Junko; Tomiyoshi, Keisuke; Hoshida, Hisashi; Akada, Rinji

2015-12-01

Mammalian gene expression constructs are generally prepared in a plasmid vector, in which a promoter and terminator are located upstream and downstream of a protein-coding sequence, respectively. In this study, we found that front terminator constructs-DNA constructs containing a terminator upstream of a promoter rather than downstream of a coding region-could sufficiently express proteins as a result of end joining of the introduced DNA fragment. By taking advantage of front terminator constructs, FLAG substitutions, and deletions were generated using mutagenesis primers to identify amino acids specifically recognized by commercial FLAG antibodies. A minimal epitope sequence for polyclonal FLAG antibody recognition was also identified. In addition, we analyzed the sequence of a C-terminal Ser-Lys-Leu peroxisome localization signal, and identified the key residues necessary for peroxisome targeting. Moreover, front terminator constructs of hepatitis B surface antigen were used for deletion analysis, leading to the identification of regions required for the particle formation. Collectively, these results indicate that front terminator constructs allow for easy manipulations of C-terminal protein-coding sequences, and suggest that direct gene expression with PCR-amplified DNA is useful for high-throughput protein analysis in mammalian cells.

Knowing what to respond in the future does not cancel the influence of past events.

PubMed

Tubau, Elisabet; López-Moliner, Joan

2009-05-29

Everyday tasks seldom involve isolate actions but sequences of them. We can see whether previous actions influence the current one by exploring the response time to controlled sequences of stimuli. Specifically, depending on the response-stimulus temporal interval (RSI), different mechanisms have been proposed to explain sequential effects in two-choice serial response tasks. Whereas an automatic facilitation mechanism is thought to produce a benefit for response repetitions at short RSIs, subjective expectancies are considered to replace the automatic facilitation at longer RSIs, producing a cost-benefit pattern: repetitions are faster after other repetitions but they are slower after alternations. However, there is not direct evidence showing the impact of subjective expectancies on sequential effects. By using a fixed sequence, the results of the reported experiment showed that the repetition effect was enhanced in participants who acquired complete knowledge of the order. Nevertheless, a similar cost-benefit pattern was observed in all participants and in all learning blocks. Therefore, results of the experiment suggest that sequential effects, including the cost-benefit pattern, are the consequence of automatic mechanisms which operate independently of (and simultaneously with) explicit knowledge of the sequence or other subjective expectancies.

Domain fusion analysis by applying relational algebra to protein sequence and domain databases

PubMed Central

Truong, Kevin; Ikura, Mitsuhiko

2003-01-01

Background Domain fusion analysis is a useful method to predict functionally linked proteins that may be involved in direct protein-protein interactions or in the same metabolic or signaling pathway. As separate domain databases like BLOCKS, PROSITE, Pfam, SMART, PRINTS-S, ProDom, TIGRFAMs, and amalgamated domain databases like InterPro continue to grow in size and quality, a computational method to perform domain fusion analysis that leverages on these efforts will become increasingly powerful. Results This paper proposes a computational method employing relational algebra to find domain fusions in protein sequence databases. The feasibility of this method was illustrated on the SWISS-PROT+TrEMBL sequence database using domain predictions from the Pfam HMM (hidden Markov model) database. We identified 235 and 189 putative functionally linked protein partners in H. sapiens and S. cerevisiae, respectively. From scientific literature, we were able to confirm many of these functional linkages, while the remainder offer testable experimental hypothesis. Results can be viewed at . Conclusion As the analysis can be computed quickly on any relational database that supports standard SQL (structured query language), it can be dynamically updated along with the sequence and domain databases, thereby improving the quality of predictions over time. PMID:12734020

Mining dynamic noteworthy functions in software execution sequences.

PubMed

Zhang, Bing; Huang, Guoyan; Wang, Yuqian; He, Haitao; Ren, Jiadong

2017-01-01

As the quality of crucial entities can directly affect that of software, their identification and protection become an important premise for effective software development, management, maintenance and testing, which thus contribute to improving the software quality and its attack-defending ability. Most analysis and evaluation on important entities like codes-based static structure analysis are on the destruction of the actual software running. In this paper, from the perspective of software execution process, we proposed an approach to mine dynamic noteworthy functions (DNFM)in software execution sequences. First, according to software decompiling and tracking stack changes, the execution traces composed of a series of function addresses were acquired. Then these traces were modeled as execution sequences and then simplified so as to get simplified sequences (SFS), followed by the extraction of patterns through pattern extraction (PE) algorithm from SFS. After that, evaluating indicators inner-importance and inter-importance were designed to measure the noteworthiness of functions in DNFM algorithm. Finally, these functions were sorted by their noteworthiness. Comparison and contrast were conducted on the experiment results from two traditional complex network-based node mining methods, namely PageRank and DegreeRank. The results show that the DNFM method can mine noteworthy functions in software effectively and precisely.

Sequence-length variation of mtDNA HVS-I C-stretch in Chinese ethnic groups.

PubMed

Chen, Feng; Dang, Yong-hui; Yan, Chun-xia; Liu, Yan-ling; Deng, Ya-jun; Fulton, David J R; Chen, Teng

2009-10-01

The purpose of this study was to investigate mitochondrial DNA (mtDNA) hypervariable segment-I (HVS-I) C-stretch variations and explore the significance of these variations in forensic and population genetics studies. The C-stretch sequence variation was studied in 919 unrelated individuals from 8 Chinese ethnic groups using both direct and clone sequencing approaches. Thirty eight C-stretch haplotypes were identified, and some novel and population specific haplotypes were also detected. The C-stretch genetic diversity (GD) values were relatively high, and probability (P) values were low. Additionally, C-stretch length heteroplasmy was observed in approximately 9% of individuals studied. There was a significant correlation (r=-0.961, P<0.01) between the expansion of the cytosine sequence length in the C-stretch of HVS-I and a reduction in the number of upstream adenines. These results indicate that the C-stretch could be a useful genetic maker in forensic identification of Chinese populations. The results from the Fst and dA genetic distance matrix, neighbor-joining tree, and principal component map also suggest that C-stretch could be used as a reliable genetic marker in population genetics.

Identification of Direct Target Genes Using Joint Sequence and Expression Likelihood with Application to DAF-16

PubMed Central

Yu, Ron X.; Liu, Jie; True, Nick; Wang, Wei

2008-01-01

A major challenge in the post-genome era is to reconstruct regulatory networks from the biological knowledge accumulated up to date. The development of tools for identifying direct target genes of transcription factors (TFs) is critical to this endeavor. Given a set of microarray experiments, a probabilistic model called TRANSMODIS has been developed which can infer the direct targets of a TF by integrating sequence motif, gene expression and ChIP-chip data. The performance of TRANSMODIS was first validated on a set of transcription factor perturbation experiments (TFPEs) involving Pho4p, a well studied TF in Saccharomyces cerevisiae. TRANSMODIS removed elements of arbitrariness in manual target gene selection process and produced results that concur with one's intuition. TRANSMODIS was further validated on a genome-wide scale by comparing it with two other methods in Saccharomyces cerevisiae. The usefulness of TRANSMODIS was then demonstrated by applying it to the identification of direct targets of DAF-16, a critical TF regulating ageing in Caenorhabditis elegans. We found that 189 genes were tightly regulated by DAF-16. In addition, DAF-16 has differential preference for motifs when acting as an activator or repressor, which awaits experimental verification. TRANSMODIS is computationally efficient and robust, making it a useful probabilistic framework for finding immediate targets. PMID:18350157

Use of micro-resistivity imaging tools in developing lower Pennsylvanian Morrow channel sandstone reservoirs, Cheyenne, Kiowa and Lincoln Counties, Colorado

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

Germinario, M.P.

1996-01-01

In southeastern Colorado, Lower Pennsylvanian Morrow channel sandstones are part of complex valley-fill sequences incised into Morrow marine deposits. Morrow valleys are approximately [1/2] to 1 mile wide. Valley-fill consists of floodplain and channel filling shales, very fine-grained estuarine sandstones and fine- to coarse-grained channel sandstones that are up to 50' thick. Channel sandstones represent a sequence of stacked fluvial bars deposited in braided, anastomosing and meandering fluvial environments. Cross-stratification in channel sandstones can be imaged by micro-resistivity wireline logging tools and interpreted interactively on various workstation software packages. Recognition, interpretation and measurement of current, stoss face, and lateral accretionmore » beds in these sandstones can result in an estimated direction of paleocurrent flow of the channel. Determination of the channel's local paleoflow direction can provide significant sand risk reduction in developmental drilling, especially in 80 acre or less spacing patterns. As the distance between offset drilling locations increases, the reliability of paleoflow prediction decreases, and the corresponding sand risk rises. Lateral accretion bedding in Morrow channel sandstones has proven to be a poor indicator of sand thickening direction, due to the complex stacking of multiple channel sandstones within any given valley-fill sequence. Micro-resistivity imaging reduces risk in Morrow channel sandstone development drilling programs. Furthermore, these interpretation techniques could be applicable in other fluvial channel sandstone plays.« less

Use of micro-resistivity imaging tools in developing lower Pennsylvanian Morrow channel sandstone reservoirs, Cheyenne, Kiowa and Lincoln Counties, Colorado

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

Germinario, M.P.

1996-12-31

In southeastern Colorado, Lower Pennsylvanian Morrow channel sandstones are part of complex valley-fill sequences incised into Morrow marine deposits. Morrow valleys are approximately {1/2} to 1 mile wide. Valley-fill consists of floodplain and channel filling shales, very fine-grained estuarine sandstones and fine- to coarse-grained channel sandstones that are up to 50` thick. Channel sandstones represent a sequence of stacked fluvial bars deposited in braided, anastomosing and meandering fluvial environments. Cross-stratification in channel sandstones can be imaged by micro-resistivity wireline logging tools and interpreted interactively on various workstation software packages. Recognition, interpretation and measurement of current, stoss face, and lateral accretionmore » beds in these sandstones can result in an estimated direction of paleocurrent flow of the channel. Determination of the channel`s local paleoflow direction can provide significant sand risk reduction in developmental drilling, especially in 80 acre or less spacing patterns. As the distance between offset drilling locations increases, the reliability of paleoflow prediction decreases, and the corresponding sand risk rises. Lateral accretion bedding in Morrow channel sandstones has proven to be a poor indicator of sand thickening direction, due to the complex stacking of multiple channel sandstones within any given valley-fill sequence. Micro-resistivity imaging reduces risk in Morrow channel sandstone development drilling programs. Furthermore, these interpretation techniques could be applicable in other fluvial channel sandstone plays.« less

Fast and Accurate Multivariate Gaussian Modeling of Protein Families: Predicting Residue Contacts and Protein-Interaction Partners

PubMed Central

Feinauer, Christoph; Procaccini, Andrea; Zecchina, Riccardo; Weigt, Martin; Pagnani, Andrea

2014-01-01

In the course of evolution, proteins show a remarkable conservation of their three-dimensional structure and their biological function, leading to strong evolutionary constraints on the sequence variability between homologous proteins. Our method aims at extracting such constraints from rapidly accumulating sequence data, and thereby at inferring protein structure and function from sequence information alone. Recently, global statistical inference methods (e.g. direct-coupling analysis, sparse inverse covariance estimation) have achieved a breakthrough towards this aim, and their predictions have been successfully implemented into tertiary and quaternary protein structure prediction methods. However, due to the discrete nature of the underlying variable (amino-acids), exact inference requires exponential time in the protein length, and efficient approximations are needed for practical applicability. Here we propose a very efficient multivariate Gaussian modeling approach as a variant of direct-coupling analysis: the discrete amino-acid variables are replaced by continuous Gaussian random variables. The resulting statistical inference problem is efficiently and exactly solvable. We show that the quality of inference is comparable or superior to the one achieved by mean-field approximations to inference with discrete variables, as done by direct-coupling analysis. This is true for (i) the prediction of residue-residue contacts in proteins, and (ii) the identification of protein-protein interaction partner in bacterial signal transduction. An implementation of our multivariate Gaussian approach is available at the website http://areeweb.polito.it/ricerca/cmp/code. PMID:24663061

Alu repeats: A source for the genesis of primate microsatellites

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

Arcot, S.S.; Batzer, M.A.; Wang, Zhenyuan

1995-09-01

As a result of their abundance, relatively uniform distribution, and high degree of polymorphism, microsatellites and minisatellites have become valuable tools in genetic mapping, forensic identity testing, and population studies. In recent years, a number of microsatellite repeats have been found to be associated with Alu interspersed repeated DNA elements. The association of an Alu element with a microsatellite repeat could result from the integration of an Alu element within a preexisting microsatellite repeat. Alternatively, Alu elements could have a direct role in the origin of microsatellite repeats. Errors introduced during reverse transcription of the primary transcript derived from anmore » Alu {open_quotes}master{close_quote} gene or the accumulation of random mutations in the middle A-rich regions and oligo(dA)-rich tails of Alu elements after insertion and subsequent expansion and contraction of these sequences could result in the genesis of a microsatellite repeat. We have tested these hypotheses by a direct evolutionary comparison of the sequences of some recent Alu elements that are found only in humans and are absent from nonhuman primates, as well as some older Alu elements that are present at orthologous positions in a number of nonhuman primates. The origin of {open_quotes}young{close_quotes} Alu insertions, absence of sequences that resemble microsatellite repeats at the orthologous loci in chimpanzees, and the gradual expansion of microsatellite repeats in some old Alu repeats at orthologous positions within the genomes of a number of nonhuman primates suggest that Alu elements are a source for the genesis of primate microsatellite repeats. 48 refs., 5 figs., 3 tabs.« less

Biophysical and structural considerations for protein sequence evolution

PubMed Central

2011-01-01

Background Protein sequence evolution is constrained by the biophysics of folding and function, causing interdependence between interacting sites in the sequence. However, current site-independent models of sequence evolutions do not take this into account. Recent attempts to integrate the influence of structure and biophysics into phylogenetic models via statistical/informational approaches have not resulted in expected improvements in model performance. This suggests that further innovations are needed for progress in this field. Results Here we develop a coarse-grained physics-based model of protein folding and binding function, and compare it to a popular informational model. We find that both models violate the assumption of the native sequence being close to a thermodynamic optimum, causing directional selection away from the native state. Sampling and simulation show that the physics-based model is more specific for fold-defining interactions that vary less among residue type. The informational model diffuses further in sequence space with fewer barriers and tends to provide less support for an invariant sites model, although amino acid substitutions are generally conservative. Both approaches produce sequences with natural features like dN/dS < 1 and gamma-distributed rates across sites. Conclusions Simple coarse-grained models of protein folding can describe some natural features of evolving proteins but are currently not accurate enough to use in evolutionary inference. This is partly due to improper packing of the hydrophobic core. We suggest possible improvements on the representation of structure, folding energy, and binding function, as regards both native and non-native conformations, and describe a large number of possible applications for such a model. PMID:22171550

A k-mer-based barcode DNA classification methodology based on spectral representation and a neural gas network.

PubMed

Fiannaca, Antonino; La Rosa, Massimo; Rizzo, Riccardo; Urso, Alfonso

2015-07-01

In this paper, an alignment-free method for DNA barcode classification that is based on both a spectral representation and a neural gas network for unsupervised clustering is proposed. In the proposed methodology, distinctive words are identified from a spectral representation of DNA sequences. A taxonomic classification of the DNA sequence is then performed using the sequence signature, i.e., the smallest set of k-mers that can assign a DNA sequence to its proper taxonomic category. Experiments were then performed to compare our method with other supervised machine learning classification algorithms, such as support vector machine, random forest, ripper, naïve Bayes, ridor, and classification tree, which also consider short DNA sequence fragments of 200 and 300 base pairs (bp). The experimental tests were conducted over 10 real barcode datasets belonging to different animal species, which were provided by the on-line resource "Barcode of Life Database". The experimental results showed that our k-mer-based approach is directly comparable, in terms of accuracy, recall and precision metrics, with the other classifiers when considering full-length sequences. In addition, we demonstrate the robustness of our method when a classification is performed task with a set of short DNA sequences that were randomly extracted from the original data. For example, the proposed method can reach the accuracy of 64.8% at the species level with 200-bp fragments. Under the same conditions, the best other classifier (random forest) reaches the accuracy of 20.9%. Our results indicate that we obtained a clear improvement over the other classifiers for the study of short DNA barcode sequence fragments. Copyright © 2015 Elsevier B.V. All rights reserved.

Assessing the Fidelity of Ancient DNA Sequences Amplified From Nuclear Genes

PubMed Central

Binladen, Jonas; Wiuf, Carsten; Gilbert, M. Thomas P.; Bunce, Michael; Barnett, Ross; Larson, Greger; Greenwood, Alex D.; Haile, James; Ho, Simon Y. W.; Hansen, Anders J.; Willerslev, Eske

2006-01-01

To date, the field of ancient DNA has relied almost exclusively on mitochondrial DNA (mtDNA) sequences. However, a number of recent studies have reported the successful recovery of ancient nuclear DNA (nuDNA) sequences, thereby allowing the characterization of genetic loci directly involved in phenotypic traits of extinct taxa. It is well documented that postmortem damage in ancient mtDNA can lead to the generation of artifactual sequences. However, as yet no one has thoroughly investigated the damage spectrum in ancient nuDNA. By comparing clone sequences from 23 fossil specimens, recovered from environments ranging from permafrost to desert, we demonstrate the presence of miscoding lesion damage in both the mtDNA and nuDNA, resulting in insertion of erroneous bases during amplification. Interestingly, no significant differences in the frequency of miscoding lesion damage are recorded between mtDNA and nuDNA despite great differences in cellular copy numbers. For both mtDNA and nuDNA, we find significant positive correlations between total sequence heterogeneity and the rates of type 1 transitions (adenine → guanine and thymine → cytosine) and type 2 transitions (cytosine → thymine and guanine → adenine), respectively. Type 2 transitions are by far the most dominant and increase relative to those of type 1 with damage load. The results suggest that the deamination of cytosine (and 5-methyl cytosine) to uracil (and thymine) is the main cause of miscoding lesions in both ancient mtDNA and nuDNA sequences. We argue that the problems presented by postmortem damage, as well as problems with contamination from exogenous sources of conserved nuclear genes, allelic variation, and the reliance on single nucleotide polymorphisms, call for great caution in studies relying on ancient nuDNA sequences. PMID:16299392

C-Terminal DxD-Containing Sequences within Paramyxovirus Nucleocapsid Proteins Determine Matrix Protein Compatibility and Can Direct Foreign Proteins into Budding Particles

PubMed Central

Ray, Greeshma; Schmitt, Phuong Tieu

2016-01-01

ABSTRACT Paramyxovirus particles are formed by a budding process coordinated by viral matrix (M) proteins. M proteins coalesce at sites underlying infected cell membranes and induce other viral components, including viral glycoproteins and viral ribonucleoprotein complexes (vRNPs), to assemble at these locations from which particles bud. M proteins interact with the nucleocapsid (NP or N) components of vRNPs, and these interactions enable production of infectious, genome-containing virions. For the paramyxoviruses parainfluenza virus 5 (PIV5) and mumps virus, M-NP interaction also contributes to efficient production of virus-like particles (VLPs) in transfected cells. A DLD sequence near the C-terminal end of PIV5 NP protein was previously found to be necessary for M-NP interaction and efficient VLP production. Here, we demonstrate that 15-residue-long, DLD-containing sequences derived from either the PIV5 or Nipah virus nucleocapsid protein C-terminal ends are sufficient to direct packaging of a foreign protein, Renilla luciferase, into budding VLPs. Mumps virus NP protein harbors DWD in place of the DLD sequence found in PIV5 NP protein, and consequently, PIV5 NP protein is incompatible with mumps virus M protein. A single amino acid change converting DLD to DWD within PIV5 NP protein induced compatibility between these proteins and allowed efficient production of mumps VLPs. Our data suggest a model in which paramyxoviruses share an overall common strategy for directing M-NP interactions but with important variations contained within DLD-like sequences that play key roles in defining M/NP protein compatibilities. IMPORTANCE Paramyxoviruses are responsible for a wide range of diseases that affect both humans and animals. Paramyxovirus pathogens include measles virus, mumps virus, human respiratory syncytial virus, and the zoonotic paramyxoviruses Nipah virus and Hendra virus. Infectivity of paramyxovirus particles depends on matrix-nucleocapsid protein interactions which enable efficient packaging of encapsidated viral RNA genomes into budding virions. In this study, we have defined regions near the C-terminal ends of paramyxovirus nucleocapsid proteins that are important for matrix protein interaction and that are sufficient to direct a foreign protein into budding particles. These results advance our basic understanding of paramyxovirus genome packaging interactions and also have implications for the potential use of virus-like particles as protein delivery tools. PMID:26792745

C-Terminal DxD-Containing Sequences within Paramyxovirus Nucleocapsid Proteins Determine Matrix Protein Compatibility and Can Direct Foreign Proteins into Budding Particles.

PubMed

Ray, Greeshma; Schmitt, Phuong Tieu; Schmitt, Anthony P

2016-01-20

Paramyxovirus particles are formed by a budding process coordinated by viral matrix (M) proteins. M proteins coalesce at sites underlying infected cell membranes and induce other viral components, including viral glycoproteins and viral ribonucleoprotein complexes (vRNPs), to assemble at these locations from which particles bud. M proteins interact with the nucleocapsid (NP or N) components of vRNPs, and these interactions enable production of infectious, genome-containing virions. For the paramyxoviruses parainfluenza virus 5 (PIV5) and mumps virus, M-NP interaction also contributes to efficient production of virus-like particles (VLPs) in transfected cells. A DLD sequence near the C-terminal end of PIV5 NP protein was previously found to be necessary for M-NP interaction and efficient VLP production. Here, we demonstrate that 15-residue-long, DLD-containing sequences derived from either the PIV5 or Nipah virus nucleocapsid protein C-terminal ends are sufficient to direct packaging of a foreign protein, Renilla luciferase, into budding VLPs. Mumps virus NP protein harbors DWD in place of the DLD sequence found in PIV5 NP protein, and consequently, PIV5 NP protein is incompatible with mumps virus M protein. A single amino acid change converting DLD to DWD within PIV5 NP protein induced compatibility between these proteins and allowed efficient production of mumps VLPs. Our data suggest a model in which paramyxoviruses share an overall common strategy for directing M-NP interactions but with important variations contained within DLD-like sequences that play key roles in defining M/NP protein compatibilities. Paramyxoviruses are responsible for a wide range of diseases that affect both humans and animals. Paramyxovirus pathogens include measles virus, mumps virus, human respiratory syncytial virus, and the zoonotic paramyxoviruses Nipah virus and Hendra virus. Infectivity of paramyxovirus particles depends on matrix-nucleocapsid protein interactions which enable efficient packaging of encapsidated viral RNA genomes into budding virions. In this study, we have defined regions near the C-terminal ends of paramyxovirus nucleocapsid proteins that are important for matrix protein interaction and that are sufficient to direct a foreign protein into budding particles. These results advance our basic understanding of paramyxovirus genome packaging interactions and also have implications for the potential use of virus-like particles as protein delivery tools. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

First Complete Genomic Sequence of a Rabies Virus from the Republic of Tajikistan Obtained Directly from a Flinders Technology Associates Card

PubMed Central

Goharriz, H.; Marston, D. A.; Sharifzoda, F.; Ellis, R. J.; Horton, D. L.; Khakimov, T.; Whatmore, A.; Khamroev, K.; Makhmadshoev, A. N.; Bazarov, M.; Fooks, A. R.

2017-01-01

ABSTRACT A brain homogenate derived from a rabid dog in the district of Tojikobod, Republic of Tajikistan, was applied to a Flinders Technology Associates (FTA) card. A full-genome sequence of rabies virus (RABV) was generated from the FTA card directly without extraction, demonstrating the utility of these cards for readily obtaining genetic data. PMID:28684566

First Complete Genomic Sequence of a Rabies Virus from the Republic of Tajikistan Obtained Directly from a Flinders Technology Associates Card.

PubMed

Goharriz, H; Marston, D A; Sharifzoda, F; Ellis, R J; Horton, D L; Khakimov, T; Whatmore, A; Khamroev, K; Makhmadshoev, A N; Bazarov, M; Fooks, A R; Banyard, A C

2017-07-06

A brain homogenate derived from a rabid dog in the district of Tojikobod, Republic of Tajikistan, was applied to a Flinders Technology Associates (FTA) card. A full-genome sequence of rabies virus (RABV) was generated from the FTA card directly without extraction, demonstrating the utility of these cards for readily obtaining genetic data. © Crown copyright 2017.

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

Gardner, Shea N.; McLoughlin, Kevin; Be, Nicholas A.

Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne alphavirus that has caused large outbreaks of severe illness in both horses and humans. New approaches are needed to rapidly infer the origin of a newly discovered VEEV strain, estimate its equine amplification and resultant epidemic potential, and predict human virulence phenotype. We performed whole genome single nucleotide polymorphism (SNP) analysis of all available VEE antigenic complex genomes, verified that a SNP-based phylogeny accurately captured the features of a phylogenetic tree based on multiple sequence alignment, and developed a high resolution genome-wide SNP microarray. We used the microarray to analyze a broadmore » panel of VEEV isolates, found excellent concordance between array- and sequence-based SNP calls, genotyped unsequenced isolates, and placed them on a phylogeny with sequenced genomes. The microarray successfully genotyped VEEV directly from tissue samples of an infected mouse, bypassing the need for viral isolation, culture and genomic sequencing. Lastly, we identified genomic variants associated with serotypes and host species, revealing a complex relationship between genotype and phenotype.« less

Rift Valley Fever, Sudan, 2007 and 2010

PubMed Central

Aradaib, Imadeldin E.; Erickson, Bobbie R.; Elageb, Rehab M.; Khristova, Marina L.; Carroll, Serena A.; Elkhidir, Isam M.; Karsany, Mubarak E.; Karrar, AbdelRahim E.; Elbashir, Mustafa I.

2013-01-01

To elucidate whether Rift Valley fever virus (RVFV) diversity in Sudan resulted from multiple introductions or from acquired changes over time from 1 introduction event, we generated complete genome sequences from RVFV strains detected during the 2007 and 2010 outbreaks. Phylogenetic analyses of small, medium, and large RNA segment sequences indicated several genetic RVFV variants were circulating in Sudan, which all grouped into Kenya-1 or Kenya-2 sublineages from the 2006–2008 eastern Africa epizootic. Bayesian analysis of sequence differences estimated that diversity among the 2007 and 2010 Sudan RVFV variants shared a most recent common ancestor circa 1996. The data suggest multiple introductions of RVFV into Sudan as part of sweeping epizootics from eastern Africa. The sequences indicate recent movement of RVFV and support the need for surveillance to recognize when and where RVFV circulates between epidemics, which can make data from prediction tools easier to interpret and preventive measures easier to direct toward high-risk areas. PMID:23347790

New nitrogen-fixing microorganisms detected in oligotrophic oceans by amplification of nitrogenase (nifH) genes

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

Zehr, J.P.; Mellon, M.T.; Zani, S.

1998-09-01

Oligotrophic oceanic waters of the central ocean gyres typically have extremely low dissolved fixed inorganic nitrogen concentrations, but few nitrogen-fixing microorganisms from the oceanic environment have been cultivated. Nitrogenase gene (nifH) sequences amplified directly from oceanic waters showed that the open ocean contains more diverse diazotrophic microbial populations and more diverse habitats for nitrogen fixers than previously observed by classical microbiological techniques. Nitrogenase genes derived from unicellular and filamentous cyanobacteria, as well as from the {alpha} and {gamma} subdivisions of the class Proteobacteria, were found in both the Atlantic and Pacific oceans. nifH sequences that cluster phylogenetically with sequences frommore » sulfate reducers or clostridia were found associated with planktonic crustaceans. Nitrogenase sequence types obtained from invertebrates represented phylotypes distinct from the phylotypes detected in the picoplankton size fraction. The results indicate that there are in the oceanic environment several distinct potentially nitrogen-fixing microbial assemblages that include representatives of diverse phylotypes.« less

High prevalence of DUOX2 mutations in Japanese patients with permanent congenital hypothyroidism or transient hypothyroidism.

PubMed

Matsuo, Kumihiro; Tanahashi, Yusuke; Mukai, Tokuo; Suzuki, Shigeru; Tajima, Toshihiro; Azuma, Hiroshi; Fujieda, Kenji

2016-07-01

Dual oxidase 2 (DUOX2) mutations are a cause of dyshormonogenesis (DH) and have been identified in patients with permanent congenital hypothyroidism (PH) and with transient hypothyroidism (TH). We aimed to elucidate the prevalence and phenotypical variations of DUOX2 mutations. Forty-eight Japanese DH patients were enroled and analysed for sequence variants of DUOX2, DUOXA2, and TPO using polymerase chain reaction-amplified direct sequencing. Fourteen sequence variants of DUOX2, including 10 novel variants, were identified in 11 patients. DUOX2 variants were more prevalent (11/48, 22.9%) than TPO (3/48, 6.3%) (p=0.020). The prevalence of DUOX2 variants in TH was slightly, but not significantly, higher than in PH. Furthermore, one patient had digenic heterozygous sequence variants of both DUOX2 and TPO. Our results suggest that DUOX2 mutations might be the most common cause of both PH and TH, and that phenotypes of these mutations might be milder than those of other causes.

Novel, non-symbiotic isolates of Neorhizobium from a dryland agricultural soil.

PubMed

Soenens, Amalia; Imperial, Juan

2018-01-01

Semi-selective enrichment, followed by PCR screening, resulted in the successful direct isolation of fast-growing Rhizobia from a dryland agricultural soil. Over 50% of these isolates belong to the genus Neorhizobium , as concluded from partial rpoB and near-complete 16S rDNA sequence analysis. Further genotypic and genomic analysis of five representative isolates confirmed that they form a coherent group within Neorhizobium , closer to N. galegae than to the remaining Neorhizobium species, but clearly differentiated from the former, and constituting at least one new genomospecies within Neorhizobium. All the isolates lacked nod and nif symbiotic genes but contained a repABC replication/maintenance region, characteristic of rhizobial plasmids, within large contigs from their draft genome sequences. These repABC sequences were related, but not identical, to repABC sequences found in symbiotic plasmids from N. galegae , suggesting that the non-symbiotic isolates have the potential to harbor symbiotic plasmids. This is the first report of non-symbiotic members of Neorhizobium from soil.

PHYLOSCANNER: Inferring Transmission from Within- and Between-Host Pathogen Genetic Diversity

PubMed Central

Hall, Matthew; Ratmann, Oliver; Bonsall, David; Golubchik, Tanya; de Cesare, Mariateresa; Gall, Astrid; Cornelissen, Marion; Fraser, Christophe

2018-01-01

Abstract A central feature of pathogen genomics is that different infectious particles (virions and bacterial cells) within an infected individual may be genetically distinct, with patterns of relatedness among infectious particles being the result of both within-host evolution and transmission from one host to the next. Here, we present a new software tool, phyloscanner, which analyses pathogen diversity from multiple infected hosts. phyloscanner provides unprecedented resolution into the transmission process, allowing inference of the direction of transmission from sequence data alone. Multiply infected individuals are also identified, as they harbor subpopulations of infectious particles that are not connected by within-host evolution, except where recombinant types emerge. Low-level contamination is flagged and removed. We illustrate phyloscanner on both viral and bacterial pathogens, namely HIV-1 sequenced on Illumina and Roche 454 platforms, HCV sequenced with the Oxford Nanopore MinION platform, and Streptococcus pneumoniae with sequences from multiple colonies per individual. phyloscanner is available from https://github.com/BDI-pathogens/phyloscanner. PMID:29186559

Application of genetic algorithm in integrated setup planning and operation sequencing

NASA Astrophysics Data System (ADS)

Kafashi, Sajad; Shakeri, Mohsen

2011-01-01

Process planning is an essential component for linking design and manufacturing process. Setup planning and operation sequencing is two main tasks in process planning. Many researches solved these two problems separately. Considering the fact that the two functions are complementary, it is necessary to integrate them more tightly so that performance of a manufacturing system can be improved economically and competitively. This paper present a generative system and genetic algorithm (GA) approach to process plan the given part. The proposed approach and optimization methodology analyses the TAD (tool approach direction), tolerance relation between features and feature precedence relations to generate all possible setups and operations using workshop resource database. Based on these technological constraints the GA algorithm approach, which adopts the feature-based representation, optimizes the setup plan and sequence of operations using cost indices. Case study show that the developed system can generate satisfactory results in optimizing the setup planning and operation sequencing simultaneously in feasible condition.

AFLP fragment isolation technique as a method to produce random sequences for single nucleotide polymorphism discovery in the green turtle, Chelonia mydas.

PubMed

Roden, Suzanne E; Dutton, Peter H; Morin, Phillip A

2009-01-01

The green sea turtle, Chelonia mydas, was used as a case study for single nucleotide polymorphism (SNP) discovery in a species that has little genetic sequence information available. As green turtles have a complex population structure, additional nuclear markers other than microsatellites could add to our understanding of their complex life history. Amplified fragment length polymorphism technique was used to generate sets of random fragments of genomic DNA, which were then electrophoretically separated with precast gels, stained with SYBR green, excised, and directly sequenced. It was possible to perform this method without the use of polyacrylamide gels, radioactive or fluorescent labeled primers, or hybridization methods, reducing the time, expense, and safety hazards of SNP discovery. Within 13 loci, 2547 base pairs were screened, resulting in the discovery of 35 SNPs. Using this method, it was possible to yield a sufficient number of loci to screen for SNP markers without the availability of prior sequence information.

Action-perception coupling in violinists.

PubMed

Kajihara, Takafumi; Verdonschot, Rinus G; Sparks, Joseph; Stewart, Lauren

2013-01-01

The current study investigates auditory-motor coupling in musically trained participants using a Stroop-type task that required the execution of simple finger sequences according to aurally presented number sequences (e.g., "2," "4," "5," "3," "1"). Digital remastering was used to manipulate the pitch contour of the number sequences such that they were either congruent or incongruent with respect to the resulting action sequence. Conservatoire-level violinists showed a strong effect of congruency manipulation (increased response time for incongruent vs. congruent trials), in comparison to a control group of non-musicians. In Experiment 2, this paradigm was used to determine whether pedagogical background would influence this effect in a group of young violinists. Suzuki trained violinists differed significantly from those with no musical background, while traditionally trained violinists did not. The findings extend previous research in this area by demonstrating that obligatory audio-motor coupling is directly related to a musicians' expertise on their instrument of study and is influenced by pedagogy.

Flexible manipulation of terahertz wave reflection using polarization insensitive coding metasurfaces.

PubMed

Jiu-Sheng, Li; Ze-Jiang, Zhao; Jian-Quan, Yao

2017-11-27

In order to extend to 3-bit encoding, we propose notched-wheel structures as polarization insensitive coding metasurfaces to control terahertz wave reflection and suppress backward scattering. By using a coding sequence of "00110011…" along x-axis direction and 16 × 16 random coding sequence, we investigate the polarization insensitive properties of the coding metasurfaces. By designing the coding sequences of the basic coding elements, the terahertz wave reflection can be flexibly manipulated. Additionally, radar cross section (RCS) reduction in the backward direction is less than -10dB in a wide band. The present approach can offer application for novel terahertz manipulation devices.

[Prospects of molecular breeding in medical plants].

PubMed

Ma, Xiao-Jun; Mo, Chang-Ming

2017-06-01

The molecular-assisted breeding, transgenic breeding and molecular designing breeding are three development directions of plant molecular breeding. Base on these three development directions, this paper summarizes developing status and new tendency of research field of genetic linkage mapping, QTL mapping, association mapping, molecular-assisted selections, pollen-mediated transformations, agrobacterium-mediated transformations, particle gun-mediated transformations, genome editing technologies, whole-genome sequencing, transcriptome sequencing, proteome sequencing and varietal molecular designing. The objective and existing problem of medical plant molecular breeding were discussed the prospect of these three molecular breeding technologies application on medical plant molecular breeding was outlooked. Copyright© by the Chinese Pharmaceutical Association.

NEBNext Direct: A Novel, Rapid, Hybridization-Based Approach for the Capture and Library Conversion of Genomic Regions of Interest.

PubMed

Emerman, Amy B; Bowman, Sarah K; Barry, Andrew; Henig, Noa; Patel, Kruti M; Gardner, Andrew F; Hendrickson, Cynthia L

2017-07-05

Next-generation sequencing (NGS) is a powerful tool for genomic studies, translational research, and clinical diagnostics that enables the detection of single nucleotide polymorphisms, insertions and deletions, copy number variations, and other genetic variations. Target enrichment technologies improve the efficiency of NGS by only sequencing regions of interest, which reduces sequencing costs while increasing coverage of the selected targets. Here we present NEBNext Direct ® , a hybridization-based, target-enrichment approach that addresses many of the shortcomings of traditional target-enrichment methods. This approach features a simple, 7-hr workflow that uses enzymatic removal of off-target sequences to achieve a high specificity for regions of interest. Additionally, unique molecular identifiers are incorporated for the identification and filtering of PCR duplicates. The same protocol can be used across a wide range of input amounts, input types, and panel sizes, enabling NEBNext Direct to be broadly applicable across a wide variety of research and diagnostic needs. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

PERFORMANCE OF CONVENTIONAL PCRs BASED ON PRIMERS DIRECTED TO NUCLEAR AND MITOCHONDRIAL GENES FOR THE DETECTION AND IDENTIFICATION OF Leishmania spp.

PubMed Central

LOPES, Estela Gallucci; GERALDO, Carlos Alberto; MARCILI, Arlei; SILVA, Ricardo Duarte; KEID, Lara Borges; OLIVEIRA, Trícia Maria Ferreira da Silva; SOARES, Rodrigo Martins

2016-01-01

In visceral leishmaniasis, the detection of the agent is of paramount importance to identify reservoirs of infection. Here, we evaluated the diagnostic attributes of PCRs based on primers directed to cytochrome-B (cytB), cytochrome-oxidase-subunit II (coxII), cytochrome-C (cytC), and the minicircle-kDNA. Although PCRs directed to cytB, coxII, cytC were able to detect different species of Leishmania, and the nucleotide sequence of their amplicons allowed the unequivocal differentiation of species, the analytical and diagnostic sensitivity of these PCRs were much lower than the analytical and diagnostic sensitivity of the kDNA-PCR. Among the 73 seropositive animals, the asymptomatic dogs had spleen and bone marrow samples collected and tested; only two animals were positive by PCRs based on cytB, coxII, and cytC, whereas 18 were positive by the kDNA-PCR. Considering the kDNA-PCR results, six dogs had positive spleen and bone marrow samples, eight dogs had positive bone marrow results but negative results in spleen samples and, in four dogs, the reverse situation occurred. We concluded that PCRs based on cytB, coxII, and cytC can be useful tools to identify Leishmania species when used in combination with automated sequencing. The discordance between the results of the kDNA-PCR in bone marrow and spleen samples may indicate that conventional PCR lacks sensitivity for the detection of infected dogs. Thus, primers based on the kDNA should be preferred for the screening of infected dogs. PMID:27253743

Triplex technology in studies of DNA damage, DNA repair, and mutagenesis.

PubMed

Mukherjee, Anirban; Vasquez, Karen M

2011-08-01

Triplex-forming oligonucleotides (TFOs) can bind to the major groove of homopurine-homopyrimidine stretches of double-stranded DNA in a sequence-specific manner through Hoogsteen hydrogen bonding to form DNA triplexes. TFOs by themselves or conjugated to reactive molecules can be used to direct sequence-specific DNA damage, which in turn results in the induction of several DNA metabolic activities. Triplex technology is highly utilized as a tool to study gene regulation, molecular mechanisms of DNA repair, recombination, and mutagenesis. In addition, TFO targeting of specific genes has been exploited in the development of therapeutic strategies to modulate DNA structure and function. In this review, we discuss advances made in studies of DNA damage, DNA repair, recombination, and mutagenesis by using triplex technology to target specific DNA sequences. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Intact coding region of the serotonin transporter gene in obsessive-compulsive disorder

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

Altemus, M.; Murphy, D.L.; Greenberg, B.

1996-07-26

Epidemiologic studies indicate that obsessive-compulsive disorder is genetically transmitted in some families, although no genetic abnormalities have been identified in individuals with this disorder. The selective response of obsessive-compulsive disorder to treatment with agents which block serotonin reuptake suggests the gene coding for the serotonin transporter as a candidate gene. The primary structure of the serotonin-transporter coding region was sequenced in 22 patients with obsessive-compulsive disorder, using direct PCR sequencing of cDNA synthesized from platelet serotonin-transporter mRNA. No variations in amino acid sequence were found among the obsessive-compulsive disorder patients or healthy controls. These results do not support a rolemore » for alteration in the primary structure of the coding region of the serotonin-transporter gene in the pathogenesis of obsessive-compulsive disorder. 27 refs.« less

Multiple stage miniature stepping motor

DOEpatents

Niven, William A.; Shikany, S. David; Shira, Michael L.

1981-01-01

A stepping motor comprising a plurality of stages which may be selectively activated to effect stepping movement of the motor, and which are mounted along a common rotor shaft to achieve considerable reduction in motor size and minimum diameter, whereby sequential activation of the stages results in successive rotor steps with direction being determined by the particular activating sequence followed.

Utilizing Spectrum Efficiently (USE)

DTIC Science & Technology

2011-02-28

18 4.8 Space-Time Coded Asynchronous DS - CDMA with Decentralized MAI Suppression: Performance and...numerical results. 4.8 Space-Time Coded Asynchronous DS - CDMA with Decentralized MAI Suppression: Performance and Spectral Efficiency In [60] multiple...supported at a given signal-to-interference ratio in asynchronous direct-sequence code-division multiple-access ( DS - CDMA ) sys- tems was examined. It was

Molecular characterization of long direct repeat (LDR) sequences expressing a stable mRNA encoding for a 35-amino-acid cell-killing peptide and a cis-encoded small antisense RNA in Escherichia coli.

PubMed

Kawano, Mitsuoki; Oshima, Taku; Kasai, Hiroaki; Mori, Hirotada

2002-07-01

Genome sequence analyses of Escherichia coli K-12 revealed four copies of long repetitive elements. These sequences are designated as long direct repeat (LDR) sequences. Three of the repeats (LDR-A, -B, -C), each approximately 500 bp in length, are located as tandem repeats at 27.4 min on the genetic map. Another copy (LDR-D), 450 bp in length and nearly identical to LDR-A, -B and -C, is located at 79.7 min, a position that is directly opposite the position of LDR-A, -B and -C. In this study, we demonstrate that LDR-D encodes a 35-amino-acid peptide, LdrD, the overexpression of which causes rapid cell killing and nucleoid condensation of the host cell. Northern blot and primer extension analysis showed constitutive transcription of a stable mRNA (approximately 370 nucleotides) encoding LdrD and an unstable cis-encoded antisense RNA (approximately 60 nucleotides), which functions as a trans-acting regulator of ldrD translation. We propose that LDR encodes a toxin-antitoxin module. LDR-homologous sequences are not pre-sent on any known plasmids but are conserved in Salmonella and other enterobacterial species.

Complete plastid genome sequence of Daucus carota: implications for biotechnology and phylogeny of angiosperms.

PubMed

Ruhlman, Tracey; Lee, Seung-Bum; Jansen, Robert K; Hostetler, Jessica B; Tallon, Luke J; Town, Christopher D; Daniell, Henry

2006-08-31

Carrot (Daucus carota) is a major food crop in the US and worldwide. Its capacity for storage and its lifecycle as a biennial make it an attractive species for the introduction of foreign genes, especially for oral delivery of vaccines and other therapeutic proteins. Until recently efforts to express recombinant proteins in carrot have had limited success in terms of protein accumulation in the edible tap roots. Plastid genetic engineering offers the potential to overcome this limitation, as demonstrated by the accumulation of BADH in chromoplasts of carrot taproots to confer exceedingly high levels of salt resistance. The complete plastid genome of carrot provides essential information required for genetic engineering. Additionally, the sequence data add to the rapidly growing database of plastid genomes for assessing phylogenetic relationships among angiosperms. The complete carrot plastid genome is 155,911 bp in length, with 115 unique genes and 21 duplicated genes within the IR. There are four ribosomal RNAs, 30 distinct tRNA genes and 18 intron-containing genes. Repeat analysis reveals 12 direct and 2 inverted repeats > or = 30 bp with a sequence identity > or = 90%. Phylogenetic analysis of nucleotide sequences for 61 protein-coding genes using both maximum parsimony (MP) and maximum likelihood (ML) were performed for 29 angiosperms. Phylogenies from both methods provide strong support for the monophyly of several major angiosperm clades, including monocots, eudicots, rosids, asterids, eurosids II, euasterids I, and euasterids II. The carrot plastid genome contains a number of dispersed direct and inverted repeats scattered throughout coding and non-coding regions. This is the first sequenced plastid genome of the family Apiaceae and only the second published genome sequence of the species-rich euasterid II clade. Both MP and ML trees provide very strong support (100% bootstrap) for the sister relationship of Daucus with Panax in the euasterid II clade. These results provide the best taxon sampling of complete chloroplast genomes and the strongest support yet for the sister relationship of Caryophyllales to the asterids. The availability of the complete plastid genome sequence should facilitate improved transformation efficiency and foreign gene expression in carrot through utilization of endogenous flanking sequences and regulatory elements.

Hemichordates and the Origin of Chordates

NASA Technical Reports Server (NTRS)

Gerhart, John; Kirschner, Marc; Lowe, Chris

2002-01-01

At the start of the period of the NASA grant three years ago, we had no information on the organization and development of the body axis of the hemichordate, Saccoglossus kowalevskii. Now we have substantial findings about the anteroposterior axis and dorsoventral axis, and based on this information, we have new insights about the origin of chordates from ancestral deuterostomes. We found ways to obtain and preserve large numbers of embryos and hatched juveniles. We can now collect about 40,000 embryos in the month of September, the time of S. kowalevskii spawning at Woods Hole. Excellent cDNA libraries were prepared from three developmental stages. From these libraries, we directly isolated about 30 gene ortholog sequences by screening and pcr techniques, all of these sequences of interest in the inquiry about the animal's organization and development. We also performed a mid-sized EST project (60,000 randomly picked clones, many of these arrayed). About half of these have been analyzed so far by blastx and are suitable for direct use of clones. We have obtained about 50 interesting sequences from this set. The rest still await analysis. Thus, at this time we have isolated orthologs of 80 genes that are known to be expressed in chordates in conserved domains and known to have interesting roles in chordate organization and development. The orthology of the S. kowalevskii sequences has been verified by neighbor joining and parsimony methods, with bootstrap estimates of validity. The S. kowalevskii sequences cluster with other deuterostome sequences, namely, other hemichordates, echinoderms, ascidians, amphioxus, or vertebrates, depending on what sequences are available in the database for comparison. We have used these sequences to do high quality in situ hybridization on S. kowalevskii embryos, and the results can be divided into three sections-those concerning the anteroposterior axis of S. kowalevskii in comparison to the same axis of chordates, those concerning the dorsoventral axis of S. kowalevskii in comparison to the same axis of chordates, and those concerning the signals and transcription factors found in the endoderm, of S. kowalevskii compared to the signals and transcription factors in the endo-mesodermal cells of Spemann's organizer of chordates.

Selection dynamic of Escherichia coli host in M13 combinatorial peptide phage display libraries.

PubMed

Zanconato, Stefano; Minervini, Giovanni; Poli, Irene; De Lucrezia, Davide

2011-01-01

Phage display relies on an iterative cycle of selection and amplification of random combinatorial libraries to enrich the initial population of those peptides that satisfy a priori chosen criteria. The effectiveness of any phage display protocol depends directly on library amino acid sequence diversity and the strength of the selection procedure. In this study we monitored the dynamics of the selective pressure exerted by the host organism on a random peptide library in the absence of any additional selection pressure. The results indicate that sequence censorship exerted by Escherichia coli dramatically reduces library diversity and can significantly impair phage display effectiveness.

Problems in turbidite research: A need for COMFAN

USGS Publications Warehouse

Normark, W.R.; Mutti, E.; Bouma, A.H.

1984-01-01

Comparison of modern submarine fans and ancient turbidite sequences is still in its infancy, mainly because of the incompatibility of study approaches. Research on modern fan systems mainly deals with morphologic aspects and surficial sediments, while observations on ancient turbidite formations are mostly directed to vertical sequences. The lack of a common data set also results from different scales of observation. To review the current status of modern and ancient turbidite research, an international group of specialists formed COMFAN (Committee on Fans) and met in September 1982 at the Gulf Research and Development Company research facilities in Pennsylvania. ?? 1984 Springer-Verlag New York Inc.

Interuser Interference Analysis for Direct-Sequence Spread-Spectrum Systems Part I: Partial-Period Cross-Correlation

NASA Technical Reports Server (NTRS)

Ni, Jianjun (David)

2012-01-01

This presentation discusses an analysis approach to evaluate the interuser interference for Direct-Sequence Spread-Spectrum (DSSS) Systems for Space Network (SN) Users. Part I of this analysis shows that the correlation property of pseudo noise (PN) sequences is the critical factor which determines the interuser interference performance of the DSSS system. For non-standard DSSS systems in which PN sequence s period is much larger than one data symbol duration, it is the partial-period cross-correlation that determines the system performance. This study reveals through an example that a well-designed PN sequence set (e.g. Gold Sequence, in which the cross-correlation for a whole-period is well controlled) may have non-controlled partial-period cross-correlation which could cause severe interuser interference for a DSSS system. Since the analytical derivation of performance metric (bit error rate or signal-to-noise ratio) based on partial-period cross-correlation is prohibitive, the performance degradation due to partial-period cross-correlation will be evaluated using simulation in Part II of this analysis in the future.

Combining protein sequence, structure, and dynamics: A novel approach for functional evolution analysis of PAS domain superfamily.

PubMed

Dong, Zheng; Zhou, Hongyu; Tao, Peng

2018-02-01

PAS domains are widespread in archaea, bacteria, and eukaryota, and play important roles in various functions. In this study, we aim to explore functional evolutionary relationship among proteins in the PAS domain superfamily in view of the sequence-structure-dynamics-function relationship. We collected protein sequences and crystal structure data from RCSB Protein Data Bank of the PAS domain superfamily belonging to three biological functions (nucleotide binding, photoreceptor activity, and transferase activity). Protein sequences were aligned and then used to select sequence-conserved residues and build phylogenetic tree. Three-dimensional structure alignment was also applied to obtain structure-conserved residues. The protein dynamics were analyzed using elastic network model (ENM) and validated by molecular dynamics (MD) simulation. The result showed that the proteins with same function could be grouped by sequence similarity, and proteins in different functional groups displayed statistically significant difference in their vibrational patterns. Interestingly, in all three functional groups, conserved amino acid residues identified by sequence and structure conservation analysis generally have a lower fluctuation than other residues. In addition, the fluctuation of conserved residues in each biological function group was strongly correlated with the corresponding biological function. This research suggested a direct connection in which the protein sequences were related to various functions through structural dynamics. This is a new attempt to delineate functional evolution of proteins using the integrated information of sequence, structure, and dynamics. © 2017 The Protein Society.

Taxator-tk: precise taxonomic assignment of metagenomes by fast approximation of evolutionary neighborhoods

PubMed Central

Dröge, J.; Gregor, I.; McHardy, A. C.

2015-01-01

Motivation: Metagenomics characterizes microbial communities by random shotgun sequencing of DNA isolated directly from an environment of interest. An essential step in computational metagenome analysis is taxonomic sequence assignment, which allows identifying the sequenced community members and reconstructing taxonomic bins with sequence data for the individual taxa. For the massive datasets generated by next-generation sequencing technologies, this cannot be performed with de-novo phylogenetic inference methods. We describe an algorithm and the accompanying software, taxator-tk, which performs taxonomic sequence assignment by fast approximate determination of evolutionary neighbors from sequence similarities. Results: Taxator-tk was precise in its taxonomic assignment across all ranks and taxa for a range of evolutionary distances and for short as well as for long sequences. In addition to the taxonomic binning of metagenomes, it is well suited for profiling microbial communities from metagenome samples because it identifies bacterial, archaeal and eukaryotic community members without being affected by varying primer binding strengths, as in marker gene amplification, or copy number variations of marker genes across different taxa. Taxator-tk has an efficient, parallelized implementation that allows the assignment of 6 Gb of sequence data per day on a standard multiprocessor system with 10 CPU cores and microbial RefSeq as the genomic reference data. Availability and implementation: Taxator-tk source and binary program files are publicly available at http://algbio.cs.uni-duesseldorf.de/software/. Contact: Alice.McHardy@uni-duesseldorf.de Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25388150

ssHMM: extracting intuitive sequence-structure motifs from high-throughput RNA-binding protein data

PubMed Central

Krestel, Ralf; Ohler, Uwe; Vingron, Martin; Marsico, Annalisa

2017-01-01

Abstract RNA-binding proteins (RBPs) play an important role in RNA post-transcriptional regulation and recognize target RNAs via sequence-structure motifs. The extent to which RNA structure influences protein binding in the presence or absence of a sequence motif is still poorly understood. Existing RNA motif finders either take the structure of the RNA only partially into account, or employ models which are not directly interpretable as sequence-structure motifs. We developed ssHMM, an RNA motif finder based on a hidden Markov model (HMM) and Gibbs sampling which fully captures the relationship between RNA sequence and secondary structure preference of a given RBP. Compared to previous methods which output separate logos for sequence and structure, it directly produces a combined sequence-structure motif when trained on a large set of sequences. ssHMM’s model is visualized intuitively as a graph and facilitates biological interpretation. ssHMM can be used to find novel bona fide sequence-structure motifs of uncharacterized RBPs, such as the one presented here for the YY1 protein. ssHMM reaches a high motif recovery rate on synthetic data, it recovers known RBP motifs from CLIP-Seq data, and scales linearly on the input size, being considerably faster than MEMERIS and RNAcontext on large datasets while being on par with GraphProt. It is freely available on Github and as a Docker image. PMID:28977546

Benchmarking of the Oxford Nanopore MinION sequencing for quantitative and qualitative assessment of cDNA populations.

PubMed

Oikonomopoulos, Spyros; Wang, Yu Chang; Djambazian, Haig; Badescu, Dunarel; Ragoussis, Jiannis

2016-08-24

To assess the performance of the Oxford Nanopore Technologies MinION sequencing platform, cDNAs from the External RNA Controls Consortium (ERCC) RNA Spike-In mix were sequenced. This mix mimics mammalian mRNA species and consists of 92 polyadenylated transcripts with known concentration. cDNA libraries were generated using a template switching protocol to facilitate the direct comparison between different sequencing platforms. The MinION performance was assessed for its ability to sequence the cDNAs directly with good accuracy in terms of abundance and full length. The abundance of the ERCC cDNA molecules sequenced by MinION agreed with their expected concentration. No length or GC content bias was observed. The majority of cDNAs were sequenced as full length. Additionally, a complex cDNA population derived from a human HEK-293 cell line was sequenced on an Illumina HiSeq 2500, PacBio RS II and ONT MinION platforms. We observed that there was a good agreement in the measured cDNA abundance between PacBio RS II and ONT MinION (rpearson = 0.82, isoforms with length more than 700bp) and between Illumina HiSeq 2500 and ONT MinION (rpearson = 0.75). This indicates that the ONT MinION can sequence quantitatively both long and short full length cDNA molecules.

Dielectrophoretic isolation and detection of cancer-related circulating cell-free DNA biomarkers from blood and plasma

PubMed Central

Sonnenberg, Avery; Marciniak, Jennifer Y.; Skowronski, Elaine A.; Manouchehri, Sareh; Rassenti, Laura; Ghia, Emanuela M.; Widhopf, George F.; Kipps, Thomas J.; Heller, Michael J.

2014-01-01

Conventional methods for the isolation of cancer-related circulating cell-free (ccf) DNA from patient blood (plasma) are time consuming and laborious. A DEP approach utilizing a microarray device now allows rapid isolation of ccf-DNA directly from a small volume of unprocessed blood. In this study, the DEP device is used to compare the ccf-DNA isolated directly from whole blood and plasma from 11 chronic lymphocytic leukemia (CLL) patients and one normal individual. Ccf-DNA from both blood and plasma samples was separated into DEP high-field regions, after which cells (blood), proteins, and other biomolecules were removed by a fluidic wash. The concentrated ccf-DNA was detected on-chip by fluorescence, and then eluted for PCR and DNA sequencing. The complete process from blood to PCR required less than 10 min; an additional 15 min was required to obtain plasma from whole blood. Ccf-DNA from the equivalent of 5 µL of CLL blood and 5 µL of plasma was amplified by PCR using Ig heavy-chain variable (IGHV) specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone. The PCR and DNA sequencing results obtained by DEP from all 11 CLL blood samples and from 8 of the 11 CLL plasma samples were exactly comparable to the DNA sequencing results obtained from genomic DNA isolated from CLL patient leukemic B cells (gold standard). PMID:24723219

Dielectrophoretic isolation and detection of cancer-related circulating cell-free DNA biomarkers from blood and plasma.

PubMed

Sonnenberg, Avery; Marciniak, Jennifer Y; Skowronski, Elaine A; Manouchehri, Sareh; Rassenti, Laura; Ghia, Emanuela M; Widhopf, George F; Kipps, Thomas J; Heller, Michael J

2014-07-01

Conventional methods for the isolation of cancer-related circulating cell-free (ccf) DNA from patient blood (plasma) are time consuming and laborious. A DEP approach utilizing a microarray device now allows rapid isolation of ccf-DNA directly from a small volume of unprocessed blood. In this study, the DEP device is used to compare the ccf-DNA isolated directly from whole blood and plasma from 11 chronic lymphocytic leukemia (CLL) patients and one normal individual. Ccf-DNA from both blood and plasma samples was separated into DEP high-field regions, after which cells (blood), proteins, and other biomolecules were removed by a fluidic wash. The concentrated ccf-DNA was detected on-chip by fluorescence, and then eluted for PCR and DNA sequencing. The complete process from blood to PCR required less than 10 min; an additional 15 min was required to obtain plasma from whole blood. Ccf-DNA from the equivalent of 5 μL of CLL blood and 5 μL of plasma was amplified by PCR using Ig heavy-chain variable (IGHV) specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone. The PCR and DNA sequencing results obtained by DEP from all 11 CLL blood samples and from 8 of the 11 CLL plasma samples were exactly comparable to the DNA sequencing results obtained from genomic DNA isolated from CLL patient leukemic B cells (gold standard). © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of efficiency of nested multiplex allele-specific PCR assay for detection of multidrug resistant tuberculosis directly from sputum samples.

PubMed

Mistri, S K; Sultana, M; Kamal, S M M; Alam, M M; Irin, F; Nessa, J; Ahsan, C R; Yasmin, M

2016-05-01

For an effective control of tuberculosis, rapid detection of multidrug resistant tuberculosis (MDR-TB) is necessary. Therefore, we developed a modified nested multiplex allele-specific polymerase chain reaction (MAS-PCR) method that enables rapid MDR-TB detection directly from sputum samples. The efficacy of this method was evaluated using 79 sputum samples collected from suspected tuberculosis patients. The performance of nested MAS-PCR method was compared with other MDR-TB detection methods like drug susceptibility testing (DST) and DNA sequencing. As rifampicin (RIF) resistance conforms to MDR-TB in greater than 90% cases, only the presence of RIF-associated mutations in rpoB gene was determined by DNA sequencing and nested MAS-PCR to detect MDR-TB. The concordance between nested MAS-PCR and DNA sequencing results was found to be 96·3%. When compared with DST, the sensitivity and specificity of nested MAS-PCR for RIF-resistance detection were determined to be 92·9 and 100% respectively. For developing- and high-TB burden countries, molecular-based tests have been recommended by the World Health Organization for rapid detection of MDR-TB. The results of this study indicate that, nested MAS-PCR assay might be a practical and relatively cost effective molecular method for rapid detection of MDR-TB from suspected sputum samples in developing countries with resource poor settings. © 2016 The Society for Applied Microbiology.

Implied alignment: a synapomorphy-based multiple-sequence alignment method and its use in cladogram search

NASA Technical Reports Server (NTRS)

Wheeler, Ward C.

2003-01-01

A method to align sequence data based on parsimonious synapomorphy schemes generated by direct optimization (DO; earlier termed optimization alignment) is proposed. DO directly diagnoses sequence data on cladograms without an intervening multiple-alignment step, thereby creating topology-specific, dynamic homology statements. Hence, no multiple-alignment is required to generate cladograms. Unlike general and globally optimal multiple-alignment procedures, the method described here, implied alignment (IA), takes these dynamic homologies and traces them back through a single cladogram, linking the unaligned sequence positions in the terminal taxa via DO transformation series. These "lines of correspondence" link ancestor-descendent states and, when displayed as linearly arrayed columns without hypothetical ancestors, are largely indistinguishable from standard multiple alignment. Since this method is based on synapomorphy, the treatment of certain classes of insertion-deletion (indel) events may be different from that of other alignment procedures. As with all alignment methods, results are dependent on parameter assumptions such as indel cost and transversion:transition ratios. Such an IA could be used as a basis for phylogenetic search, but this would be questionable since the homologies derived from the implied alignment depend on its natal cladogram and any variance, between DO and IA + Search, due to heuristic approach. The utility of this procedure in heuristic cladogram searches using DO and the improvement of heuristic cladogram cost calculations are discussed. c2003 The Willi Hennig Society. Published by Elsevier Science (USA). All rights reserved.

Transcription initiation from the dihydrofolate reductase promoter is positioned by HIP1 binding at the initiation site.

PubMed

Means, A L; Farnham, P J

1990-02-01

We have identified a sequence element that specifies the position of transcription initiation for the dihydrofolate reductase gene. Unlike the functionally analogous TATA box that directs RNA polymerase II to initiate transcription 30 nucleotides downstream, the positioning element of the dihydrofolate reductase promoter is located directly at the site of transcription initiation. By using DNase I footprint analysis, we have shown that a protein binds to this initiator element. Transcription initiated at the dihydrofolate reductase initiator element when 28 nucleotides were inserted between it and all other upstream sequences, or when it was placed on either side of the DNA helix, suggesting that there is no strict spatial requirement between the initiator and an upstream element. Although neither a single Sp1-binding site nor a single initiator element was sufficient for transcriptional activity, the combination of one Sp1-binding site and the dihydrofolate reductase initiator element cloned into a plasmid vector resulted in transcription starting at the initiator element. We have also shown that the simian virus 40 late major initiation site has striking sequence homology to the dihydrofolate reductase initiation site and that the same, or a similar, protein binds to both sites. Examination of the sequences at other RNA polymerase II initiation sites suggests that we have identified an element that is important in the transcription of other housekeeping genes. We have thus named the protein that binds to the initiator element HIP1 (Housekeeping Initiator Protein 1).

Accommodation of end-state comfort reveals subphonemic planning in speech

PubMed Central

Gick, Bryan

2015-01-01

Applying Rosenbaum’s “end-state comfort” hypothesis (Rosenbaum et al., 1992, 1996) to tongue motion provides evidence of long-distance subphonemic planning in speech. Speakers’ tongue postures may anticipate upcoming speech up to three segments, two syllables, and a morpheme or word boundary later. We used m-mode ultrasound imaging to measure the direction of tongue tip/blade movements for known variants of flap/tap allophones of North American English /t/ and /d/. Results show that speakers produce different flap variants early in words or word sequences so as to facilitate the kinematic needs of flap/tap or other /r/ variants that appear later in the word or word sequence. Similar results were also observed across word boundaries, indicating that this is not a lexical effect. PMID:25790787

A cost-effectiveness analysis of first-line induction and maintenance treatment sequences in patients with advanced nonsquamous non-small-cell lung cancer in France

PubMed Central

Taipale, Kaisa; Winfree, Katherine B; Boye, Mark; Basson, Mickael; Sleilaty, Ghassan; Eaton, James; Evans, Rachel; Chouaid, Christos

2017-01-01

Background Comparative effectiveness and cost-effectiveness data for induction–maintenance (I–M) sequences for the treatment of patients with nonsquamous non-small-cell lung cancer (nsqNSCLC) are limited because of a lack of direct evidence. This analysis aimed to compare the cost-effectiveness of I–M pemetrexed with those of other I–M regimens used for the treatment of patients with advanced nsqNSCLC in the French health-care setting. Materials and methods A previously developed global partitioned survival model was adapted to the France-only setting by restricting treatment sequences to include 12 I–M regimens most relevant to France, and incorporating French costs and resource-use data. Following a systematic literature review, network meta-analyses were performed to obtain hazard ratios for progression-free survival (PFS) and overall survival (OS) relative to gemcitabine + cisplatin (induction sequences) or best supportive care (BSC) (maintenance sequences). Modeled health-care benefits were expressed as life-years (LYs) and quality-adjusted LYs (QALYs) (estimated using French EuroQol five-dimension questionnaire tariffs). The study was conducted from the payer perspective (National Health Insurance). Cost- and benefit-model inputs were discounted at an annual rate of 4%. Results Base-case results showed pemetrexed + cisplatin induction followed by (→) pemetrexed maintenance had the longest mean OS and PFS and highest LYs and QALYs. Costs ranged from €12,762 for paclitaxel + carboplatin → BSC to €35,617 for pemetrexed + cisplatin → pemetrexed (2015 values). Gemcitabine + cisplatin → BSC, pemetrexed + cisplatin → BSC, and pemetrexed + cisplatin → pemetrexed were associated with fully incremental cost-effectiveness ratios (ICERs) of €16,593, €80,656, and €102,179, respectively, per QALY gained versus paclitaxel + carboplatin → BSC. All other treatment sequences were either dominated (ie, another sequence had lower costs and better/equivalent outcomes) or extendedly dominated (ie, the comparator had a higher ICER than a more effective comparator) in the model. Sensitivity analyses showed the model to be relatively insensitive to plausible changes in the main assumptions, with none increasing or decreasing the ICER by more than ~€20,000 per QALY gained. Conclusion In the absence of direct comparative trial evidence, this cost-effectiveness analysis indicated that of a large number of I–M sequences used for the treatment of patients with nsqNSCLC in France, pemetrexed + cisplatin → pemetrexed achieved the best clinical outcomes (0.28 incremental QALYs gained) versus paclitaxel + carboplatin → BSC. PMID:28860832

Isolation and mapping of telomeric pentanucleotide (TAACC)n repeats of the Pacific whiteleg shrimp, Penaeus vannamei, using fluorescence in situ hybridization.

PubMed

Alcivar-Warren, Acacia; Meehan-Meola, Dawn; Wang, Yongping; Guo, Ximing; Zhou, Linghua; Xiang, Jianhai; Moss, Shaun; Arce, Steve; Warren, William; Xu, Zhenkang; Bell, Kireina

2006-01-01

To develop genetic and physical maps for shrimp, accurate information on the actual number of chromosomes and a large number of genetic markers is needed. Previous reports have shown two different chromosome numbers for the Pacific whiteleg shrimp, Penaeus vannamei, the most important penaeid shrimp species cultured in the Western hemisphere. Preliminary results obtained by direct sequencing of clones from a Sau3A-digested genomic library of P. vannamei ovary identified a large number of (TAACC/GGTTA)-containing SSRs. The objectives of this study were to (1) examine the frequency of (TAACC)n repeats in 662 P. vannamei genomic clones that were directly sequenced, and perform homology searches of these clones, (2) confirm the number of chromosomes in testis of P. vannamei, and (3) localize the TAACC repeats in P. vannamei chromosome spreads using fluorescence in situ hybridization (FISH). Results for objective 1 showed that 395 out of the 662 clones sequenced contained single or multiple SSRs with three or more repeat motifs, 199 of which contained variable tandem repeats of the pentanucleotide (TAACC/GGTTA)n, with 3 to 14 copies per sequence. The frequency of (TAACC)n repeats in P. vannamei is 4.68 kb for SSRs with five or more repeat motifs. Sequence comparisons using the BLASTN nonredundant and expressed sequence tag (EST) databases indicated that most of the TAACC-containing clones were similar to either the core pentanucleotide repeat in PVPENTREP locus (GenBank accession no. X82619) or portions of 28S rRNA. Transposable elements (transposase for Tn1000 and reverse transcriptase family members), hypothetical or unnamed protein products, and genes of known function such as 18S and 28S rRNAs, heat shock protein 70, and thrombospondin were identified in non-TAACC-containing clones. For objective 2, the meiotic chromosome number of P. vannamei was confirmed as N = 44. For objective 3, four FISH probes (P1 to P4) containing different numbers of TAACC repeats produced positive signals on telomeres of P. vannamei chromosomes. A few chromosomes had positive signals interstitially. Probe signal strength and chromosome coverage differed in the general order of P1>P2>P3>P4, which correlated with the length of TAACC repeats within the probes: 83, 66, 35, and 30 bp, respectively, suggesting that the TAACC repeats, and not the flanking sequences, produced the TAACC signals at chromosome ends and TAACC is likely the telomere sequence for P. vannamei.

Targeted isolation, sequence assembly and characterization of two white spruce (Picea glauca) BAC clones for terpenoid synthase and cytochrome P450 genes involved in conifer defence reveal insights into a conifer genome

PubMed Central

2009-01-01

Background Conifers are a large group of gymnosperm trees which are separated from the angiosperms by more than 300 million years of independent evolution. Conifer genomes are extremely large and contain considerable amounts of repetitive DNA. Currently, conifer sequence resources exist predominantly as expressed sequence tags (ESTs) and full-length (FL)cDNAs. There is no genome sequence available for a conifer or any other gymnosperm. Conifer defence-related genes often group into large families with closely related members. The goals of this study are to assess the feasibility of targeted isolation and sequence assembly of conifer BAC clones containing specific genes from two large gene families, and to characterize large segments of genomic DNA sequence for the first time from a conifer. Results We used a PCR-based approach to identify BAC clones for two target genes, a terpene synthase (3-carene synthase; 3CAR) and a cytochrome P450 (CYP720B4) from a non-arrayed genomic BAC library of white spruce (Picea glauca). Shotgun genomic fragments isolated from the BAC clones were sequenced to a depth of 15.6- and 16.0-fold coverage, respectively. Assembly and manual curation yielded sequence scaffolds of 172 kbp (3CAR) and 94 kbp (CYP720B4) long. Inspection of the genomic sequences revealed the intron-exon structures, the putative promoter regions and putative cis-regulatory elements of these genes. Sequences related to transposable elements (TEs), high complexity repeats and simple repeats were prevalent and comprised approximately 40% of the sequenced genomic DNA. An in silico simulation of the effect of sequencing depth on the quality of the sequence assembly provides direction for future efforts of conifer genome sequencing. Conclusion We report the first targeted cloning, sequencing, assembly, and annotation of large segments of genomic DNA from a conifer. We demonstrate that genomic BAC clones for individual members of multi-member gene families can be isolated in a gene-specific fashion. The results of the present work provide important new information about the structure and content of conifer genomic DNA that will guide future efforts to sequence and assemble conifer genomes. PMID:19656416

Setting up a probe based, closed tube real-time PCR assay for focused detection of variable sequence alterations.

PubMed

Becságh, Péter; Szakács, Orsolya

2014-10-01

During diagnostic workflow when detecting sequence alterations, sometimes it is important to design an algorithm that includes screening and direct tests in combination. Normally the use of direct test, which is mainly sequencing, is limited. There is an increased need for effective screening tests, with "closed tube" during the whole process and therefore decreasing the risk of PCR product contamination. The aim of this study was to design such a closed tube, detection probe based screening assay to detect different kind of sequence alterations in the exon 11 of the human c-kit gene region. Inside this region there are variable possible deletions and single nucleotide changes. During assay setup, more probe chemistry formats were screened and tested. After some optimization steps the taqman probe format was selected.

Local Renyi entropic profiles of DNA sequences

PubMed Central

Vinga, Susana; Almeida, Jonas S

2007-01-01

Background In a recent report the authors presented a new measure of continuous entropy for DNA sequences, which allows the estimation of their randomness level. The definition therein explored was based on the Rényi entropy of probability density estimation (pdf) using the Parzen's window method and applied to Chaos Game Representation/Universal Sequence Maps (CGR/USM). Subsequent work proposed a fractal pdf kernel as a more exact solution for the iterated map representation. This report extends the concepts of continuous entropy by defining DNA sequence entropic profiles using the new pdf estimations to refine the density estimation of motifs. Results The new methodology enables two results. On the one hand it shows that the entropic profiles are directly related with the statistical significance of motifs, allowing the study of under and over-representation of segments. On the other hand, by spanning the parameters of the kernel function it is possible to extract important information about the scale of each conserved DNA region. The computational applications, developed in Matlab m-code, the corresponding binary executables and additional material and examples are made publicly available at . Conclusion The ability to detect local conservation from a scale-independent representation of symbolic sequences is particularly relevant for biological applications where conserved motifs occur in multiple, overlapping scales, with significant future applications in the recognition of foreign genomic material and inference of motif structures. PMID:17939871

Expert cognition in the production sequence of Acheulian cleavers at Gesher Benot Ya'aqov, Israel: A lithic and cognitive analysis

PubMed Central

Wynn, Thomas; Goren-Inbar, Naama

2017-01-01

Stone cleavers are one of the most distinctive components of the Acheulian toolkit. These tools were produced as part of a long and complex reduction sequence and they provide indications for planning and remarkable knapping skill. These aspects hold implications regarding the cognitive complexity and abilities of their makers and users. In this study we have analyzed a cleaver assemblage originating from the Acheulian site of Gesher Benot Ya‘aqov, Israel, to provide a reconstruction of the chaîne opératoire which structured their production. This reduction sequence was taken as the basis for a cognitive analysis which allowed us to draw conclusion regarding numerous behavioral and cognitive aspects of the GBY hominins. The results indicate that the cleavers production incorporated a highly specific sequence of decisions and actions which resulted in three distinct modes of cleavers modification. Furthermore, the decision to produce a cleaver must have been taken very early in the sequence, thus differentiating its production from that of handaxes. The substantial predetermination and the specific reduction sequence provide evidence that the Gesher Benot Ya‘aqov hominins had a number of cognitive categories such as a general ‘tool concept’ and a more specific ‘cleaver concept’, setting them apart from earlier tool-producing hominins and extant tool-using non-human primates. Furthermore, it appears that the Gesher Benot Ya‘aqov lithic technology was governed by expert cognition, which is the kind of thinking typical of modern human experts in their various domains. Thus, the results provide direct indications that important components of modern cognition have been well established in the minds of the Gesher Benot Ya‘aqov hominins. PMID:29145489

Expert cognition in the production sequence of Acheulian cleavers at Gesher Benot Ya'aqov, Israel: A lithic and cognitive analysis.

PubMed

Herzlinger, Gadi; Wynn, Thomas; Goren-Inbar, Naama

2017-01-01

Stone cleavers are one of the most distinctive components of the Acheulian toolkit. These tools were produced as part of a long and complex reduction sequence and they provide indications for planning and remarkable knapping skill. These aspects hold implications regarding the cognitive complexity and abilities of their makers and users. In this study we have analyzed a cleaver assemblage originating from the Acheulian site of Gesher Benot Ya'aqov, Israel, to provide a reconstruction of the chaîne opératoire which structured their production. This reduction sequence was taken as the basis for a cognitive analysis which allowed us to draw conclusion regarding numerous behavioral and cognitive aspects of the GBY hominins. The results indicate that the cleavers production incorporated a highly specific sequence of decisions and actions which resulted in three distinct modes of cleavers modification. Furthermore, the decision to produce a cleaver must have been taken very early in the sequence, thus differentiating its production from that of handaxes. The substantial predetermination and the specific reduction sequence provide evidence that the Gesher Benot Ya'aqov hominins had a number of cognitive categories such as a general 'tool concept' and a more specific 'cleaver concept', setting them apart from earlier tool-producing hominins and extant tool-using non-human primates. Furthermore, it appears that the Gesher Benot Ya'aqov lithic technology was governed by expert cognition, which is the kind of thinking typical of modern human experts in their various domains. Thus, the results provide direct indications that important components of modern cognition have been well established in the minds of the Gesher Benot Ya'aqov hominins.

A nonadaptive origin of a beneficial trait: in silico selection for free energy of folding leads to the neutral emergence of mutational robustness in single domain proteins.

PubMed

Pagan, Rafael F; Massey, Steven E

2014-02-01

Proteins are regarded as being robust to the deleterious effects of mutations. Here, the neutral emergence of mutational robustness in a population of single domain proteins is explored using computer simulations. A pairwise contact model was used to calculate the ΔG of folding (ΔG folding) using the three dimensional protein structure of leech eglin C. A random amino acid sequence with low mutational robustness, defined as the average ΔΔG resulting from a point mutation (ΔΔG average), was threaded onto the structure. A population of 1,000 threaded sequences was evolved under selection for stability, using an upper and lower energy threshold. Under these conditions, mutational robustness increased over time in the most common sequence in the population. In contrast, when the wild type sequence was used it did not show an increase in robustness. This implies that the emergence of mutational robustness is sequence specific and that wild type sequences may be close to maximal robustness. In addition, an inverse relationship between ∆∆G average and protein stability is shown, resulting partly from a larger average effect of point mutations in more stable proteins. The emergence of mutational robustness was also observed in the Escherichia coli colE1 Rop and human CD59 proteins, implying that the property may be common in single domain proteins under certain simulation conditions. The results indicate that at least a portion of mutational robustness in small globular proteins might have arisen by a process of neutral emergence, and could be an example of a beneficial trait that has not been directly selected for, termed a "pseudaptation."

Deep sequencing reveals double mutations in cis of MPL exon 10 in myeloproliferative neoplasms.

PubMed

Pietra, Daniela; Brisci, Angela; Rumi, Elisa; Boggi, Sabrina; Elena, Chiara; Pietrelli, Alessandro; Bordoni, Roberta; Ferrari, Maurizio; Passamonti, Francesco; De Bellis, Gianluca; Cremonesi, Laura; Cazzola, Mario

2011-04-01

Somatic mutations of MPL exon 10, mainly involving a W515 substitution, have been described in JAK2 (V617F)-negative patients with essential thrombocythemia and primary myelofibrosis. We used direct sequencing and high-resolution melt analysis to identify mutations of MPL exon 10 in 570 patients with myeloproliferative neoplasms, and allele specific PCR and deep sequencing to further characterize a subset of mutated patients. Somatic mutations were detected in 33 of 221 patients (15%) with JAK2 (V617F)-negative essential thrombocythemia or primary myelofibrosis. Only one patient with essential thrombocythemia carried both JAK2 (V617F) and MPL (W515L). High-resolution melt analysis identified abnormal patterns in all the MPL mutated cases, while direct sequencing did not detect the mutant MPL in one fifth of them. In 3 cases carrying double MPL mutations, deep sequencing analysis showed identical load and location in cis of the paired lesions, indicating their simultaneous occurrence on the same chromosome.

DS-SS with de Bruijn sequences for secure Inter Satellite Links

NASA Astrophysics Data System (ADS)

Spinsante, S.; Warty, C.; Gambi, E.

Today, both the military and commercial sectors are placing an increased emphasis on global communications. This has prompted the development of several Low Earth Orbit satellite systems that promise a worldwide connectivity and real-time voice, data and video communications. Constellations that avoid repeated uplink and downlink work by exploiting Inter Satellite Links have proved to be very economical in space routing. However, traditionally Inter Satellite Links were considered to be out of reach for any malicious activity and thus little, or no security was employed. This paper proposes a secured Inter Satellite Links based network, built upon the adoption of the Direct Sequence Spread Spectrum technique, with binary de Bruijn sequences used as spreading codes. Selected sequences from the de Bruijn family may be used over directional spot beams. The main intent of the paper is to propose a secure and robust communication link for the next generation of satellite communications, relying on a classical spread spectrum approach employing innovative sequences.

Single-cell genomic sequencing using Multiple Displacement Amplification.

PubMed

Lasken, Roger S

2007-10-01

Single microbial cells can now be sequenced using DNA amplified by the Multiple Displacement Amplification (MDA) reaction. The few femtograms of DNA in a bacterium are amplified into micrograms of high molecular weight DNA suitable for DNA library construction and Sanger sequencing. The MDA-generated DNA also performs well when used directly as template for pyrosequencing by the 454 Life Sciences method. While MDA from single cells loses some of the genomic sequence, this approach will greatly accelerate the pace of sequencing from uncultured microbes. The genetically linked sequences from single cells are also a powerful tool to be used in guiding genomic assembly of shotgun sequences of multiple organisms from environmental DNA extracts (metagenomic sequences).

Novel primer specific false terminations during DNA sequencing reactions: danger of inaccuracy of mutation analysis in molecular diagnostics

PubMed Central

Anwar, R; Booth, A; Churchill, A J; Markham, A F

1996-01-01

The determination of nucleotide sequence is fundamental to the identification and molecular analysis of genes. Direct sequencing of PCR products is now becoming a commonplace procedure for haplotype analysis, and for defining mutations and polymorphism within genes, particularly for diagnostic purposes. A previously unrecognised phenomenon, primer related variability, observed in sequence data generated using Taq cycle sequencing and T7 Sequenase sequencing, is reported. This suggests that caution is necessary when interpreting DNA sequence data. This is particularly important in situations where treatment may be dependent on the accuracy of the molecular diagnosis. Images PMID:16696096

Variations on a theme of Lander and Waterman

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

Speed, T.

1997-12-01

The original Lander and Waterman mathematical analysis was for fingerprinting random clones. Since that time, a number of variants of their theory have appeared, including ones which apply to mapping by anchoring random clones, and to non-random or directed clone mapping. The same theory is now widely used to devise random sequencing strategies. In this talk I will review these developments, and go on the discuss the theory required for directed sequencing strategies.

Coherent direct sequence optical code multiple access encoding-decoding efficiency versus wavelength detuning.

PubMed

Pastor, D; Amaya, W; García-Olcina, R; Sales, S

2007-07-01

We present a simple theoretical model of and the experimental verification for vanishing of the autocorrelation peak due to wavelength detuning on the coding-decoding process of coherent direct sequence optical code multiple access systems based on a superstructured fiber Bragg grating. Moreover, the detuning vanishing effect has been explored to take advantage of this effect and to provide an additional degree of multiplexing and/or optical code tuning.

Universal Sequence Replication, Reversible Polymerization and Early Functional Biopolymers: A Model for the Initiation of Prebiotic Sequence Evolution

PubMed Central

Walker, Sara Imari; Grover, Martha A.; Hud, Nicholas V.

2012-01-01

Many models for the origin of life have focused on understanding how evolution can drive the refinement of a preexisting enzyme, such as the evolution of efficient replicase activity. Here we present a model for what was, arguably, an even earlier stage of chemical evolution, when polymer sequence diversity was generated and sustained before, and during, the onset of functional selection. The model includes regular environmental cycles (e.g. hydration-dehydration cycles) that drive polymers between times of replication and functional activity, which coincide with times of different monomer and polymer diffusivity. Template-directed replication of informational polymers, which takes place during the dehydration stage of each cycle, is considered to be sequence-independent. New sequences are generated by spontaneous polymer formation, and all sequences compete for a finite monomer resource that is recycled via reversible polymerization. Kinetic Monte Carlo simulations demonstrate that this proposed prebiotic scenario provides a robust mechanism for the exploration of sequence space. Introduction of a polymer sequence with monomer synthetase activity illustrates that functional sequences can become established in a preexisting pool of otherwise non-functional sequences. Functional selection does not dominate system dynamics and sequence diversity remains high, permitting the emergence and spread of more than one functional sequence. It is also observed that polymers spontaneously form clusters in simulations where polymers diffuse more slowly than monomers, a feature that is reminiscent of a previous proposal that the earliest stages of life could have been defined by the collective evolution of a system-wide cooperation of polymer aggregates. Overall, the results presented demonstrate the merits of considering plausible prebiotic polymer chemistries and environments that would have allowed for the rapid turnover of monomer resources and for regularly varying monomer/polymer diffusivities. PMID:22493682

Next-generation sequencing: the future of molecular genetics in poultry production and food safety.

PubMed

Diaz-Sanchez, S; Hanning, I; Pendleton, Sean; D'Souza, Doris

2013-02-01

The era of molecular biology and automation of the Sanger chain-terminator sequencing method has led to discovery and advances in diagnostics and biotechnology. The Sanger methodology dominated research for over 2 decades, leading to significant accomplishments and technological improvements in DNA sequencing. Next-generation high-throughput sequencing (HT-NGS) technologies were developed subsequently to overcome the limitations of this first generation technology that include higher speed, less labor, and lowered cost. Various platforms developed include sequencing-by-synthesis 454 Life Sciences, Illumina (Solexa) sequencing, SOLiD sequencing (among others), and the Ion Torrent semiconductor sequencing technologies that use different detection principles. As technology advances, progress made toward third generation sequencing technologies are being reported, which include Nanopore Sequencing and real-time monitoring of PCR activity through fluorescent resonant energy transfer. The advantages of these technologies include scalability, simplicity, with increasing DNA polymerase performance and yields, being less error prone, and even more economically feasible with the eventual goal of obtaining real-time results. These technologies can be directly applied to improve poultry production and enhance food safety. For example, sequence-based (determination of the gut microbial community, genes for metabolic pathways, or presence of plasmids) and function-based (screening for function such as antibiotic resistance, or vitamin production) metagenomic analysis can be carried out. Gut microbialflora/communities of poultry can be sequenced to determine the changes that affect health and disease along with efficacy of methods to control pathogenic growth. Thus, the purpose of this review is to provide an overview of the principles of these current technologies and their potential application to improve poultry production and food safety as well as public health.

Rooting gene trees without outgroups: EP rooting.

PubMed

Sinsheimer, Janet S; Little, Roderick J A; Lake, James A

2012-01-01

Gene sequences are routinely used to determine the topologies of unrooted phylogenetic trees, but many of the most important questions in evolution require knowing both the topologies and the roots of trees. However, general algorithms for calculating rooted trees from gene and genomic sequences in the absence of gene paralogs are few. Using the principles of evolutionary parsimony (EP) (Lake JA. 1987a. A rate-independent technique for analysis of nucleic acid sequences: evolutionary parsimony. Mol Biol Evol. 4:167-181) and its extensions (Cavender, J. 1989. Mechanized derivation of linear invariants. Mol Biol Evol. 6:301-316; Nguyen T, Speed TP. 1992. A derivation of all linear invariants for a nonbalanced transversion model. J Mol Evol. 35:60-76), we explicitly enumerate all linear invariants that solely contain rooting information and derive algorithms for rooting gene trees directly from gene and genomic sequences. These new EP linear rooting invariants allow one to determine rooted trees, even in the complete absence of outgroups and gene paralogs. EP rooting invariants are explicitly derived for three taxon trees, and rules for their extension to four or more taxa are provided. The method is demonstrated using 18S ribosomal DNA to illustrate how the new animal phylogeny (Aguinaldo AMA et al. 1997. Evidence for a clade of nematodes, arthropods, and other moulting animals. Nature 387:489-493; Lake JA. 1990. Origin of the metazoa. Proc Natl Acad Sci USA 87:763-766) may be rooted directly from sequences, even when they are short and paralogs are unavailable. These results are consistent with the current root (Philippe H et al. 2011. Acoelomorph flatworms are deuterostomes related to Xenoturbella. Nature 470:255-260).

Rooting Gene Trees without Outgroups: EP Rooting

PubMed Central

Sinsheimer, Janet S.; Little, Roderick J. A.; Lake, James A.

2012-01-01

Gene sequences are routinely used to determine the topologies of unrooted phylogenetic trees, but many of the most important questions in evolution require knowing both the topologies and the roots of trees. However, general algorithms for calculating rooted trees from gene and genomic sequences in the absence of gene paralogs are few. Using the principles of evolutionary parsimony (EP) (Lake JA. 1987a. A rate-independent technique for analysis of nucleic acid sequences: evolutionary parsimony. Mol Biol Evol. 4:167–181) and its extensions (Cavender, J. 1989. Mechanized derivation of linear invariants. Mol Biol Evol. 6:301–316; Nguyen T, Speed TP. 1992. A derivation of all linear invariants for a nonbalanced transversion model. J Mol Evol. 35:60–76), we explicitly enumerate all linear invariants that solely contain rooting information and derive algorithms for rooting gene trees directly from gene and genomic sequences. These new EP linear rooting invariants allow one to determine rooted trees, even in the complete absence of outgroups and gene paralogs. EP rooting invariants are explicitly derived for three taxon trees, and rules for their extension to four or more taxa are provided. The method is demonstrated using 18S ribosomal DNA to illustrate how the new animal phylogeny (Aguinaldo AMA et al. 1997. Evidence for a clade of nematodes, arthropods, and other moulting animals. Nature 387:489–493; Lake JA. 1990. Origin of the metazoa. Proc Natl Acad Sci USA 87:763–766) may be rooted directly from sequences, even when they are short and paralogs are unavailable. These results are consistent with the current root (Philippe H et al. 2011. Acoelomorph flatworms are deuterostomes related to Xenoturbella. Nature 470:255–260). PMID:22593551

Molecular Detection, Isolation, and Physiological Characterization of Functionally Dominant Phenol-Degrading Bacteria in Activated Sludge

PubMed Central

Watanabe, Kazuya; Teramoto, Maki; Futamata, Hiroyuki; Harayama, Shigeaki

1998-01-01

DNA was isolated from phenol-digesting activated sludge, and partial fragments of the 16S ribosomal DNA (rDNA) and the gene encoding the largest subunit of multicomponent phenol hydroxylase (LmPH) were amplified by PCR. An analysis of the amplified fragments by temperature gradient gel electrophoresis (TGGE) demonstrated that two major 16S rDNA bands (bands R2 and R3) and two major LmPH gene bands (bands P2 and P3) appeared after the activated sludge became acclimated to phenol. The nucleotide sequences of these major bands were determined. In parallel, bacteria were isolated from the activated sludge by direct plating or by plating after enrichment either in batch cultures or in a chemostat culture. The bacteria isolated were classified into 27 distinct groups by a repetitive extragenic palindromic sequence PCR analysis. The partial nucleotide sequences of 16S rDNAs and LmPH genes of members of these 27 groups were then determined. A comparison of these nucleotide sequences with the sequences of the major TGGE bands indicated that the major bacterial populations, R2 and R3, possessed major LmPH genes P2 and P3, respectively. The dominant populations could be isolated either by direct plating or by chemostat culture enrichment but not by batch culture enrichment. One of the dominant strains (R3) which contained a novel type of LmPH (P3), was closely related to Valivorax paradoxus, and the result of a kinetic analysis of its phenol-oxygenating activity suggested that this strain was the principal phenol digester in the activated sludge. PMID:9797297

Systematic elucidation and in vivo validation of sequences enriched in hindbrain transcriptional control

PubMed Central

Burzynski, Grzegorz M.; Reed, Xylena; Taher, Leila; Stine, Zachary E.; Matsui, Takeshi; Ovcharenko, Ivan; McCallion, Andrew S.

2012-01-01

Illuminating the primary sequence encryption of enhancers is central to understanding the regulatory architecture of genomes. We have developed a machine learning approach to decipher motif patterns of hindbrain enhancers and identify 40,000 sequences in the human genome that we predict display regulatory control that includes the hindbrain. Consistent with their roles in hindbrain patterning, MEIS1, NKX6-1, as well as HOX and POU family binding motifs contributed strongly to this enhancer model. Predicted hindbrain enhancers are overrepresented at genes expressed in hindbrain and associated with nervous system development, and primarily reside in the areas of open chromatin. In addition, 77 (0.2%) of these predictions are identified as hindbrain enhancers on the VISTA Enhancer Browser, and 26,000 (60%) overlap enhancer marks (H3K4me1 or H3K27ac). To validate these putative hindbrain enhancers, we selected 55 elements distributed throughout our predictions and six low scoring controls for evaluation in a zebrafish transgenic assay. When assayed in mosaic transgenic embryos, 51/55 elements directed expression in the central nervous system. Furthermore, 30/34 (88%) predicted enhancers analyzed in stable zebrafish transgenic lines directed expression in the larval zebrafish hindbrain. Subsequent analysis of sequence fragments selected based upon motif clustering further confirmed the critical role of the motifs contributing to the classifier. Our results demonstrate the existence of a primary sequence code characteristic to hindbrain enhancers. This code can be accurately extracted using machine-learning approaches and applied successfully for de novo identification of hindbrain enhancers. This study represents a critical step toward the dissection of regulatory control in specific neuronal subtypes. PMID:22759862

Rupture directivity of microseismic events recorded during hydraulic fracture stimulations.

NASA Astrophysics Data System (ADS)

Urbancic, T.; Smith-Boughner, L.; Baig, A.; Viegas, G.

2016-12-01

We model the dynamics of a complex rupture sequence with four sub-events. These events were recorded during hydraulic fracture stimulations in a gas-bearing shale formation. With force-balance accelerometers, 4.5Hz and 15Hz instruments recording the failure history, we study the directivity of the entire rupture sequence and each sub-event. Two models are considered: unilateral and bi-lateral failures of penny shaped cracks. From the seismic moment tensors of these sub-events, we consider different potential failure planes and rupture directions. Using numerical wave-propagation codes, we generate synthetic rupture sequences with both unilateral and bi-lateral ruptures. These are compared to the four sub-events to determine the directionality of the observed failures and the sensitivity of our recording bandwidth and geometry to distinguishing between different rupture processes. The frequency of unilateral and bilateral rupture processes throughout the fracture stimulation is estimated by comparing the directivity characteristics of the modeled sub-events to other high-quality microseismic events recorded during the same stimulation program. Understanding the failure processes of these microseismic events can provide great insight into the changes in the rock mass responsible for these complex rupture processes.

Multiplex PCR method for MinION and Illumina sequencing of Zika and other virus genomes directly from clinical samples.

PubMed

Quick, Joshua; Grubaugh, Nathan D; Pullan, Steven T; Claro, Ingra M; Smith, Andrew D; Gangavarapu, Karthik; Oliveira, Glenn; Robles-Sikisaka, Refugio; Rogers, Thomas F; Beutler, Nathan A; Burton, Dennis R; Lewis-Ximenez, Lia Laura; de Jesus, Jaqueline Goes; Giovanetti, Marta; Hill, Sarah C; Black, Allison; Bedford, Trevor; Carroll, Miles W; Nunes, Marcio; Alcantara, Luiz Carlos; Sabino, Ester C; Baylis, Sally A; Faria, Nuno R; Loose, Matthew; Simpson, Jared T; Pybus, Oliver G; Andersen, Kristian G; Loman, Nicholas J

2017-06-01

Genome sequencing has become a powerful tool for studying emerging infectious diseases; however, genome sequencing directly from clinical samples (i.e., without isolation and culture) remains challenging for viruses such as Zika, for which metagenomic sequencing methods may generate insufficient numbers of viral reads. Here we present a protocol for generating coding-sequence-complete genomes, comprising an online primer design tool, a novel multiplex PCR enrichment protocol, optimized library preparation methods for the portable MinION sequencer (Oxford Nanopore Technologies) and the Illumina range of instruments, and a bioinformatics pipeline for generating consensus sequences. The MinION protocol does not require an Internet connection for analysis, making it suitable for field applications with limited connectivity. Our method relies on multiplex PCR for targeted enrichment of viral genomes from samples containing as few as 50 genome copies per reaction. Viral consensus sequences can be achieved in 1-2 d by starting with clinical samples and following a simple laboratory workflow. This method has been successfully used by several groups studying Zika virus evolution and is facilitating an understanding of the spread of the virus in the Americas. The protocol can be used to sequence other viral genomes using the online Primal Scheme primer designer software. It is suitable for sequencing either RNA or DNA viruses in the field during outbreaks or as an inexpensive, convenient method for use in the lab.

The implication of DNA bending energy for nucleosome positioning and sliding.

PubMed

Liu, Guoqing; Xing, Yongqiang; Zhao, Hongyu; Cai, Lu; Wang, Jianying

2018-06-11

Nucleosome not only directly affects cellular processes, such as DNA replication, recombination, and transcription, but also severs as a fundamentally important target of epigenetic modifications. Our previous study indicated that the bending property of DNA is important in nucleosome formation, particularly in predicting the dyad positions of nucleosomes on a DNA segment. Here, we investigated the role of bending energy in nucleosome positioning and sliding in depth to decipher sequence-directed mechanism. The results show that bending energy is a good physical index to predict the free energy in the process of nucleosome reconstitution in vitro. Our data also imply that there are at least 20% of the nucleosomes in budding yeast do not adopt canonical positioning, in which underlying sequences wrapped around histones are structurally symmetric. We also revealed distinct patterns of bending energy profile for distinctly organized chromatin structures, such as well-positioned nucleosomes, fuzzy nucleosomes, and linker regions and discussed nucleosome sliding in terms of bending energy. We proposed that the stability of a nucleosome is positively correlated with the strength of the bending anisotropy of DNA segment, and both accessibility and directionality of nucleosome sliding is likely to be modulated by diverse patterns of DNA bending energy profile.

DNA damage induced by ascorbate in the presence of Cu2+.

PubMed

Kobayashi, S; Ueda, K; Morita, J; Sakai, H; Komano, T

1988-01-25

DNA damage induced by ascorbate in the presence of Cu2+ was investigated by use of bacteriophage phi X174 double-stranded supercoiled DNA and linear restriction fragments as substrates. Single-strand cleavage was induced when supercoiled DNA was incubated with 5 microM-10 mM ascorbate and 50 microM Cu2+ at 37 degrees C for 10 min. The induced DNA damage was analyzed by sequencing of fragments singly labeled at their 5'- or 3'-end. DNA was cleaved directly and almost uniformly at every nucleotide by ascorbate and Cu2+. Piperidine treatment after the reaction showed that ascorbate and Cu2+ induced another kind of DNA damage different from the direct cleavage. The damage proceeded to DNA cleavage by piperidine treatment and was sequence-specific rather than random. These results indicate that ascorbate induces two classes of DNA damage in the presence of Cu2+, one being direct strand cleavage, probably via damage to the DNA backbone, and the other being a base modification labile to alkali treatment. These two classes of DNA damage were inhibited by potassium iodide, catalase and metal chelaters, suggesting the involvement of radicals generated from ascorbate hydroperoxide.

Using directed evolution to improve hydrogen production in chimeric hydrogenases from Clostridia species.

PubMed

Plummer, Scott M; Plummer, Mark A; Merkel, Patricia A; Hagen, Moira; Biddle, Jennifer F; Waidner, Lisa A

2016-11-01

Hydrogenases are enzymes that play a key role in controlling excess reducing equivalents in both photosynthetic and anaerobic organisms. This enzyme is viewed as potentially important for the industrial generation of hydrogen gas; however, insufficient hydrogen production has impeded its use in a commercial process. Here, we explore the potential to circumvent this problem by directly evolving the Fe-Fe hydrogenase genes from two species of Clostridia bacteria. In addition, a computational model based on these mutant sequences was developed and used as a predictive aid for the isolation of enzymes with even greater efficiency in hydrogen production. Two of the improved mutants have a logarithmic increase in hydrogen production in our in vitro assay. Furthermore, the model predicts hydrogenase sequences with hydrogen productions as high as 540-fold over the positive control. Taken together, these results demonstrate the potential of directed evolution to improve the native bacterial hydrogenases as a first step for improvement of hydrogenase activity, further in silico prediction, and finally, construction and demonstration of an improved algal hydrogenase in an in vivo assay of C. reinhardtii hydrogen production. Copyright © 2016 Elsevier Inc. All rights reserved.

In silico site-directed mutagenesis informs species-specific predictions of chemical susceptibility derived from the Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool

EPA Science Inventory

The Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS) tool was developed to address needs for rapid, cost effective methods of species extrapolation of chemical susceptibility. Specifically, the SeqAPASS tool compares the primary sequence (Level 1), functiona...

Hadoop-BAM: directly manipulating next generation sequencing data in the cloud

PubMed Central

Niemenmaa, Matti; Kallio, Aleksi; Schumacher, André; Klemelä, Petri; Korpelainen, Eija; Heljanko, Keijo

2012-01-01

Summary: Hadoop-BAM is a novel library for the scalable manipulation of aligned next-generation sequencing data in the Hadoop distributed computing framework. It acts as an integration layer between analysis applications and BAM files that are processed using Hadoop. Hadoop-BAM solves the issues related to BAM data access by presenting a convenient API for implementing map and reduce functions that can directly operate on BAM records. It builds on top of the Picard SAM JDK, so tools that rely on the Picard API are expected to be easily convertible to support large-scale distributed processing. In this article we demonstrate the use of Hadoop-BAM by building a coverage summarizing tool for the Chipster genome browser. Our results show that Hadoop offers good scalability, and one should avoid moving data in and out of Hadoop between analysis steps. Availability: Available under the open-source MIT license at http://sourceforge.net/projects/hadoop-bam/ Contact: matti.niemenmaa@aalto.fi Supplementary information: Supplementary material is available at Bioinformatics online. PMID:22302568

MyLabStocks: a web-application to manage molecular biology materials

PubMed Central

Chuffart, Florent; Yvert, Gaël

2014-01-01

Laboratory stocks are the hardware of research. They must be stored and managed with mimimum loss of material and information. Plasmids, oligonucleotides and strains are regularly exchanged between collaborators within and between laboratories. Managing and sharing information about every item is crucial for retrieval of reagents, for planning experiments and for reproducing past experimental results. We have developed a web-based application to manage stocks commonly used in a molecular biology laboratory. Its functionalities include user-defined privileges, visualization of plasmid maps directly from their sequence and the capacity to search items from fields of annotation or directly from a query sequence using BLAST. It is designed to handle records of plasmids, oligonucleotides, yeast strains, antibodies, pipettes and notebooks. Based on PHP/MySQL, it can easily be extended to handle other types of stocks and it can be installed on any server architecture. MyLabStocks is freely available from: https://forge.cbp.ens-lyon.fr/redmine/projects/mylabstocks under an open source licence. PMID:24643870

Code-Time Diversity for Direct Sequence Spread Spectrum Systems

PubMed Central

Hassan, A. Y.

2014-01-01

Time diversity is achieved in direct sequence spread spectrum by receiving different faded delayed copies of the transmitted symbols from different uncorrelated channel paths when the transmission signal bandwidth is greater than the coherence bandwidth of the channel. In this paper, a new time diversity scheme is proposed for spread spectrum systems. It is called code-time diversity. In this new scheme, N spreading codes are used to transmit one data symbol over N successive symbols interval. The diversity order in the proposed scheme equals to the number of the used spreading codes N multiplied by the number of the uncorrelated paths of the channel L. The paper represents the transmitted signal model. Two demodulators structures will be proposed based on the received signal models from Rayleigh flat and frequency selective fading channels. Probability of error in the proposed diversity scheme is also calculated for the same two fading channels. Finally, simulation results are represented and compared with that of maximal ration combiner (MRC) and multiple-input and multiple-output (MIMO) systems. PMID:24982925

Nontrivial paths and periodic orbits of the T-fractal billiard table

NASA Astrophysics Data System (ADS)

Lapidus, Michel L.; Miller, Robyn L.; Niemeyer, Robert G.

2016-07-01

We introduce and prove numerous new results about the orbits of the T-fractal billiard. Specifically, in section 3, we give a variety of sufficient conditions for the existence of a sequence of compatible periodic orbits. In section 4, we examine the limiting behavior of particular sequences of compatible periodic orbits. Additionally, sufficient conditions for the existence of particular nontrivial paths are given in section 4. The proofs of two results of Lapidus and Niemeyer (2013 The current state of fractal billiards Fractal Geometry and Dynamical Systems in Pure and Applied Mathematics II: Fractals in Applied Mathematics (Contemporary Mathematics vol 601) ed D Carfi et al (Providence, RI: American Mathematical Society) pp 251-88 (e-print: arXiv:math.DS.1210.0282v2, 2013) appear here for the first time, as well. In section 5, an orbit with an irrational initial direction reaches an elusive point in a way that yields a nontrivial path of finite length, yet, by our convention, constitutes a singular orbit of the fractal billiard table. The existence of such an orbit seems to indicate that the classification of orbits may not be so straightforward. A discussion of our results and directions for future research is then given in section 6.

Precision of working memory for visual motion sequences and transparent motion surfaces.

PubMed

Zokaei, Nahid; Gorgoraptis, Nikos; Bahrami, Bahador; Bays, Paul M; Husain, Masud

2011-12-01

Recent studies investigating working memory for location, color, and orientation support a dynamic resource model. We examined whether this might also apply to motion, using random dot kinematograms (RDKs) presented sequentially or simultaneously. Mean precision for motion direction declined as sequence length increased, with precision being lower for earlier RDKs. Two alternative models of working memory were compared specifically to distinguish between the contributions of different sources of error that corrupt memory (W. Zhang & S. J. Luck, 2008 vs. P. M. Bays, R. F. G. Catalao, & M. Husain, 2009). The latter provided a significantly better fit for the data, revealing that decrease in memory precision for earlier items is explained by an increase in interference from other items in a sequence rather than random guessing or a temporal decay of information. Misbinding feature attributes is an important source of error in working memory. Precision of memory for motion direction decreased when two RDKs were presented simultaneously as transparent surfaces, compared to sequential RDKs. However, precision was enhanced when one motion surface was prioritized, demonstrating that selective attention can improve recall precision. These results are consistent with a resource model that can be used as a general conceptual framework for understanding working memory across a range of visual features.

Rapid detection of microbial DNA by a novel isothermal genome exponential amplification reaction (GEAR) assay.

PubMed

Prithiviraj, Jothikumar; Hill, Vincent; Jothikumar, Narayanan

2012-04-20

In this study we report the development of a simple target-specific isothermal nucleic acid amplification technique, termed genome exponential amplification reaction (GEAR). Escherichia coli was selected as the microbial target to demonstrate the GEAR technique as a proof of concept. The GEAR technique uses a set of four primers; in the present study these primers targeted 5 regions on the 16S rRNA gene of E. coli. The outer forward and reverse Tab primer sequences are complementary to each other at their 5' end, whereas their 3' end sequences are complementary to their respective target nucleic acid sequences. The GEAR assay was performed at a constant temperature 60 °C and monitored continuously in a real-time PCR instrument in the presence of an intercalating dye (SYTO 9). The GEAR assay enabled amplification of as few as one colony forming units of E. coli per reaction within 30 min. We also evaluated the GEAR assay for rapid identification of bacterial colonies cultured on agar media directly in the reaction without DNA extraction. Cells from E. coli colonies were picked and added directly to GEAR assay mastermix without prior DNA extraction. DNA in the cells could be amplified, yielding positive results within 15 min. Published by Elsevier Inc.

Human action recognition based on spatial-temporal descriptors using key poses

NASA Astrophysics Data System (ADS)

Hu, Shuo; Chen, Yuxin; Wang, Huaibao; Zuo, Yaqing

2014-11-01

Human action recognition is an important area of pattern recognition today due to its direct application and need in various occasions like surveillance and virtual reality. In this paper, a simple and effective human action recognition method is presented based on the key poses of human silhouette and the spatio-temporal feature. Firstly, the contour points of human silhouette have been gotten, and the key poses are learned by means of K-means clustering based on the Euclidean distance between each contour point and the centre point of the human silhouette, and then the type of each action is labeled for further match. Secondly, we obtain the trajectories of centre point of each frame, and create a spatio-temporal feature value represented by W to describe the motion direction and speed of each action. The value W contains the information of location and temporal order of each point on the trajectories. Finally, the matching stage is performed by comparing the key poses and W between training sequences and test sequences, the nearest neighbor sequences is found and its label supplied the final result. Experiments on the public available Weizmann datasets show the proposed method can improve accuracy by distinguishing amphibious poses and increase suitability for real-time applications by reducing the computational cost.

Genomic analysis suggests that mRNA destabilization by the microprocessor is specialized for the auto-regulation of Dgcr8.

PubMed

Shenoy, Archana; Blelloch, Robert

2009-09-11

The Microprocessor, containing the RNA binding protein Dgcr8 and RNase III enzyme Drosha, is responsible for processing primary microRNAs to precursor microRNAs. The Microprocessor regulates its own levels by cleaving hairpins in the 5'UTR and coding region of the Dgcr8 mRNA, thereby destabilizing the mature transcript. To determine whether the Microprocessor has a broader role in directly regulating other coding mRNA levels, we integrated results from expression profiling and ultra high-throughput deep sequencing of small RNAs. Expression analysis of mRNAs in wild-type, Dgcr8 knockout, and Dicer knockout mouse embryonic stem (ES) cells uncovered mRNAs that were specifically upregulated in the Dgcr8 null background. A number of these transcripts had evolutionarily conserved predicted hairpin targets for the Microprocessor. However, analysis of deep sequencing data of 18 to 200nt small RNAs in mouse ES, HeLa, and HepG2 indicates that exonic sequence reads that map in a pattern consistent with Microprocessor activity are unique to Dgcr8. We conclude that the Microprocessor's role in directly destabilizing coding mRNAs is likely specifically targeted to Dgcr8 itself, suggesting a specialized cellular mechanism for gene auto-regulation.

Cloning and expression of 130-kd mosquito-larvicidal delta-endotoxin gene of Bacillus thuringiensis var. Israelensis in Escherichia coli.

PubMed

Angsuthanasombat, C; Chungjatupornchai, W; Kertbundit, S; Luxananil, P; Settasatian, C; Wilairat, P; Panyim, S

1987-07-01

Five recombinant E. coli clones exhibiting toxicity to Aedes aegypti larvae were obtained from a library of 800 clones containing XbaI DNA fragments of 110 kb plasmid from B. thuringiensis var. israelensis. All the five clones (pMU 14/258/303/388/679) had the same 3.8-kb insert and encoded a major protein of 130 kDa which was highly toxic to A. aegypti larvae. Three clones (pMU 258/303/388) transcribed the 130 kD a gene in the same direction as that of lac Z promoter of pUC12 vector whereas the transcription of the other two (pMU 14/679) was in the opposite direction. A 1.9-kb fragment of the 3.8 kb insert coded for a protein of 65 kDa. Partial DNA sequence of the 3.8 kb insert, corresponding to the 5'-terminal of the 130 kDa gene, revealed a continuous reading frame, a Shine-Dalgarno sequence and a tentative 5'-regulatory region. These results demonstrated that the 3.8 kb insert is a minimal DNA fragment containing a regulatory region plus the coding sequence of the 130 kDa protein that is highly toxic to mosquito larvae.

FREQ-Seq: A Rapid, Cost-Effective, Sequencing-Based Method to Determine Allele Frequencies Directly from Mixed Populations

PubMed Central

Delaney, Nigel F.; Marx, Christopher J.

2012-01-01

Understanding evolutionary dynamics within microbial populations requires the ability to accurately follow allele frequencies through time. Here we present a rapid, cost-effective method (FREQ-Seq) that leverages Illumina next-generation sequencing for localized, quantitative allele frequency detection. Analogous to RNA-Seq, FREQ-Seq relies upon counts from the >105 reads generated per locus per time-point to determine allele frequencies. Loci of interest are directly amplified from a mixed population via two rounds of PCR using inexpensive, user-designed oligonucleotides and a bar-coded bridging primer system that can be regenerated in-house. The resulting bar-coded PCR products contain the adapters needed for Illumina sequencing, eliminating further library preparation. We demonstrate the utility of FREQ-Seq by determining the order and dynamics of beneficial alleles that arose as a microbial population, founded with an engineered strain of Methylobacterium, evolved to grow on methanol. Quantifying allele frequencies with minimal bias down to 1% abundance allowed effective analysis of SNPs, small in-dels and insertions of transposable elements. Our data reveal large-scale clonal interference during the early stages of adaptation and illustrate the utility of FREQ-Seq as a cost-effective tool for tracking allele frequencies in populations. PMID:23118913

Direct Problem-Based Learning (DPBL): A Framework for Integrating Direct Instruction and Problem-Based Learning Approach

ERIC Educational Resources Information Center

Winarno, Sri; Muthu, Kalaiarasi Sonai; Ling, Lew Sook

2018-01-01

Direct instruction approach has been widely used in higher education. Many studies revealed that direct instruction improved students' knowledge. The characteristics of direct instruction include the subject delivered through face-to-face interaction with the lecturers and materials that sequenced deliberately and taught explicitly. However,…

Sedimentation studies reveal a direct role of phosphorylation in Smad3:Smad4 homo- and hetero-trimerization.

PubMed

Correia, J J; Chacko, B M; Lam, S S; Lin, K

2001-02-06

SMAD proteins are known to oligomerize and hetero-associate during their activation and translocation to the nucleus for transcriptional control. Analytical ultracentrifuge studies on Smad3 and Smad4 protein constructs are presented to clarify the model of homo- and hetero-oligomerization and the role of phosphorylation in the activation process. These constructs all exhibit a tendency to form disulfide cross-linked aggregates, primarily dimers, and a strong reducing agent, TCEP, was found to be required to determine the best estimates for reversible association models and equilibrium constants. A Smad4 construct, S4AF, consisting of the middle linker (L) domain and the C-terminal (C) domain, is shown to be a monomer, while a Smad3 construct, S3LC, consisting of the LC domains, is shown to form a trimer with an affinity K(3) = (1.2-3.1) x 10(9) M(-2). A Smad3 construct that mimics phosphorylation at the C-terminal target sequence, S3LC(3E), has 17--35-fold enhanced ability to form trimer over that of the wild-type construct, S3LC. S4AF associates with either S3LC or S3LC(3E) to form a hetero-trimer. In each case, the hetero-trimer is favored over the formation of the homo-trimer. Despite high sequence homology between Smad3 and Smad4, a chimeric Smad4 construct with an engineered Smad3 C-terminal pseudo-phosphorylation sequence, S4AF(3E), shows no tendency to form trimer. This suggests a Smad4-specific sequence insert inhibits homo-trimer formation, or other domains or sequences in S3LC are required in addition to the target sequence to mediate the formation of trimer. These results represent a direct molecular measure of the importance of hetero-trimerization and phosphorylation in the TGF-beta-activated Smad protein signal transduction process.

Genetic Characterization of a Sinorhizobium meliloti Chromosomal Region Involved in Lipopolysaccharide Biosynthesis

PubMed Central

Lagares, Antonio; Hozbor, Daniela F.; Niehaus, Karsten; Otero, Augusto J. L. Pich; Lorenzen, Jens; Arnold, Walter; Pühler, Alfred

2001-01-01

The genetic characterization of a 5.5-kb chromosomal region of Sinorhizobium meliloti 2011 that contains lpsB, a gene required for the normal development of symbiosis with Medicago spp., is presented. The nucleotide sequence of this DNA fragment revealed the presence of six genes: greA and lpsB, transcribed in the forward direction; and lpsE, lpsD, lpsC, and lrp, transcribed in the reverse direction. Except for lpsB, none of the lps genes were relevant for nodulation and nitrogen fixation. Analysis of the transcriptional organization of lpsB showed that greA and lpsB are part of separate transcriptional units, which is in agreement with the finding of a DNA stretch homologous to a “nonnitrogen” promoter consensus sequence between greA and lpsB. The opposite orientation of lpsB with respect to its first downstream coding sequence, lpsE, indicated that the altered LPS and the defective symbiosis of lpsB mutants are both consequences of a primary nonpolar defect in a single gene. Global sequence comparisons revealed that the greA-lpsB and lrp genes of S. meliloti have a genetic organization similar to that of their homologous loci in R. leguminosarum bv. viciae. In particular, high sequence similarity was found between the translation product of lpsB and a core-related biosynthetic mannosyltransferase of R. leguminosarum bv. viciae encoded by the lpcC gene. The functional relationship between these two genes was demonstrated in genetic complementation experiments in which the S. meliloti lpsB gene restored the wild-type LPS phenotype when introduced into lpcC mutants of R. leguminosarum. These results support the view that S. meliloti lpsB also encodes a mannosyltransferase that participates in the biosynthesis of the LPS core. Evidence is provided for the presence of other lpsB-homologous sequences in several members of the family Rhizobiaceae. PMID:11157937

Variants of the D{sub 5} dopamine receptor gene found in patients with schizophrenia: Identification of a nonsense mutation and multiple missense changes

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

Sobell, J.L.; Lind, T.J.; Sommer, S.S.

To determine whether mutations in the D{sub 5} dopamine receptor (D{sub 5}DR) gene are associated with schizophrenia, the gene was examined in 78 unrelated schizophrenic individuals. After amplification by the polymerase chain reaction, products were examined by dideoxy fingerprinting (ddF), a highly sensitive screening method related to single strand conformational polymorphism analysis. All samples with unusual ddF patterns were sequenced to precisely identify the sequence change. In the 156 D{sub 5}DR alleles examined, nine sequence changes were identified. Four of the nine did not affect protein structure; of these, three were silent changes and one was a transition in themore » 3{prime} untranslated region. The remaining five sequence changes result in protein alterations: of these, one is a missense change in a non-conserved amino acid, 3 are missense changes in amino acids that are conserved in some dopamine D{sub 5} receptors and the last is a nonsense mutation. To investigate whether the nonsense mutation was associated with schizophrenia, 400 additional schizophrenic cases of western European descent and 1914 ethnically-similar controls were screened for the change. One additional schizophrenic carrier was identified and verified by direct genomic sequencing (allele frequency: .0013), but eight carriers also were found and confirmed among the non-schizophrenics (allele frequency: .0021)(p>.25). The gene was re-examined in all newly identified carriers of the nonsense mutation by direct sequencing and/or ddF in search of additional mutations. None were identified. Family studies also were conducted to investigate possible cosegregation of the mutation with other neuropsychiatric diseases, but this was not demonstrated. Thus, the mutation does not appear to be associated with an increased risk of schizophrenia nor does an initial analysis suggest cosegregation with other neuropsychiatric disorders or symptom complexes.« less

The nonamer UUAUUUAUU is the key AU-rich sequence motif that mediates mRNA degradation.

PubMed Central

Zubiaga, A M; Belasco, J G; Greenberg, M E

1995-01-01

Labile mRNAs that encode cytokine and immediate-early gene products often contain AU-rich sequences within their 3' untranslated region (UTR). These AU-rich sequences appear to be key determinants of the short half-lives of these mRNAs, although the sequence features of these elements and the mechanism by which they target mRNAs for rapid decay have not been fully defined. We have examined the features of AU-rich elements (AREs) that are crucial for their function as determinants of mRNA instability in mammalian cells by testing the ability of various mutant c-fos AREs and synthetic AREs to direct rapid mRNA deadenylation and decay when inserted within the 3' UTR of the normally stable beta-globin mRNA. Evidence is presented that the pentamer AUUUA, which previously was suggested to be the minimal determinant of instability present in mammalian AREs, cannot direct rapid mRNA deadenylation and decay. Instead, the nonomer UUAUUUAUU is the elemental AU-rich sequence motif that destabilizes mRNA. Removal of one uridine residue from either end of the nonamer (UUAUUUAU or UAUUUAUU) results in a decrease of potency of the element, while removal of a uridine residue from both ends of the nonamer (UAUUUAU) eliminates detectable destabilizing activity. The inclusion of an additional uridine residue at both ends of the nonamer (UUUAUUUAUUU) does not further increase the efficacy of the element. Taken together, these findings suggest that the nonamer UUAUUUAUU is the minimal AU-rich motif that effectively destabilizes mRNA. Additional ARE potency is achieved by combining multiple copies of this nonamer in a single mRNA 3' UTR. Furthermore, analysis of poly(A) shortening rates for ARE-containing mRNAs reveals that the UUAUUUAUU sequence also accelerates mRNA deadenylation and suggests that the UUAUUUAUU motif targets mRNA for rapid deadenylation as an early step in the mRNA decay process. PMID:7891716

Vertebral Osteomyelitis Caused by Helicobacter cinaedi Identified Using Broad-range Polymerase Chain Reaction with Sequencing of the Biopsied Specimen.

PubMed

Hase, Ryota; Hirooka, Takuya; Itabashi, Takashi; Endo, Yasunobu; Otsuka, Yoshihito

2018-05-15

A 65-year-old man presented with gradually exacerbating low back pain. Magnetic resonance imaging revealed vertebral osteomyelitis in the Th11-L2 vertebral bodies and discs. The patient showed negative findings on conventional cultures. Direct broad-range polymerase chain reaction (PCR) with sequencing of the biopsied specimen had the highest similarity to the 16S rRNA gene of Helicobacter cinaedi. This case suggests that direct broad-range PCR with sequencing should be considered when conventional cultures cannot identify the causative organism of vertebral osteomyelitis, and that this method may be particularly useful when the pathogen is a fastidious organism, such as H. cinaedi.

Enhancing genomic laboratory reports from the patients' view: A qualitative analysis.

PubMed

Stuckey, Heather; Williams, Janet L; Fan, Audrey L; Rahm, Alanna Kulchak; Green, Jamie; Feldman, Lynn; Bonhag, Michele; Zallen, Doris T; Segal, Michael M; Williams, Marc S

2015-10-01

The purpose of this study was to develop a family genomic laboratory report designed to communicate genome sequencing results to parents of children who were participating in a whole genome sequencing clinical research study. Semi-structured interviews were conducted with parents of children who participated in a whole genome sequencing clinical research study to address the elements, language and format of a sample family-directed genome laboratory report. The qualitative interviews were followed by two focus groups aimed at evaluating example presentations of information about prognosis and next steps related to the whole genome sequencing result. Three themes emerged from the qualitative data: (i) Parents described a continual search for valid information and resources regarding their child's condition, a need that prior reports did not meet for parents; (ii) Parents believed that the Family Report would help facilitate communication with physicians and family members; and (iii) Parents identified specific items they appreciated in a genomics Family Report: simplicity of language, logical flow, visual appeal, information on what to expect in the future and recommended next steps. Parents affirmed their desire for a family genomic results report designed for their use and reference. They articulated the need for clear, easy to understand language that provided information with temporal detail and specific recommendations regarding relevant findings consistent with that available to clinicians. © 2015 Wiley Periodicals, Inc.

Enhancing genomic laboratory reports from the patients' view: A qualitative analysis

PubMed Central

Stuckey, Heather; Fan, Audrey L.; Rahm, Alanna Kulchak; Green, Jamie; Feldman, Lynn; Bonhag, Michele; Zallen, Doris T.; Segal, Michael M.; Williams, Marc S.

2015-01-01

The purpose of this study was to develop a family genomic laboratory report designed to communicate genome sequencing results to parents of children who were participating in a whole genome sequencing clinical research study. Semi‐structured interviews were conducted with parents of children who participated in a whole genome sequencing clinical research study to address the elements, language and format of a sample family‐directed genome laboratory report. The qualitative interviews were followed by two focus groups aimed at evaluating example presentations of information about prognosis and next steps related to the whole genome sequencing result. Three themes emerged from the qualitative data: (i) Parents described a continual search for valid information and resources regarding their child's condition, a need that prior reports did not meet for parents; (ii) Parents believed that the Family Report would help facilitate communication with physicians and family members; and (iii) Parents identified specific items they appreciated in a genomics Family Report: simplicity of language, logical flow, visual appeal, information on what to expect in the future and recommended next steps. Parents affirmed their desire for a family genomic results report designed for their use and reference. They articulated the need for clear, easy to understand language that provided information with temporal detail and specific recommendations regarding relevant findings consistent with that available to clinicians. PMID:26086630

Meta sequence analysis of human blood peptides and their parent proteins.

PubMed

Bowden, Peter; Pendrak, Voitek; Zhu, Peihong; Marshall, John G

2010-04-18

Sequence analysis of the blood peptides and their qualities will be key to understanding the mechanisms that contribute to error in LC-ESI-MS/MS. Analysis of peptides and their proteins at the level of sequences is much more direct and informative than the comparison of disparate accession numbers. A portable database of all blood peptide and protein sequences with descriptor fields and gene ontology terms might be useful for designing immunological or MRM assays from human blood. The results of twelve studies of human blood peptides and/or proteins identified by LC-MS/MS and correlated against a disparate array of genetic libraries were parsed and matched to proteins from the human ENSEMBL, SwissProt and RefSeq databases by SQL. The reported peptide and protein sequences were organized into an SQL database with full protein sequences and up to five unique peptides in order of prevalence along with the peptide count for each protein. Structured query language or BLAST was used to acquire descriptive information in current databases. Sampling error at the level of peptides is the largest source of disparity between groups. Chi Square analysis of peptide to protein distributions confirmed the significant agreement between groups on identified proteins. Copyright 2010. Published by Elsevier B.V.

Quantification of turbulence and velocity in stenotic flow using spiral three-dimensional phase-contrast MRI.

PubMed

Petersson, Sven; Dyverfeldt, Petter; Sigfridsson, Andreas; Lantz, Jonas; Carlhäll, Carl-Johan; Ebbers, Tino

2016-03-01

Evaluate spiral three-dimensional (3D) phase contrast MRI for the assessment of turbulence and velocity in stenotic flow. A-stack-of-spirals 3D phase contrast MRI sequence was evaluated in vitro against a conventional Cartesian sequence. Measurements were made in a flow phantom with a 75% stenosis. Both spiral and Cartesian imaging were performed using different scan orientations and flow rates. Volume flow rate, maximum velocity and turbulent kinetic energy (TKE) were computed for both methods. Moreover, the estimated TKE was compared with computational fluid dynamics (CFD) data. There was good agreement between the turbulent kinetic energy from the spiral, Cartesian and CFD data. Flow rate and maximum velocity from the spiral data agreed well with Cartesian data. As expected, the short echo time of the spiral sequence resulted in less prominent displacement artifacts compared with the Cartesian sequence. However, both spiral and Cartesian flow rate estimates were sensitive to displacement when the flow was oblique to the encoding directions. Spiral 3D phase contrast MRI appears favorable for the assessment of stenotic flow. The spiral sequence was more than three times faster and less sensitive to displacement artifacts when compared with a conventional Cartesian sequence. © 2015 Wiley Periodicals, Inc.

Variation in the genomic locations and sequence conservation of STAR elements among staphylococcal species provides insight into DNA repeat evolution

PubMed Central

2012-01-01

Background Staphylococcus aureus Repeat (STAR) elements are a type of interspersed intergenic direct repeat. In this study the conservation and variation in these elements was explored by bioinformatic analyses of published staphylococcal genome sequences and through sequencing of specific STAR element loci from a large set of S. aureus isolates. Results Using bioinformatic analyses, we found that the STAR elements were located in different genomic loci within each staphylococcal species. There was no correlation between the number of STAR elements in each genome and the evolutionary relatedness of staphylococcal species, however higher levels of repeats were observed in both S. aureus and S. lugdunensis compared to other staphylococcal species. Unexpectedly, sequencing of the internal spacer sequences of individual repeat elements from multiple isolates showed conservation at the sequence level within deep evolutionary lineages of S. aureus. Whilst individual STAR element loci were demonstrated to expand and contract, the sequences associated with each locus were stable and distinct from one another. Conclusions The high degree of lineage and locus-specific conservation of these intergenic repeat regions suggests that STAR elements are maintained due to selective or molecular forces with some of these elements having an important role in cell physiology. The high prevalence in two of the more virulent staphylococcal species is indicative of a potential role for STAR elements in pathogenesis. PMID:23020678

Differential expression profiles of miRNAs induced by vaccination followed by Marek’s disease virus challenge at cytolytic stage in chickens resistant or susceptible to Marek’s disease

USDA-ARS?s Scientific Manuscript database

Mounting evidence shows microRNAs (miRNAs) directly regulate gene expression post-transcriptionally through base-pairing with regions in the 3’-untranslated sequences of target gene mRNAs, which results in dysregulation of gene expression/translation and subsequently modulates cellular processes. We...

Multiple bidirectional initiations and terminations of transcription in the Marek's disease virus long repeat regions.

PubMed Central

Chen, X B; Velicer, L F

1991-01-01

Marek's disease is an oncogenic disease of chickens caused by a herpesvirus, Marek's disease virus (MDV). Serial in vitro passage of pathogenic MDV results in amplification of a 132-bp direct repeat in the MDV genome's TRL and IRL repeat regions and loss of tumorigenicity. This led to the hypothesis that upon such expansion, one or more tumor-inducing genes fail to be expressed. In this report a group of cDNAs mapping in the expanded regions were isolated from a pathogenic MDV strain in which the 132-bp direct repeat number was found to range between one and seven. Partial cDNA sequencing and S1 nuclease protection analysis revealed that the corresponding transcripts are either initiated or terminated within or near the expanded regions at multiple sites in both rightward and leftward directions. Furthermore, each 132-bp repeat contains one TATA box and two polyadenylation consensus sequences in each direction. These RNAs contain a partial copy or one or more full copies of the 132-bp direct repeat at either their 5' or 3' end. Northern (RNA) blot analysis showed that the majority of transcripts are 1.8 kb in size, while the minor species range in size from 0.67 to 3.1 kb. Together, these data raise the possibility that the 132-bp direct repeat, and indirectly its copy number, may be involved in the regulation of transcriptional initiation and termination and therefore in the generation of four groups of transcripts from the TRL and IRL, although this remains to be demonstrated. Images PMID:1850022

Secretion of alpha 2-plasmin inhibitor is impaired by amino acid deletion in a small region of the molecule.

PubMed

Toyota, S; Hirosawa, S; Aoki, N

1994-02-01

Alpha 2-plasmin inhibitor (alpha 2PI) deficiency Okinawa results from defective secretion of the inhibitor from the liver and appears to be a direct consequence of the deletion of Glu137 in the amino acid sequence of alpha 2PI. To examine the effects of replacing the amino acid occupying position 137 or deleting its neighboring amino acid on alpha 2PI secretion, we used oligonucleotide-directed mutagenesis of alpha 2PI cDNA to change the codon specifying Glu137 or delete a codon specifying its neighboring amino acid. The effects were determined by pulse-chase experiments and by enzyme-linked immunosorbent assay of media from transiently transfected COS-7 cells. Replacement of Glu137 with an amino acid other than Cys had little effect on alpha 2PI secretion. In contrast, deletion of an amino acid in a region spanning a sequence of less than 30 amino acids including positions 127 and 137 severely impaired the secretion. The results suggest that structural integrity of the region, rather than its component amino acids, is important for the intracellular transport and secretion of alpha 2PI.

Determining orientation and direction of DNA sequences

DOEpatents

Goodwin, Edwin H.; Meyne, Julianne

2000-01-01

Determining orientation and direction of DNA sequences. A method by which fluorescence in situ hybridization can be made strand specific is described. Cell cultures are grown in a medium containing a halogenated nucleotide. The analog is partially incorporated in one DNA strand of each chromatid. This substitution takes place in opposite strands of the two sister chromatids. After staining with the fluorescent DNA-binding dye Hoechst 33258, cells are exposed to long-wavelength ultraviolet light which results in numerous strand nicks. These nicks enable the substituted strand to be denatured and solubilized by heat, treatment with high or low pH aqueous solutions, or by immersing the strands in 2.times.SSC (0.3M NaCl+0.03M sodium citrate), to name three procedures. It is unnecessary to enzymatically digest the strands using Exo III or another exonuclease in order to excise and solubilize nucleotides starting at the sites of the nicks. The denaturing/solubilizing process removes most of the substituted strand while leaving the prereplication strand largely intact. Hybridization of a single-stranded probe of a tandem repeat arranged in a head-to-tail orientation will result in hybridization only to the chromatid with the complementary strand present.

A novel mutation of the Keratin 12 gene responsible for a severe phenotype of Meesmann's corneal dystrophy

PubMed Central

Sullivan, Lori S.; Baylin, Eric B.; Font, Ramon; Daiger, Stephen P.; Pepose, Jay S.; Clinch, Thomas E.; Nakamura, Hisashi; Zhao, Xinping C.

2007-01-01

Purpose To determine if a mutation within the coding region of the keratin 12 gene (KRT12) is responsible for a severe form of Meesmann's corneal dystrophy. Methods A family with clinically identified Meesmann's corneal dystrophy was recruited and studied. Electron microscopy was performed on scrapings of corneal epithelial cells from the proband. Mutations in the KRT12 gene were sought using direct genomic sequencing of leukocyte DNA from two affected and two unaffected family members. Subsequently, the observed mutation was screened in all available family members using polymerase chain reaction and direct sequencing. Results A heterozygous missense mutation (Arg430Pro) was found in exon 6 of KRT12 in all 14 affected individuals studied. Unaffected family members and 100 normal controls were negative for this mutation. Conclusions We have identified a novel mutation in the KRT12 gene that is associated with a symptomatic phenotype of Meesmann's corneal dystrophy. This mutation results in a substitution of proline for arginine in the helix termination motif that may disrupt the normal helix, leading to a dramatic structural change of the keratin 12 protein. PMID:17653038

Determination of haplotypes at structurally complex regions using emulsion haplotype fusion PCR

PubMed Central

2012-01-01

Background Genotyping and massively-parallel sequencing projects result in a vast amount of diploid data that is only rarely resolved into its constituent haplotypes. It is nevertheless this phased information that is transmitted from one generation to the next and is most directly associated with biological function and the genetic causes of biological effects. Despite progress made in genome-wide sequencing and phasing algorithms and methods, problems assembling (and reconstructing linear haplotypes in) regions of repetitive DNA and structural variation remain. These dynamic and structurally complex regions are often poorly understood from a sequence point of view. Regions such as these that are highly similar in their sequence tend to be collapsed onto the genome assembly. This is turn means downstream determination of the true sequence haplotype in these regions poses a particular challenge. For structurally complex regions, a more focussed approach to assembling haplotypes may be required. Results In order to investigate reconstruction of spatial information at structurally complex regions, we have used an emulsion haplotype fusion PCR approach to reproducibly link sequences of up to 1kb in length to allow phasing of multiple variants from neighbouring loci, using allele-specific PCR and sequencing to detect the phase. By using emulsion systems linking flanking regions to amplicons within the CNV, this led to the reconstruction of a 59kb haplotype across the DEFA1A3 CNV in HapMap individuals. Conclusion This study has demonstrated a novel use for emulsion haplotype fusion PCR in addressing the issue of reconstructing structural haplotypes at multiallelic copy variable regions, using the DEFA1A3 locus as an example. PMID:23231411

Primary motor and premotor cortex in implicit sequence learning--evidence for competition between implicit and explicit human motor memory systems.

PubMed

Kantak, Shailesh S; Mummidisetty, Chaithanya K; Stinear, James W

2012-09-01

Implicit and explicit memory systems for motor skills compete with each other during and after motor practice. Primary motor cortex (M1) is known to be engaged during implicit motor learning, while dorsal premotor cortex (PMd) is critical for explicit learning. To elucidate the neural substrates underlying the interaction between implicit and explicit memory systems, adults underwent a randomized crossover experiment of anodal transcranial direct current stimulation (AtDCS) applied over M1, PMd or sham stimulation during implicit motor sequence (serial reaction time task, SRTT) practice. We hypothesized that M1-AtDCS during practice will enhance online performance and offline learning of the implicit motor sequence. In contrast, we also hypothesized that PMd-AtDCS will attenuate performance and retention of the implicit motor sequence. Implicit sequence performance was assessed at baseline, at the end of acquisition (EoA), and 24 h after practice (retention test, RET). M1-AtDCS during practice significantly improved practice performance and supported offline stabilization compared with Sham tDCS. Performance change from EoA to RET revealed that PMd-AtDCS during practice attenuated offline stabilization compared with M1-AtDCS and sham stimulation. The results support the role of M1 in implementing online performance gains and offline stabilization for implicit motor sequence learning. In contrast, enhancing the activity within explicit motor memory network nodes such as the PMd during practice may be detrimental to offline stabilization of the learned implicit motor sequence. These results support the notion of competition between implicit and explicit motor memory systems and identify underlying neural substrates that are engaged in this competition. © 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

High precision in protein contact prediction using fully convolutional neural networks and minimal sequence features.

PubMed

Jones, David T; Kandathil, Shaun M

2018-04-26

In addition to substitution frequency data from protein sequence alignments, many state-of-the-art methods for contact prediction rely on additional sources of information, or features, of protein sequences in order to predict residue-residue contacts, such as solvent accessibility, predicted secondary structure, and scores from other contact prediction methods. It is unclear how much of this information is needed to achieve state-of-the-art results. Here, we show that using deep neural network models, simple alignment statistics contain sufficient information to achieve state-of-the-art precision. Our prediction method, DeepCov, uses fully convolutional neural networks operating on amino-acid pair frequency or covariance data derived directly from sequence alignments, without using global statistical methods such as sparse inverse covariance or pseudolikelihood estimation. Comparisons against CCMpred and MetaPSICOV2 show that using pairwise covariance data calculated from raw alignments as input allows us to match or exceed the performance of both of these methods. Almost all of the achieved precision is obtained when considering relatively local windows (around 15 residues) around any member of a given residue pairing; larger window sizes have comparable performance. Assessment on a set of shallow sequence alignments (fewer than 160 effective sequences) indicates that the new method is substantially more precise than CCMpred and MetaPSICOV2 in this regime, suggesting that improved precision is attainable on smaller sequence families. Overall, the performance of DeepCov is competitive with the state of the art, and our results demonstrate that global models, which employ features from all parts of the input alignment when predicting individual contacts, are not strictly needed in order to attain precise contact predictions. DeepCov is freely available at https://github.com/psipred/DeepCov. d.t.jones@ucl.ac.uk.

Direct Estimation of Structure and Motion from Multiple Frames

DTIC Science & Technology

1990-03-01

sequential frames in an image sequence. As a consequence, the information that can be extracted from a single optical flow field is limited to a snapshot of...researchers have developed techniques that extract motion and structure inform.4tion without computation of the optical flow. Best known are the "direct...operated iteratively on a sequence of images to recover structure. It required feature extraction and matching. Broida and Chellappa [9] suggested the use of

Genetic differences between blood- and brain-derived viral sequences from human immunodeficiency virus type 1-infected patients: evidence of conserved elements in the V3 region of the envelope protein of brain-derived sequences.

PubMed Central

Korber, B T; Kunstman, K J; Patterson, B K; Furtado, M; McEvilly, M M; Levy, R; Wolinsky, S M

1994-01-01

Human immunodeficiency virus type 1 (HIV-1) sequences were generated from blood and from brain tissue obtained by stereotactic biopsy from six patients undergoing a diagnostic neurosurgical procedure. Proviral DNA was directly amplified by nested PCR, and 8 to 36 clones from each sample were sequenced. Phylogenetic analysis of intrapatient envelope V3-V5 region HIV-1 DNA sequence sets revealed that brain viral sequences were clustered relative to the blood viral sequences, suggestive of tissue-specific compartmentalization of the virus in four of the six cases. In the other two cases, the blood and brain virus sequences were intermingled in the phylogenetic analyses, suggesting trafficking of virus between the two tissues. Slide-based PCR-driven in situ hybridization of two of the patients' brain biopsy samples confirmed our interpretation of the intrapatient phylogenetic analyses. Interpatient V3 region brain-derived sequence distances were significantly less than blood-derived sequence distances. Relative to the tip of the loop, the set of brain-derived viral sequences had a tendency towards negative or neutral charge compared with the set of blood-derived viral sequences. Entropy calculations were used as a measure of the variability at each position in alignments of blood and brain viral sequences. A relatively conserved set of positions were found, with a significantly lower entropy in the brain-than in the blood-derived viral sequences. These sites constitute a brain "signature pattern," or a noncontiguous set of amino acids in the V3 region conserved in viral sequences derived from brain tissue. This brain-derived signature pattern was also well preserved among isolates previously characterized in vitro as macrophage tropic. Macrophage-monocyte tropism may be the biological constraint that results in the conservation of the viral brain signature pattern. Images PMID:7933130

Myelin protein zero gene sequencing diagnoses Charcot-Marie-Tooth Type 1B disease

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

Su, Y.; Zhang, H.; Madrid, R.

1994-09-01

Charcot-Marie-Tooth disease (CMT), the most common genetic neuropathy, affects about 1 in 2600 people in Norway and is found worldwide. CMT Type 1 (CMT1) has slow nerve conduction with demyelinated Schwann cells. Autosomal dominant CMT Type 1B (CMT1B) results from mutations in the myelin protein zero gene which directs the synthesis of more than half of all Schwann cell protein. This gene was mapped to the chromosome 1q22-1q23.1 borderline by fluorescence in situ hybridization. The first 7 of 7 reported CMT1B mutations are unique. Thus the most effective means to identify CMT1B mutations in at-risk family members and fetuses ismore » to sequence the entire coding sequence in dominant or sporadic CMT patients without the CMT1A duplication. Of the 19 primers used in 16 pars to uniquely amplify the entire MPZ coding sequence, 6 primer pairs were used to amplify and sequence the 6 exons. The DyeDeoxy Terminator cycle sequencing method used with four different color fluorescent lables was superior to manual sequencing because it sequences more bases unambiguously from extracted genomic DNA samples within 24 hours. This protocol was used to test 28 CMT and Dejerine-Sottas patients without CMT1A gene duplication. Sequencing MPZ gene-specific amplified fragments identified 9 polymorphic sites within the 6 exons that encode the 248 amino acid MPZ protein. The large number of major CMT1B mutations identified by single strand sequencing are being verified by reverse strand sequencing and when possible, by restriction enzyme analysis. This protocol can be used to distringuish CMT1B patients from othre CMT phenotypes and to determine the CMT1B status of relatives both presymptomatically and prenatally.« less

Comparative genomic sequence analysis of novel Helicoverpa armigera nucleopolyhedrovirus (NPV) isolated from Kenya and three other previously sequenced Helicoverpa spp. NPVs.

PubMed

Ogembo, Javier Gordon; Caoili, Barbara L; Shikata, Masamitsu; Chaeychomsri, Sudawan; Kobayashi, Michihiro; Ikeda, Motoko

2009-10-01

A newly cloned Helicoverpa armigera nucleopolyhedrovirus (HearNPV) from Kenya, HearNPV-NNg1, has a higher insecticidal activity than HearNPV-G4, which also exhibits lower insecticidal activity than HearNPV-C1. In the search for genes and/or nucleotide sequences that might be involved in the observed virulence differences among Helicoverpa spp. NPVs, the entire genome of NNg1 was sequenced and compared with previously sequenced genomes of G4, C1 and Helicoverpa zea single-nucleocapsid NPV (Hz). The NNg1 genome was 132,425 bp in length, with a total of 143 putative open reading frames (ORFs), and shared high levels of overall amino acid and nucleotide sequence identities with G4, C1 and Hz. Three NNg1 ORFs, ORF5, ORF100 and ORF124, which were shared with C1, were absent in G4 and Hz, while NNg1 and C1 were missing a homologue of G4/Hz ORF5. Another three ORFs, ORF60 (bro-b), ORF119 and ORF120, and one direct repeat sequence (dr) were unique to NNg1. Relative to the overall nucleotide sequence identity, lower sequence identities were observed between NNg1 hrs and the homologous hrs in the other three Helicoverpa spp. NPVs, despite containing the same number of hrs located at essentially the same positions on the genomes. Differences were also observed between NNg1 and each of the other three Helicoverpa spp. NPVs in the diversity of bro genes encoded on the genomes. These results indicate several putative genes and nucleotide sequences that may be responsible for the virulence differences observed among Helicoverpa spp., yet the specific genes and/or nucleotide sequences responsible have not been identified.

Genotype diversity of hepatitis C virus (HCV) in HCV-associated liver disease patients in Indonesia.

PubMed

Utama, Andi; Tania, Navessa Padma; Dhenni, Rama; Gani, Rino Alvani; Hasan, Irsan; Sanityoso, Andri; Lelosutan, Syafruddin A R; Martamala, Ruswhandi; Lesmana, Laurentius Adrianus; Sulaiman, Ali; Tai, Susan

2010-09-01

Hepatitis C virus (HCV) genotype distribution in Indonesia has been reported. However, the identification of HCV genotype was based on 5'-UTR or NS5B sequence. This study was aimed to observe HCV core sequence variation among HCV-associated liver disease patients in Jakarta, and to analyse the HCV genotype diversity based on the core sequence. Sixty-eight chronic hepatitis (CH), 48 liver cirrhosis (LC) and 34 hepatocellular carcinoma (HCC) were included in this study. HCV core variation was analysed by direct sequencing. Alignment of HCV core sequences demonstrated that the core sequence was relatively varied among the genotype. Indeed, 237 bases of the core sequence could classify the HCV subtype; however, 236 bases failed to differentiate several subtypes. Based on 237 bases of the core sequences, the HCV strains were classified into genotypes 1 (subtypes 1a, 1b and 1c), 2 (subtypes 2a, 2e and 2f) and 3 (subtypes 3a and 3k). The HCV 1b (47.3%) was the most prevalent, followed by subtypes 1c (18.7%), 3k (10.7%), 2a (10.0%), 1a (6.7%), 2e (5.3%), 2f (0.7%) and 3a (0.7%). HCV 1b was the most common in all patients, and the prevalence increased with the severity of liver disease (36.8% in CH, 54.2% in LC and 58.8% in HCC). These results were similar to a previous report based on NS5B sequence analysis. Hepatitis C virus core sequence (237 bases) could identify the HCV subtype and the prevalence of HCV subtype based on core sequence was similar to those based on the NS5B region.

j5 v2.8.4

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

Hillson, Nathan

j5 automates and optimizes the design of the molecular biological process of cloning/constructing DNA. j5 enables users to benefit from (combinatorial) multi-part scar-less SLIC, Gibson, CPEC, Golden Gate assembly, or variants thereof, for which automation software does not currently exist, without the intense labor currently associated with the process. j5 inputs a list of the DNA sequences to be assembled, along with a Genbank, FASTA, jbei-seq, or SBOL v1.1 format sequence file for each DNA source. Given the list of DNA sequences to be assembled, j5 first determines the cost-minimizing assembly strategy for each part (direct synthesis, PCR/SOE, or oligo-embedding),more » designs DNA oligos with Primer3, adds flanking homology sequences (SLIC, Gibson, and CPEC; optimized with Primer3 for CPEC) or optimized overhang sequences (Golden Gate) to the oligos and direct synthesis pieces, and utilizes BLAST to check against oligo mis-priming and assembly piece incompatibility events. After identifying DNA oligos that are already contained within a local collection for reuse, the program estimates the total cost of direct synthesis and new oligos to be ordered. In the instance that j5 identifies putative assembly piece incompatibilities (multiple pieces with high flanking sequence homology), the program suggests hierarchical subassemblies where possible. The program outputs a comma-separated value (CSV) file, viewable via Excel or other spreadsheet software, that contains assembly design information (such as the PCR/SOE reactions to perform, their anticipated sizes and sequences, etc.) as well as a properly annotated genbank file containing the sequence resulting from the assembly, and appends the local oligo library with the oligos to be ordered j5 condenses multiple independent assembly projects into 96-well format for high-throughput liquid-handling robotics platforms, and generates configuration files for the PR-PR biology-friendly robot programming language. j5 thus provides a new way to design DNA assembly procedures much more productively and efficiently, not only in terms of time, but also in terms of cost. To a large extent, however, j5 does not allow people to do something that could not be done before by hand given enough time and effort. An exception to this is that, since the very act of using j5 to design the DNA assembly process standardizes the experimental details and workflow, j5 enables a single person to concurrently perform the independent DNA construction tasks of an entire group of researchers. Currently, this is not readily possible, since separate researchers employ disparate design strategies and workflows, and furthermore, their designs and workflows are very infrequently fully captured in an electronic format which is conducive to automation.« less

Fluorogenic DNA Sequencing in PDMS Microreactors

PubMed Central

Sims, Peter A.; Greenleaf, William J.; Duan, Haifeng; Xie, X. Sunney

2012-01-01

We have developed a multiplex sequencing-by-synthesis method combining terminal-phosphate labeled fluorogenic nucleotides (TPLFNs) and resealable microreactors. In the presence of phosphatase, the incorporation of a non-fluorescent TPLFN into a DNA primer by DNA polymerase results in a fluorophore. We immobilize DNA templates within polydimethylsiloxane (PDMS) microreactors, sequentially introduce one of the four identically labeled TPLFNs, seal the microreactors, allow template-directed TPLFN incorporation, and measure the signal from the fluorophores trapped in the microreactors. This workflow allows sequencing in a manner akin to pyrosequencing but without constant monitoring of each microreactor. With cycle times of <10 minutes, we demonstrate 30 base reads with ∼99% raw accuracy. “Fluorogenic pyrosequencing” combines benefits of pyrosequencing, such as rapid turn-around, native DNA generation, and single-color detection, with benefits of fluorescence-based approaches, such as highly sensitive detection and simple parallelization. PMID:21666670

The Degradome database: mammalian proteases and diseases of proteolysis.

PubMed

Quesada, Víctor; Ordóñez, Gonzalo R; Sánchez, Luis M; Puente, Xose S; López-Otín, Carlos

2009-01-01

The degradome is defined as the complete set of proteases present in an organism. The recent availability of whole genomic sequences from multiple organisms has led us to predict the contents of the degradomes of several mammalian species. To ensure the fidelity of these predictions, our methods have included manual curation of individual sequences and, when necessary, direct cloning and sequencing experiments. The results of these studies in human, chimpanzee, mouse and rat have been incorporated into the Degradome database, which can be accessed through a web interface at http://degradome.uniovi.es. The annotations about each individual protease can be retrieved by browsing catalytic classes and families or by searching specific terms. This web site also provides detailed information about genetic diseases of proteolysis, a growing field of great importance for multiple users. Finally, the user can find additional information about protease structures, protease inhibitors, ancillary domains of proteases and differences between mammalian degradomes.

The Degradome database: mammalian proteases and diseases of proteolysis

PubMed Central

Quesada, Víctor; Ordóñez, Gonzalo R.; Sánchez, Luis M.; Puente, Xose S.; López-Otín, Carlos

2009-01-01

The degradome is defined as the complete set of proteases present in an organism. The recent availability of whole genomic sequences from multiple organisms has led us to predict the contents of the degradomes of several mammalian species. To ensure the fidelity of these predictions, our methods have included manual curation of individual sequences and, when necessary, direct cloning and sequencing experiments. The results of these studies in human, chimpanzee, mouse and rat have been incorporated into the Degradome database, which can be accessed through a web interface at http://degradome.uniovi.es. The annotations about each individual protease can be retrieved by browsing catalytic classes and families or by searching specific terms. This web site also provides detailed information about genetic diseases of proteolysis, a growing field of great importance for multiple users. Finally, the user can find additional information about protease structures, protease inhibitors, ancillary domains of proteases and differences between mammalian degradomes. PMID:18776217

Functional specificity of a Hox protein mediated by the recognition of minor groove structure.

PubMed

Joshi, Rohit; Passner, Jonathan M; Rohs, Remo; Jain, Rinku; Sosinsky, Alona; Crickmore, Michael A; Jacob, Vinitha; Aggarwal, Aneel K; Honig, Barry; Mann, Richard S

2007-11-02

The recognition of specific DNA-binding sites by transcription factors is a critical yet poorly understood step in the control of gene expression. Members of the Hox family of transcription factors bind DNA by making nearly identical major groove contacts via the recognition helices of their homeodomains. In vivo specificity, however, often depends on extended and unstructured regions that link Hox homeodomains to a DNA-bound cofactor, Extradenticle (Exd). Using a combination of structure determination, computational analysis, and in vitro and in vivo assays, we show that Hox proteins recognize specific Hox-Exd binding sites via residues located in these extended regions that insert into the minor groove but only when presented with the correct DNA sequence. Our results suggest that these residues, which are conserved in a paralog-specific manner, confer specificity by recognizing a sequence-dependent DNA structure instead of directly reading a specific DNA sequence.

No exonic mutations at GJB2, GJB3, GJB4, GJB6, ARS (Component B), and LOR genes responsible for a Chinese patient affected by progressive symmetric erythrokeratodermia with pseudoainhum.

PubMed

Zhou, Fusheng; Fu, Hongyang; Liu, Linghua; Cui, Yong; Zhang, Zhengzhong; Chang, Ruixue; Yue, Zhen; Yang, Sen; Zhang, Xuejun

2014-09-01

Progressive symmetric erythrokeratodermia (PSEK) is characterized by symmetric and growing erythematous hyperkeratotic patches over the body shortly after birth, particularly trunk and limbs, the buttocks, and the face, sometimes together with palmoplantar keratoderma (PPK). The GJB2, GJB3, GJB4, GJB6, ARS (Component B), and LOR gene mutation might contribute to PSEK manifestation. This study aimed to identify sequence alteration of these genes in a Chinese PSEK patient with pseudoainhum. Genomic DNA was purified from the patient's peripheral blood. Mutation analysis of target genes was performed by direct sequencing using ABI 3730 sequencer No exonic mutations was identified in the aforementioned genes. The result underlines the genetic heterogeneity of PSEK and other related erythrokeratodermas. © 2014 The International Society of Dermatology.

160-fold acceleration of the Smith-Waterman algorithm using a field programmable gate array (FPGA)

PubMed Central

Li, Isaac TS; Shum, Warren; Truong, Kevin

2007-01-01

Background To infer homology and subsequently gene function, the Smith-Waterman (SW) algorithm is used to find the optimal local alignment between two sequences. When searching sequence databases that may contain hundreds of millions of sequences, this algorithm becomes computationally expensive. Results In this paper, we focused on accelerating the Smith-Waterman algorithm by using FPGA-based hardware that implemented a module for computing the score of a single cell of the SW matrix. Then using a grid of this module, the entire SW matrix was computed at the speed of field propagation through the FPGA circuit. These modifications dramatically accelerated the algorithm's computation time by up to 160 folds compared to a pure software implementation running on the same FPGA with an Altera Nios II softprocessor. Conclusion This design of FPGA accelerated hardware offers a new promising direction to seeking computation improvement of genomic database searching. PMID:17555593

Rare Sequence Variation in the Genome Flanking a Short Tandem Repeat Locus Can Lead to a Question of “Nonmaternity”

PubMed Central

Deucher, Anne; Chiang, Tsoyu; Schrijver, Iris

2010-01-01

Typing of STR (short tandem repeat) alleles is used in a variety of applications in clinical molecular pathology, including evaluations for maternal cell contamination. Using a commercially available STR typing assay for maternal cell contamination performed in conjunction with prenatal diagnostic testing, we were posed with apparent nonmaternity when the two fetal samples did not demonstrate the expected maternal allele at one locus. By designing primers external to the region amplified by the primers from the commercial assay and by performing direct sequencing of the resulting amplicon, we were able to determine that a guanine to adenine sequence variation led to primer mismatch and allele dropout. This explained the apparent null allele shared between the maternal and fetal samples. Therefore, although rare, allele dropout must be considered whenever unexplained homozygosity at an STR locus is observed. PMID:20203001

Staphylococcus nepalensis in the guano of bats (Mammalia).

PubMed

Vandžurová, A; Bačkor, P; Javorský, P; Pristaš, P

2013-05-31

Thirty randomly selected mesophilic isolates from the six years old guano sample from mixed Myotis myotis and M. blythii summer roosts colony were isolated and identified as Staphylococcus nepalensis using MALDI TOF analysis. 16S rRNA gene sequencing of selected five isolates and subsequent phylogenetic analysis confirmed that all sequences showed the highest similarity to S. nepalensis sequences. Several virulence factors were produced by tested isolates, mainly capsule formation and resistance to tetracycline, ampicillin, gentamycin, and chloramphenicol antibiotics. Our experiments show that the majority of cultivable mesophilic bacteria from the guano of bats belong to the S. nepalensis species. This is the first report on the occurrence of this species in the guano of bats and our results indicate that the guano accumulated near or directly in human dwellings and buildings may represent a significant risk for human health. Copyright © 2013 Elsevier B.V. All rights reserved.

Streamwise-Localized Solutions with natural 1-fold symmetry

NASA Astrophysics Data System (ADS)

Altmeyer, Sebastian; Willis, Ashley; Hof, Björn

2014-11-01

It has been proposed in recent years that turbulence is organized around unstable invariant solutions, which provide the building blocks of the chaotic dynamics. In direct numerical simulations of pipe flow we show that when imposing a minimal symmetry constraint (reflection in an axial plane only) the formation of turbulence can indeed be explained by dynamical systems concepts. The hypersurface separating laminar from turbulent motion, the edge of turbulence, is spanned by the stable manifolds of an exact invariant solution, a periodic orbit of a spatially localized structure. The turbulent states themselves (turbulent puffs in this case) are shown to arise in a bifurcation sequence from a related localized solution (the upper branch orbit). The rather complex bifurcation sequence involves secondary Hopf bifurcations, frequency locking and a period doubling cascade until eventually turbulent puffs arise. In addition we report preliminary results of the transition sequence for pipe flow without symmetry constraints.

Detection of the free living amoeba Naegleria fowleri by using conventional and real-time PCR based on a single copy DNA sequence.

PubMed

Régoudis, Estelle; Pélandakis, Michel

2016-02-01

The amoeba-flagellate Naegleria fowleri is a causative agent of primary amoebic meningoencephalitis (PAM). This thermophilic species occurs worldwide and tends to proliferate in warm aquatic environment. The PAM cases remain rare but this infection is mostly fatal. Here, we describe a single copy region which has been cloned and sequenced, and was used for both conventional and real-time PCR. Targeting a single-copy DNA sequence allows to directly quantify the N. fowleri cells. The real-time PCR results give a detection limit of 1 copy per reaction with high reproducibility without the need of a Taqman probe. This procedure is of interest as compared to other procedures which are mostly based on the detection of multi-copy DNA associated with a Taqman probe. Copyright © 2015 Elsevier Inc. All rights reserved.

Cognitive and neural foundations of discrete sequence skill: a TMS study.

PubMed

Ruitenberg, Marit F L; Verwey, Willem B; Schutter, Dennis J L G; Abrahamse, Elger L

2014-04-01

Executing discrete movement sequences typically involves a shift with practice from a relatively slow, stimulus-based mode to a fast mode in which performance is based on retrieving and executing entire motor chunks. The dual processor model explains the performance of (skilled) discrete key-press sequences in terms of an interplay between a cognitive processor and a motor system. In the present study, we tested and confirmed the core assumptions of this model at the behavioral level. In addition, we explored the involvement of the pre-supplementary motor area (pre-SMA) in discrete sequence skill by applying inhibitory 20 min 1-Hz off-line repetitive transcranial magnetic stimulation (rTMS). Based on previous work, we predicted pre-SMA involvement in the selection/initiation of motor chunks, and this was confirmed by our results. The pre-SMA was further observed to be more involved in more complex than in simpler sequences, while no evidence was found for pre-SMA involvement in direct stimulus-response translations or associative learning processes. In conclusion, support is provided for the dual processor model, and for pre-SMA involvement in the initiation of motor chunks. Copyright © 2014 Elsevier Ltd. All rights reserved.

cDNA cloning of Brassica napus malonyl-CoA:ACP transacylase (MCAT) (fab D) and complementation of an E. coli MCAT mutant.

PubMed

Simon, J W; Slabas, A R

1998-09-18

The GenBank database was searched using the E. coli malonyl CoA:ACP transacylase (MCAT) sequence, for plant protein/cDNA sequences corresponding to MCAT, a component of plant fatty acid synthetase (FAS), for which the plant cDNA has not been isolated. A 272-bp Zea mays EST sequence (GenBank accession number: AA030706) was identified which has strong homology to the E. coli MCAT. A PCR derived cDNA probe from Zea mays was used to screen a Brassica napus (rape) cDNA library. This resulted in the isolation of a 1200-bp cDNA clone which encodes an open reading frame corresponding to a protein of 351 amino acids. The protein shows 47% homology to the E. coli MCAT amino acid sequence in the coding region for the mature protein. Expression of a plasmid (pMCATrap2) containing the plant cDNA sequence in Fab D89, an E. coli mutant, in MCAT activity restores growth demonstrating functional complementation and direct function of the cloned cDNA. This is the first functional evidence supporting the identification of a plant cDNA for MCAT.

Phylogenetic Analysis of Theileria annulata Infected Cell Line S15 Iran Vaccine Strain.

PubMed

Habibi, Gh

2012-01-01

Bovine theileriosis results from infection with obligate intracellular protozoa of the genus Theileria. The phylogenetic relationships between two isolates of Theileria annulata, and 36 Theileria spp., as well as 6 outgroup including Babesia spp. and coccidian protozoa were analyzed using the 18S rRNA gene sequence. The target DNA segment was amplified by PCR. The PCR product was used for direct sequencing. The length of the 18S rRNA gene of all Theileria spp. involved in this study was around 1,400 bp. A phylogenetic tree was inferred based on the 18S rRNA gene sequence of the Iran and Iraq isolates, and other species of Theileria available in GenBank. In the constructed tree, Theileria annulata (Iran vaccine strain) was closely related to other T. annulata from Europe, Asia, as well as T. lestoquardi, T. parva and T. taurotragi all in one clade. Phylogenetic analyses based on small subunit ribosomal RNA gene suggested that the percent identity of the sequence of Iran vaccine strain was completely the same as Iraq sequence (100% identical), but the similarity of Iran vaccine strain with other T. annulata reported from China, Spain and Italy determined the 97.9 to 99.9% identity.

Investigation of DNA sequence recognition by a streptomycete MarR family transcriptional regulator through surface plasmon resonance and X-ray crystallography

PubMed Central

Stevenson, Clare E. M.; Assaad, Aoun; Chandra, Govind; Le, Tung B. K.; Greive, Sandra J.; Bibb, Mervyn J.; Lawson, David M.

2013-01-01

Consistent with their complex lifestyles and rich secondary metabolite profiles, the genomes of streptomycetes encode a plethora of transcription factors, the vast majority of which are uncharacterized. Herein, we use Surface Plasmon Resonance (SPR) to identify and delineate putative operator sites for SCO3205, a MarR family transcriptional regulator from Streptomyces coelicolor that is well represented in sequenced actinomycete genomes. In particular, we use a novel SPR footprinting approach that exploits indirect ligand capture to vastly extend the lifetime of a standard streptavidin SPR chip. We define two operator sites upstream of sco3205 and a pseudopalindromic consensus sequence derived from these enables further potential operator sites to be identified in the S. coelicolor genome. We evaluate each of these through SPR and test the importance of the conserved bases within the consensus sequence. Informed by these results, we determine the crystal structure of a SCO3205-DNA complex at 2.8 Å resolution, enabling molecular level rationalization of the SPR data. Taken together, our observations support a DNA recognition mechanism involving both direct and indirect sequence readout. PMID:23748564

Auditory and visual sequence learning in humans and monkeys using an artificial grammar learning paradigm.

PubMed

Milne, Alice E; Petkov, Christopher I; Wilson, Benjamin

2017-07-05

Language flexibly supports the human ability to communicate using different sensory modalities, such as writing and reading in the visual modality and speaking and listening in the auditory domain. Although it has been argued that nonhuman primate communication abilities are inherently multisensory, direct behavioural comparisons between human and nonhuman primates are scant. Artificial grammar learning (AGL) tasks and statistical learning experiments can be used to emulate ordering relationships between words in a sentence. However, previous comparative work using such paradigms has primarily investigated sequence learning within a single sensory modality. We used an AGL paradigm to evaluate how humans and macaque monkeys learn and respond to identically structured sequences of either auditory or visual stimuli. In the auditory and visual experiments, we found that both species were sensitive to the ordering relationships between elements in the sequences. Moreover, the humans and monkeys produced largely similar response patterns to the visual and auditory sequences, indicating that the sequences are processed in comparable ways across the sensory modalities. These results provide evidence that human sequence processing abilities stem from an evolutionarily conserved capacity that appears to operate comparably across the sensory modalities in both human and nonhuman primates. The findings set the stage for future neurobiological studies to investigate the multisensory nature of these sequencing operations in nonhuman primates and how they compare to related processes in humans. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Phylogenomics of Phrynosomatid Lizards: Conflicting Signals from Sequence Capture versus Restriction Site Associated DNA Sequencing

PubMed Central

Leaché, Adam D.; Chavez, Andreas S.; Jones, Leonard N.; Grummer, Jared A.; Gottscho, Andrew D.; Linkem, Charles W.

2015-01-01

Sequence capture and restriction site associated DNA sequencing (RADseq) are popular methods for obtaining large numbers of loci for phylogenetic analysis. These methods are typically used to collect data at different evolutionary timescales; sequence capture is primarily used for obtaining conserved loci, whereas RADseq is designed for discovering single nucleotide polymorphisms (SNPs) suitable for population genetic or phylogeographic analyses. Phylogenetic questions that span both “recent” and “deep” timescales could benefit from either type of data, but studies that directly compare the two approaches are lacking. We compared phylogenies estimated from sequence capture and double digest RADseq (ddRADseq) data for North American phrynosomatid lizards, a species-rich and diverse group containing nine genera that began diversifying approximately 55 Ma. Sequence capture resulted in 584 loci that provided a consistent and strong phylogeny using concatenation and species tree inference. However, the phylogeny estimated from the ddRADseq data was sensitive to the bioinformatics steps used for determining homology, detecting paralogs, and filtering missing data. The topological conflicts among the SNP trees were not restricted to any particular timescale, but instead were associated with short internal branches. Species tree analysis of the largest SNP assembly, which also included the most missing data, supported a topology that matched the sequence capture tree. This preferred phylogeny provides strong support for the paraphyly of the earless lizard genera Holbrookia and Cophosaurus, suggesting that the earless morphology either evolved twice or evolved once and was subsequently lost in Callisaurus. PMID:25663487

The impact of cerebellar transcranial direct current stimulation (tDCS) on learning fine-motor sequences

PubMed Central

Wu, Allan D.; Samra, Jasmine K.

2017-01-01

The cerebellum has been shown to be important for skill learning, including the learning of motor sequences. We investigated whether cerebellar transcranial direct current stimulation (tDCS) would enhance learning of fine motor sequences. Because the ability to generalize or transfer to novel task variations or circumstances is a crucial goal of real world training, we also examined the effect of tDCS on performance of novel sequences after training. In Study 1, participants received either anodal, cathodal or sham stimulation while simultaneously practising three eight-element key press sequences in a non-repeating, interleaved order. Immediately after sequence practice with concurrent tDCS, a transfer session was given in which participants practised three interleaved novel sequences. No stimulation was given during transfer. An inhibitory effect of cathodal tDCS was found during practice, such that the rate of learning was slowed in comparison to the anodal and sham groups. In Study 2, participants received anodal or sham stimulation and a 24 h delay was added between the practice and transfer sessions to reduce mental fatigue. Although this consolidation period benefitted subsequent transfer for both tDCS groups, anodal tDCS enhanced transfer performance. Together, these studies demonstrate polarity-specific effects on fine motor sequence learning and generalization. This article is part of the themed issue ‘New frontiers for statistical learning in the cognitive sciences’. PMID:27872369

The impact of cerebellar transcranial direct current stimulation (tDCS) on learning fine-motor sequences.

PubMed

Shimizu, Renee E; Wu, Allan D; Samra, Jasmine K; Knowlton, Barbara J

2017-01-05

The cerebellum has been shown to be important for skill learning, including the learning of motor sequences. We investigated whether cerebellar transcranial direct current stimulation (tDCS) would enhance learning of fine motor sequences. Because the ability to generalize or transfer to novel task variations or circumstances is a crucial goal of real world training, we also examined the effect of tDCS on performance of novel sequences after training. In Study 1, participants received either anodal, cathodal or sham stimulation while simultaneously practising three eight-element key press sequences in a non-repeating, interleaved order. Immediately after sequence practice with concurrent tDCS, a transfer session was given in which participants practised three interleaved novel sequences. No stimulation was given during transfer. An inhibitory effect of cathodal tDCS was found during practice, such that the rate of learning was slowed in comparison to the anodal and sham groups. In Study 2, participants received anodal or sham stimulation and a 24 h delay was added between the practice and transfer sessions to reduce mental fatigue. Although this consolidation period benefitted subsequent transfer for both tDCS groups, anodal tDCS enhanced transfer performance. Together, these studies demonstrate polarity-specific effects on fine motor sequence learning and generalization.This article is part of the themed issue 'New frontiers for statistical learning in the cognitive sciences'. © 2016 The Author(s).

Guiding principles for peptide nanotechnology through directed discovery.

PubMed

Lampel, A; Ulijn, R V; Tuttle, T

2018-05-21

Life's diverse molecular functions are largely based on only a small number of highly conserved building blocks - the twenty canonical amino acids. These building blocks are chemically simple, but when they are organized in three-dimensional structures of tremendous complexity, new properties emerge. This review explores recent efforts in the directed discovery of functional nanoscale systems and materials based on these same amino acids, but that are not guided by copying or editing biological systems. The review summarises insights obtained using three complementary approaches of searching the sequence space to explore sequence-structure relationships for assembly, reactivity and complexation, namely: (i) strategic editing of short peptide sequences; (ii) computational approaches to predicting and comparing assembly behaviours; (iii) dynamic peptide libraries that explore the free energy landscape. These approaches give rise to guiding principles on controlling order/disorder, complexation and reactivity by peptide sequence design.

Sequencing of hepatitis C virus for detection of resistance to direct-acting antiviral therapy: A systematic review.

PubMed

Bartlett, Sofia R; Grebely, Jason; Eltahla, Auda A; Reeves, Jacqueline D; Howe, Anita Y M; Miller, Veronica; Ceccherini-Silberstein, Francesca; Bull, Rowena A; Douglas, Mark W; Dore, Gregory J; Harrington, Patrick; Lloyd, Andrew R; Jacka, Brendan; Matthews, Gail V; Wang, Gary P; Pawlotsky, Jean-Michel; Feld, Jordan J; Schinkel, Janke; Garcia, Federico; Lennerstrand, Johan; Applegate, Tanya L

2017-07-01

The significance of the clinical impact of direct-acting antiviral (DAA) resistance-associated substitutions (RASs) in hepatitis C virus (HCV) on treatment failure is unclear. No standardized methods or guidelines for detection of DAA RASs in HCV exist. To facilitate further evaluations of the impact of DAA RASs in HCV, we conducted a systematic review of RAS sequencing protocols, compiled a comprehensive public library of sequencing primers, and provided expert guidance on the most appropriate methods to screen and identify RASs. The development of standardized RAS sequencing protocols is complicated due to a high genetic variability and the need for genotype- and subtype-specific protocols for multiple regions. We have identified several limitations of the available methods and have highlighted areas requiring further research and development. The development, validation, and sharing of standardized methods for all genotypes and subtypes should be a priority. ( Hepatology Communications 2017;1:379-390).

A Pan-HIV Strategy for Complete Genome Sequencing

PubMed Central

Yamaguchi, Julie; Alessandri-Gradt, Elodie; Tell, Robert W.; Brennan, Catherine A.

2015-01-01

Molecular surveillance is essential to monitor HIV diversity and track emerging strains. We have developed a universal library preparation method (HIV-SMART [i.e., switching mechanism at 5′ end of RNA transcript]) for next-generation sequencing that harnesses the specificity of HIV-directed priming to enable full genome characterization of all HIV-1 groups (M, N, O, and P) and HIV-2. Broad application of the HIV-SMART approach was demonstrated using a panel of diverse cell-cultured virus isolates. HIV-1 non-subtype B-infected clinical specimens from Cameroon were then used to optimize the protocol to sequence directly from plasma. When multiplexing 8 or more libraries per MiSeq run, full genome coverage at a median ∼2,000× depth was routinely obtained for either sample type. The method reproducibly generated the same consensus sequence, consistently identified viral sequence heterogeneity present in specimens, and at viral loads of ≤4.5 log copies/ml yielded sufficient coverage to permit strain classification. HIV-SMART provides an unparalleled opportunity to identify diverse HIV strains in patient specimens and to determine phylogenetic classification based on the entire viral genome. Easily adapted to sequence any RNA virus, this technology illustrates the utility of next-generation sequencing (NGS) for viral characterization and surveillance. PMID:26699702

Rapid Electrokinetic Isolation of Cancer-Related Circulating Cell-Free DNA Directly from Blood

PubMed Central

Sonnenberg, Avery; Marciniak, Jennifer Y.; Rassenti, Laura; Ghia, Emanuela M.; Skowronski, Elaine A.; Manouchehri, Sareh; McCanna, James; Widhopf, George F.; Kipps, Thomas J.; Heller, Michael J.

2014-01-01

BACKGROUND Circulating cell-free DNA (ccf-DNA) is becoming an important biomarker for cancer diagnostics and therapy monitoring. The isolation of ccf-DNA from plasma as a “liquid biopsy” may begin to replace more invasive tissue biopsies for the detection and analysis of cancer-related mutations. Conventional methods for the isolation of ccf-DNA from plasma are costly, time-consuming, and complex, preventing the use of ccf-DNA biomarkers for point-of-care diagnostics and limiting other biomedical research applications. METHODS We used an AC electrokinetic device to rapidly isolate ccf-DNA from 25 μL unprocessed blood. ccf-DNA from 15 chronic lymphocytic leukemia (CLL) patients and 3 healthy individuals was separated into dielectrophoretic (DEP) high-field regions, after which other blood components were removed by a fluidic wash. Concentrated ccf-DNA was detected by fluorescence and eluted for quantification,PCR,and DNA sequencing. The complete process, blood to PCR, required <10 min. ccf-DNA was amplified by PCR with immunoglobulin heavy chain variable region (IGHV)-specific primers to identify the unique IGHV gene expressed by the leukemic B-cell clone, and then sequenced. RESULTS PCR and DNA sequencing results obtained by DEP from 25 μL CLL blood matched results obtained by use of conventional methods for ccf-DNA isolation from 1 mL plasma and for genomic DNA isolation from CLL patient leukemic B cells isolated from 15–20 mL blood. CONCLUSIONS Rapid isolation of ccf-DNA directly from a drop of blood will advance disease-related biomarker research, accelerate the transition from tissue to liquid biopsies, and enable point-of-care diagnostic systems for patient monitoring. PMID:24270796

Human Pupillary Dilation Response to Deviant Auditory Stimuli: Effects of Stimulus Properties and Voluntary Attention

PubMed Central

Liao, Hsin-I; Yoneya, Makoto; Kidani, Shunsuke; Kashino, Makio; Furukawa, Shigeto

2016-01-01

A unique sound that deviates from a repetitive background sound induces signature neural responses, such as mismatch negativity and novelty P3 response in electro-encephalography studies. Here we show that a deviant auditory stimulus induces a human pupillary dilation response (PDR) that is sensitive to the stimulus properties and irrespective whether attention is directed to the sounds or not. In an auditory oddball sequence, we used white noise and 2000-Hz tones as oddballs against repeated 1000-Hz tones. Participants' pupillary responses were recorded while they listened to the auditory oddball sequence. In Experiment 1, they were not involved in any task. Results show that pupils dilated to the noise oddballs for approximately 4 s, but no such PDR was found for the 2000-Hz tone oddballs. In Experiments 2, two types of visual oddballs were presented synchronously with the auditory oddballs. Participants discriminated the auditory or visual oddballs while trying to ignore stimuli from the other modality. The purpose of this manipulation was to direct attention to or away from the auditory sequence. In Experiment 3, the visual oddballs and the auditory oddballs were always presented asynchronously to prevent residuals of attention on to-be-ignored oddballs due to the concurrence with the attended oddballs. Results show that pupils dilated to both the noise and 2000-Hz tone oddballs in all conditions. Most importantly, PDRs to noise were larger than those to the 2000-Hz tone oddballs regardless of the attention condition in both experiments. The overall results suggest that the stimulus-dependent factor of the PDR appears to be independent of attention. PMID:26924959

Rapid Drug Susceptibility Testing of Drug-Resistant Mycobacterium tuberculosis Isolates Directly from Clinical Samples by Use of Amplicon Sequencing: a Proof-of-Concept Study

PubMed Central

Anderson, Julia; Lemmer, Darrin; Lehmkuhl, Erik; Georghiou, Sophia B.; Heaton, Hannah; Wiggins, Kristin; Gillece, John D.; Schupp, James M.; Catanzaro, Donald G.; Crudu, Valeriu; Cohen, Ted; Rodwell, Timothy C.; Engelthaler, David M.

2016-01-01

Increasingly complex drug-resistant tuberculosis (DR-TB) is a major global health concern and one of the primary reasons why TB is now the leading infectious cause of death worldwide. Rapid characterization of a DR-TB patient's complete drug resistance profile would facilitate individualized treatment in place of empirical treatment, improve treatment outcomes, prevent amplification of resistance, and reduce the transmission of DR-TB. The use of targeted next-generation sequencing (NGS) to obtain drug resistance profiles directly from patient sputum samples has the potential to enable comprehensive evidence-based treatment plans to be implemented quickly, rather than in weeks to months, which is currently needed for phenotypic drug susceptibility testing (DST) results. In this pilot study, we evaluated the performance of amplicon sequencing of Mycobacterium tuberculosis DNA from patient sputum samples using a tabletop NGS technology and automated data analysis to provide a rapid DST solution (the Next Gen-RDST assay). One hundred sixty-six out of 176 (94.3%) sputum samples from the Republic of Moldova yielded complete Next Gen-RDST assay profiles for 7 drugs of interest. We found a high level of concordance of our Next Gen-RDST assay results with phenotypic DST (97.0%) and pyrosequencing (97.8%) results from the same clinical samples. Our Next Gen-RDST assay was also able to estimate the proportion of resistant-to-wild-type alleles down to mixtures of ≤1%, which demonstrates the ability to detect very low levels of resistant variants not detected by pyrosequencing and possibly below the threshold for phenotypic growth methods. The assay as described here could be used as a clinical or surveillance tool. PMID:27225403

High-Throughput Sequencing of microRNAs in Glucocorticoid Sensitive Paediatric Inflammatory Bowel Disease Patients.

PubMed

De Iudicibus, Sara; Lucafò, Marianna; Vitulo, Nicola; Martelossi, Stefano; Zimbello, Rosanna; De Pascale, Fabio; Forcato, Claudio; Naviglio, Samuele; Di Silvestre, Alessia; Gerdol, Marco; Stocco, Gabriele; Valle, Giorgio; Ventura, Alessandro; Bramuzzo, Matteo; Decorti, Giuliana

2018-05-08

The aim of this research was the identification of novel pharmacogenomic biomarkers for better understanding the complex gene regulation mechanisms underpinning glucocorticoid (GC) action in paediatric inflammatory bowel disease (IBD). This goal was achieved by evaluating high-throughput microRNA (miRNA) profiles during GC treatment, integrated with the assessment of expression changes in GC receptor (GR) heterocomplex genes. Furthermore, we tested the hypothesis that differentially expressed miRNAs could be directly regulated by GCs through investigating the presence of GC responsive elements (GREs) in their gene promoters. Ten IBD paediatric patients responding to GCs were enrolled. Peripheral blood was obtained at diagnosis (T0) and after four weeks of steroid treatment (T4). MicroRNA profiles were analyzed using next generation sequencing, and selected significantly differentially expressed miRNAs were validated by quantitative reverse transcription-polymerase chain reaction. In detail, 18 miRNAs were differentially expressed from T0 to T4, 16 of which were upregulated and 2 of which were downregulated. Out of these, three miRNAs (miR-144, miR-142, and miR-96) could putatively recognize the 3’UTR of the GR gene and three miRNAs (miR-363, miR-96, miR-142) contained GREs sequences, thereby potentially enabling direct regulation by the GR. In conclusion, we identified miRNAs differently expressed during GC treatment and miRNAs which could be directly regulated by GCs in blood cells of young IBD patients. These results could represent a first step towards their translation as pharmacogenomic biomarkers.

Establishing a comprehensive genetic diagnosis strategy for hemophilia B and its application in Chinese population.

PubMed

Lin, X Y; Wang, J; Xiao, X; Xu, Y W; Yan, Q J; Jiang, W Y

2018-04-01

To reduce the incidence of hemophilia B (HB) which with no complete cure currently, prenatal diagnosis and preimplantation genetic diagnosis (PGD) are effective and feasible means. However, previous studies about genetic diagnosis in HB mostly just focused on the detection of patients and carriers. Here, we established a comprehensive genetic diagnosis strategy for HB and worked it out in Chinese population. The strategy includes the detection of patients and carriers, prenatal diagnosis, and PGD. Seven unrelated HB families from Chinese population involved in this study. Firstly, probands and available members were carried out coagulation laboratory assays, and the clinical information has been recorded. Secondly, we used DNA direct sequencing to screen the whole FIX gene of them. The pathogenicity of novel mutations was verified according to 2015 ACMG-AM guidelines. For prenatal diagnosis, a mix of DNA direct sequencing and STR linkage analysis was employed. To explore a better PGD protocol, Karyomapping was first applied in PGD of HB, comparing with conventional PCR-based methods. Six different pathogenic mutations including 1 novel duplication (c.660_661dup ATCA) were identified. The results of prenatal diagnosis were consistent with birth outcomes. In the PGD case, 4 of 11 embryos were confirmed to be normal and one of them was transferred and led to a healthy birth. The established genetic diagnosis strategy for HB in our study was comprehensive and well applied in clinic practice. Besides, we recommended that DNA direct sequencing combined with Karyomapping was a better PGD protocol. © 2017 John Wiley & Sons Ltd.

A direct repeat of E-box-like elements is required for cell-autonomous circadian rhythm of clock genes

PubMed Central

Nakahata, Yasukazu; Yoshida, Mayumi; Takano, Atsuko; Soma, Haruhiko; Yamamoto, Takuro; Yasuda, Akio; Nakatsu, Toru; Takumi, Toru

2008-01-01

Background The circadian expression of the mammalian clock genes is based on transcriptional feedback loops. Two basic helix-loop-helix (bHLH) PAS (for Period-Arnt-Sim) domain-containing transcriptional activators, CLOCK and BMAL1, are known to regulate gene expression by interacting with a promoter element termed the E-box (CACGTG). The non-canonical E-boxes or E-box-like sequences have also been reported to be necessary for circadian oscillation. Results We report a new cis-element required for cell-autonomous circadian transcription of clock genes. This new element consists of a canonical E-box or a non-canonical E-box and an E-box-like sequence in tandem with the latter with a short interval, 6 base pairs, between them. We demonstrate that both E-box or E-box-like sequences are needed to generate cell-autonomous oscillation. We also verify that the spacing nucleotides with constant length between these 2 E-elements are crucial for robust oscillation. Furthermore, by in silico analysis we conclude that several clock and clock-controlled genes possess a direct repeat of the E-box-like elements in their promoter region. Conclusion We propose a novel possible mechanism regulated by double E-box-like elements, not to a single E-box, for circadian transcriptional oscillation. The direct repeat of the E-box-like elements identified in this study is the minimal required element for the generation of cell-autonomous transcriptional oscillation of clock and clock-controlled genes. PMID:18177499