Dbf4-dependent kinase and the Rtt107 scaffold promote Mus81-Mms4 resolvase activation during mitosis.

PubMed

Princz, Lissa N; Wild, Philipp; Bittmann, Julia; Aguado, F Javier; Blanco, Miguel G; Matos, Joao; Pfander, Boris

2017-03-01

DNA repair by homologous recombination is under stringent cell cycle control. This includes the last step of the reaction, disentanglement of DNA joint molecules (JMs). Previous work has established that JM resolving nucleases are activated specifically at the onset of mitosis. In case of budding yeast Mus81-Mms4, this cell cycle stage-specific activation is known to depend on phosphorylation by CDK and Cdc5 kinases. Here, we show that a third cell cycle kinase, Cdc7-Dbf4 (DDK), targets Mus81-Mms4 in conjunction with Cdc5-both kinases bind to as well as phosphorylate Mus81-Mms4 in an interdependent manner. Moreover, DDK-mediated phosphorylation of Mms4 is strictly required for Mus81 activation in mitosis, establishing DDK as a novel regulator of homologous recombination. The scaffold protein Rtt107, which binds the Mus81-Mms4 complex, interacts with Cdc7 and thereby targets DDK and Cdc5 to the complex enabling full Mus81 activation. Therefore, Mus81 activation in mitosis involves at least three cell cycle kinases, CDK, Cdc5 and DDK Furthermore, tethering of the kinases in a stable complex with Mus81 is critical for efficient JM resolution. © 2017 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

A Novel Non-canonical Forkhead-associated (FHA) Domain-binding Interface Mediates the Interaction between Rad53 and Dbf4 Proteins*

PubMed Central

Matthews, Lindsay A.; Selvaratnam, Rajeevan; Jones, Darryl R.; Akimoto, Madoka; McConkey, Brendan J.; Melacini, Giuseppe; Duncker, Bernard P.; Guarné, Alba

2014-01-01

Forkhead-associated (FHA) and BRCA1 C-terminal (BRCT) domains are overrepresented in DNA damage and replication stress response proteins. They function primarily as phosphoepitope recognition modules but can also mediate non-canonical interactions. The latter are rare, and only a few have been studied at a molecular level. We have identified a crucial non-canonical interaction between the N-terminal FHA1 domain of the checkpoint effector kinase Rad53 and the BRCT domain of the regulatory subunit of the Dbf4-dependent kinase that is critical to suppress late origin firing and to stabilize stalled forks during replication stress. The Rad53-Dbf4 interaction is phosphorylation-independent and involves a novel non-canonical interface on the FHA1 domain. Mutations within this surface result in hypersensitivity to genotoxic stress. Importantly, this surface is not conserved in the FHA2 domain of Rad53, suggesting that the FHA domains of Rad53 gain specificity by engaging additional interaction interfaces beyond their phosphoepitope-binding site. In general, our results point to FHA domains functioning as complex logic gates rather than mere phosphoepitope-targeting modules. PMID:24285546

Hierarchical MFMO Circuit Modules for an Energy-Efficient SDR DBF

NASA Astrophysics Data System (ADS)

Mar, Jeich; Kuo, Chi-Cheng; Wu, Shin-Ru; Lin, You-Rong

The hierarchical multi-function matrix operation (MFMO) circuit modules are designed using coordinate rotations digital computer (CORDIC) algorithm for realizing the intensive computation of matrix operations. The paper emphasizes that the designed hierarchical MFMO circuit modules can be used to develop a power-efficient software-defined radio (SDR) digital beamformer (DBF). The formulas of the processing time for the scalable MFMO circuit modules implemented in field programmable gate array (FPGA) are derived to allocate the proper logic resources for the hardware reconfiguration. The hierarchical MFMO circuit modules are scalable to the changing number of array branches employed for the SDR DBF to achieve the purpose of power saving. The efficient reuse of the common MFMO circuit modules in the SDR DBF can also lead to energy reduction. Finally, the power dissipation and reconfiguration function in the different modes of the SDR DBF are observed from the experiment results.

Development and validation of a Database Forensic Metamodel (DBFM)

PubMed Central

Al-dhaqm, Arafat; Razak, Shukor; Othman, Siti Hajar; Ngadi, Asri; Ahmed, Mohammed Nazir; Ali Mohammed, Abdulalem

2017-01-01

Database Forensics (DBF) is a widespread area of knowledge. It has many complex features and is well known amongst database investigators and practitioners. Several models and frameworks have been created specifically to allow knowledge-sharing and effective DBF activities. However, these are often narrow in focus and address specified database incident types. We have analysed 60 such models in an attempt to uncover how numerous DBF activities are really public even when the actions vary. We then generate a unified abstract view of DBF in the form of a metamodel. We identified, extracted, and proposed a common concept and reconciled concept definitions to propose a metamodel. We have applied a metamodelling process to guarantee that this metamodel is comprehensive and consistent. PMID:28146585

External validity of two nomograms for predicting distant brain failure after radiosurgery for brain metastases in a bi-institutional independent patient cohort.

PubMed

Prabhu, Roshan S; Press, Robert H; Boselli, Danielle M; Miller, Katherine R; Lankford, Scott P; McCammon, Robert J; Moeller, Benjamin J; Heinzerling, John H; Fasola, Carolina E; Patel, Kirtesh R; Asher, Anthony L; Sumrall, Ashley L; Curran, Walter J; Shu, Hui-Kuo G; Burri, Stuart H

2018-03-01

Patients treated with stereotactic radiosurgery (SRS) for brain metastases (BM) are at increased risk of distant brain failure (DBF). Two nomograms have been recently published to predict individualized risk of DBF after SRS. The goal of this study was to assess the external validity of these nomograms in an independent patient cohort. The records of consecutive patients with BM treated with SRS at Levine Cancer Institute and Emory University between 2005 and 2013 were reviewed. Three validation cohorts were generated based on the specific nomogram or recursive partitioning analysis (RPA) entry criteria: Wake Forest nomogram (n = 281), Canadian nomogram (n = 282), and Canadian RPA (n = 303) validation cohorts. Freedom from DBF at 1-year in the Wake Forest study was 30% compared with 50% in the validation cohort. The validation c-index for both the 6-month and 9-month freedom from DBF Wake Forest nomograms was 0.55, indicating poor discrimination ability, and the goodness-of-fit test for both nomograms was highly significant (p < 0.001), indicating poor calibration. The 1-year actuarial DBF in the Canadian nomogram study was 43.9% compared with 50.9% in the validation cohort. The validation c-index for the Canadian 1-year DBF nomogram was 0.56, and the goodness-of-fit test was also highly significant (p < 0.001). The validation accuracy and c-index of the Canadian RPA classification was 53% and 0.61, respectively. The Wake Forest and Canadian nomograms for predicting risk of DBF after SRS were found to have limited predictive ability in an independent bi-institutional validation cohort. These results reinforce the importance of validating predictive models in independent patient cohorts.

Structural changes in Mcm5 protein bypass Cdc7-Dbf4 function and reduce replication origin efficiency in Saccharomyces cerevisiae.

PubMed

Hoang, Margaret L; Leon, Ronald P; Pessoa-Brandao, Luis; Hunt, Sonia; Raghuraman, M K; Fangman, Walton L; Brewer, Bonita J; Sclafani, Robert A

2007-11-01

Eukaryotic chromosomal replication is a complicated process with many origins firing at different efficiencies and times during S phase. Prereplication complexes are assembled on all origins in G(1) phase, and yet only a subset of complexes is activated during S phase by DDK (for Dbf4-dependent kinase) (Cdc7-Dbf4). The yeast mcm5-bob1 (P83L) mutation bypasses DDK but results in reduced intrinsic firing efficiency at 11 endogenous origins and at origins located on minichromosomes. Origin efficiency may result from Mcm5 protein assuming an altered conformation, as predicted from the atomic structure of an archaeal MCM (for minichromosome maintenance) homologue. Similarly, an intragenic mutation in a residue predicted to interact with P83L suppresses the mcm5-bob1 bypass phenotype. We propose DDK phosphorylation of the MCM complex normally results in a single, highly active conformation of Mcm5, whereas the mcm5-bob1 mutation produces a number of conformations, only one of which is permissive for origin activation. Random adoption of these alternate states by the mcm5-bob1 protein can explain both how origin firing occurs independently of DDK and why origin efficiency is reduced. Because similar mutations in mcm2 and mcm4 cannot bypass DDK, Mcm5 protein may be a unique Mcm protein that is the final target of DDK regulation.

Hydrophobic Motif Phosphorylation Coordinates Activity and Polar Localization of the Neurospora crassa Nuclear Dbf2-Related Kinase COT1

PubMed Central

Maerz, Sabine; Dettmann, Anne

2012-01-01

Nuclear Dbf2p-related (NDR) kinases and associated proteins are recognized as a conserved network that regulates eukaryotic cell polarity. NDR kinases require association with MOB adaptor proteins and phosphorylation of two conserved residues in the activation segment and hydrophobic motif for activity and function. We demonstrate that the Neurospora crassa NDR kinase COT1 forms inactive dimers via a conserved N-terminal extension, which is also required for the interaction of the kinase with MOB2 to generate heterocomplexes with basal activity. Basal kinase activity also requires autophosphorylation of the COT1-MOB2 complex in the activation segment, while hydrophobic motif phosphorylation of COT1 by the germinal center kinase POD6 fully activates COT1 through induction of a conformational change. Hydrophobic motif phosphorylation is also required for plasma membrane association of the COT1-MOB2 complex. MOB2 further restricts the membrane-associated kinase complex to the hyphal apex to promote polar cell growth. These data support an integrated mechanism of NDR kinase regulation in vivo, in which kinase activation and cellular localization of COT1 are coordinated by dual phosphorylation and interaction with MOB2. PMID:22451488

Prediction of new brain metastases after radiosurgery: validation and analysis of performance of a multi-institutional nomogram.

PubMed

Ayala-Peacock, Diandra N; Attia, Albert; Braunstein, Steve E; Ahluwalia, Manmeet S; Hepel, Jaroslaw; Chung, Caroline; Contessa, Joseph; McTyre, Emory; Peiffer, Ann M; Lucas, John T; Isom, Scott; Pajewski, Nicholas M; Kotecha, Rupesh; Stavas, Mark J; Page, Brandi R; Kleinberg, Lawrence; Shen, Colette; Taylor, Robert B; Onyeuku, Nasarachi E; Hyde, Andrew T; Gorovets, Daniel; Chao, Samuel T; Corso, Christopher; Ruiz, Jimmy; Watabe, Kounosuke; Tatter, Stephen B; Zadeh, Gelareh; Chiang, Veronica L S; Fiveash, John B; Chan, Michael D

2017-11-01

Stereotactic radiosurgery (SRS) without whole brain radiotherapy (WBRT) for brain metastases can avoid WBRT toxicities, but with risk of subsequent distant brain failure (DBF). Sole use of number of metastases to triage patients may be an unrefined method. Data on 1354 patients treated with SRS monotherapy from 2000 to 2013 for new brain metastases was collected across eight academic centers. The cohort was divided into training and validation datasets and a prognostic model was developed for time to DBF. We then evaluated the discrimination and calibration of the model within the validation dataset, and confirmed its performance with an independent contemporary cohort. Number of metastases (≥8, HR 3.53 p = 0.0001), minimum margin dose (HR 1.07 p = 0.0033), and melanoma histology (HR 1.45, p = 0.0187) were associated with DBF. A prognostic index derived from the training dataset exhibited ability to discriminate patients' DBF risk within the validation dataset (c-index = 0.631) and Heller's explained relative risk (HERR) = 0.173 (SE = 0.048). Absolute number of metastases was evaluated for its ability to predict DBF in the derivation and validation datasets, and was inferior to the nomogram. A nomogram high-risk threshold yielding a 2.1-fold increased need for early WBRT was identified. Nomogram values also correlated to number of brain metastases at time of failure (r = 0.38, p < 0.0001). We present a multi-institutionally validated prognostic model and nomogram to predict risk of DBF and guide risk-stratification of patients who are appropriate candidates for radiosurgery versus upfront WBRT.

Infant feeding patterns and eczema in children in the first 6 years of life.

PubMed

Soto-Ramírez, N; Kar, S; Zhang, H; Karmaus, W

2017-10-01

Modes of infant feeding such as direct and indirect breastfeeding, and formula feeding, and their combinations may play a role in child health. The aim was to investigate which feeding patterns in the first 6 months pose risks of eczema/skin allergy in children up to 6 years compared to direct breastfeeding for at least 3 months. The Infant Feeding Practices Study II in the United States and its 6-year follow-up provided data on feeding modes in infancy and doctor's diagnosed eczema/skin allergy in the first 6 years of life (1387 infants), based on parental reports. Different feeding patterns were identified. Log-linear models were used to estimate prevalence ratios (PRs) of feeding patterns for doctor's diagnosed eczema/skin allergy in the first 6 years of life, adjusting for confounders. Compared to "direct breastfeeding for at least 3 months" (DBF3m), the combination of "direct feeding at the breast (DBF), pumping and feeding breast milk (BM), and formula (FF) in the first months" (DBF/BM/FF) showed a statistically significant higher risk of eczema/skin allergy in the first 6 years of life (PR = 1.46), adjusting for confounders. DBF combined with BM for the first 3 months followed by mixed feeding also had an increased risk (PR = 1.26), although not statistically significant. Formula feeding introduced since birth had no effect on eczema. Among the confounders, paternal eczema and race/ethnicity (Hispanic vs White) were associated with a higher risk of eczema/skin allergy. Mixed infant feeding may carry a higher risk of eczema/skin allergy compared to direct feeding at the breast. The recent epidemic of pumping and feeding in the United States and the use of mixed infant feeding modes requires additional studies to provide appropriate and renewed assessments of the risks of feeding modes for the future development of allergies. © 2017 John Wiley & Sons Ltd.

Determination of torasemide by fluorescence quenching method with some dihalogenated fluorescein dyes as probes

NASA Astrophysics Data System (ADS)

Cui, Zhiping; Liu, Shaopu; Liu, Zhongfang; Li, Yuanfang; Hu, Xiaoli; Tian, Jing

2013-10-01

A novel fluorescence quenching method for the determination of torasemide (TOR) with some dihalogenated fluorescein dyes as fluorescence probes was developed. In acidulous medium, TOR could interact with some dihalogenated fluorescein dyes such as dichlorofluorescein (DCF), dibromofluorescein (DBF) and diiodofluorescein (DIF) to form binary complexes, which could lead to fluorescence quenching of above dihalogenated fluorescein dyes. The maximum fluorescence emission wavelengths were located at 532 nm (TOR-DCF), 535 nm (TOR-DBF) and 554 nm (TOR-DIF). The relative fluorescence intensities (ΔF = F0 - F) were proportional to the concentration of TOR in certain ranges. The detection limits were 4.8 ng mL-1 for TOR-DCF system, 9.8 ng mL-1 for TOR-DBF system and 35.1 ng mL-1 for TOR-DIF system. The optimum reaction conditions, influencing factors were studied; and the effect of coexisting substances was investigated owing to the highest sensitivity of TOR-DCF system. In addition, the reaction mechanism, composition and structure of the complex were discussed by quantum chemical calculation and Job's method. The fluorescence quenching of dihalogenated fluorescein dyes by TOR was a static quenching process judging from the effect of temperature and the Stern-Volmer plots. The method was satisfactorily applied to the determination of TOR in tablets and human urine samples.

Bridge effects on light harvesting of a DBfA type polymer system

NASA Astrophysics Data System (ADS)

Sun, Sam-Shajing; Hasib, Muhammad; Gavrilenko, Alexander V.; Devan, Joshua; Gavrilenko, Vladimir

2016-09-01

Plastic optoelectronic materials and thin film devices are very attractive in future optical sensor and solar energy applications due to their lightweight, flexible shape, high photon absorption coefficients, low cost, and environmental benefits. In this study, optoelectronic properties of D, D/fA blend, DfA, and a series of DBfA type of conjugated block copolymers has been investigated, where D is a donor type PPV conjugated block, B is a non-conjugated and flexible aliphatic hydrocarbon bridge chain containing different number of aliphatic methylene units, and fA is a fluorinated acceptor type PPV conjugated block. The optical absorptions of the D/fA blend, DfA, and DBfAs are typical overlaps of individual absorptions of D and fA blocks, while the solution steady state photoluminescence (PL) emission of D were quenched to different levels in blends and block copolymers, with DBfAs containing one methylene unit bridge (DB1fA) quenched most. This could be attributed to an intra-molecular photo induced electron transfer or charge separation in DBfA systems. Theoretical first principles study of the equilibrium atomic configuration of DfA reveals the existence of twisting angles between the D and fA blocks in DfA stable states which may account for a less PL quenching of DfA as compared to DB1fA. These results are important for designing and developing high efficiency polymer based optoelectronic systems.

Deep bottleneck features for spoken language identification.

PubMed

Jiang, Bing; Song, Yan; Wei, Si; Liu, Jun-Hua; McLoughlin, Ian Vince; Dai, Li-Rong

2014-01-01

A key problem in spoken language identification (LID) is to design effective representations which are specific to language information. For example, in recent years, representations based on both phonotactic and acoustic features have proven their effectiveness for LID. Although advances in machine learning have led to significant improvements, LID performance is still lacking, especially for short duration speech utterances. With the hypothesis that language information is weak and represented only latently in speech, and is largely dependent on the statistical properties of the speech content, existing representations may be insufficient. Furthermore they may be susceptible to the variations caused by different speakers, specific content of the speech segments, and background noise. To address this, we propose using Deep Bottleneck Features (DBF) for spoken LID, motivated by the success of Deep Neural Networks (DNN) in speech recognition. We show that DBFs can form a low-dimensional compact representation of the original inputs with a powerful descriptive and discriminative capability. To evaluate the effectiveness of this, we design two acoustic models, termed DBF-TV and parallel DBF-TV (PDBF-TV), using a DBF based i-vector representation for each speech utterance. Results on NIST language recognition evaluation 2009 (LRE09) show significant improvements over state-of-the-art systems. By fusing the output of phonotactic and acoustic approaches, we achieve an EER of 1.08%, 1.89% and 7.01% for 30 s, 10 s and 3 s test utterances respectively. Furthermore, various DBF configurations have been extensively evaluated, and an optimal system proposed.

SEDIMENT DATA - COMMENCEMENT BAY HYLEBOS WATERWAY - TACOMA, WA - PRE-REMEDIAL DESIGN PROGRAM

EPA Science Inventory

Event 1A/1B Data Files URL address: http://www.epa.gov/r10earth/datalib/superfund/hybos1ab.htm. Sediment Chemistry Data (Database Format): HYBOS1AB.EXE is a self-extracting file which expands to the single-value per record .DBF format database file HYBOS1AB.DBF. This file contai...

Characterization of the Upper Pathway Genes for Fluorene Metabolism in Terrabacter sp. Strain DBF63

PubMed Central

Habe, Hiroshi; Chung, Jin-Sung; Kato, Hiroyuki; Ayabe, Yuko; Kasuga, Kano; Yoshida, Takako; Nojiri, Hideaki; Yamane, Hisakazu; Omori, Toshio

2004-01-01

Genes involved in the degradation of fluorene to phthalate were characterized in the fluorene degrader Terrabacter sp. strain DBF63. The initial attack on both fluorene and 9-fluorenone was catalyzed by DbfA to yield 9-fluorenol and 1,1a-dihydroxy-1-hydro-9-fluorenone, respectively. The FlnB protein exhibited activities against both 9-fluorenol and 1,1a-dihydroxy-1-hydro-9-fluorenone to produce 9-fluorenone and 2′-carboxy-2,3-dihydroxybiphenyl, respectively. FlnD is a heteromeric protein encoded by flnD1 and ORF16, being a member of the class III two-subunit extradiol dioxygenase. FlnE was identified as a serine hydrolase for the meta-cleavage products that yield phthalate. PMID:15317800

Interannual influence of spring phenological transitions on the water use efficiency of forest ecosystem

NASA Astrophysics Data System (ADS)

Jin, Jiaxin; Wang, Ying

2017-04-01

Climate change has significantly influenced the productivity of terrestrial ecosystems through water cycles. Understanding the phenological regulation mechanisms underlying coupled carbon-water cycles is important for improving ecological assessments and projecting terrestrial ecosystem responses and feedback to climate change. In this study, we present an analysis of the interannual relationships among flux-based spring phenological transitions (referred as photosynthetic onset) and water use efficiency (WUE) in North America and Europe using 166 site-years of data from 22 flux sites, including 10 deciduous broadleaf forest (DBF) and 12 evergreen needleleaf forest (ENF) ecosystems. We found that the WUE responses to variations in spring phenological transitions differed substantially across plant functional types (PFTs) and growth periods. During the early spring (defined as one month from spring onset) in the DBF ecosystem, photosynthetic onset dominated changes in WUE by dominating gross primary production (GPP), with one day of advanced onset increasing the WUE by 0.037 gC kg-1H2O in early spring. For the ENF sites, although advanced photosynthetic onset also significantly promoted GPP, earlier onset did not have a significant positive impact on WUE in early spring because it was not significantly correlated to evapotranspiration (ET), which is a more dominant factor for WUE than GPP across the ENF sites. Statistically significant correlations were not observed between interannual variability in photosynthetic onset and WUE for either the DBF or ENF ecosystems following a prolonged period after photosynthetic onset. For the DBF sites, the interannual variability of photosynthetic onset provided a better explanation of the variations in WUE (ca. 51.4%) compared with climatic factors, although this was only applicable to the early spring. For the ENF sites, photosynthetic onset variations did not provide a better explanation of the interannual WUE variations compared with climatic factors within any growth period. Notably, the negative correlation between the interannual variability of early spring WUE and photosynthetic onset gradually declined from boreal forests (r = -0.73) to subtropical Mediterranean forests (r = 0.35), indicating that the positive effect of earlier spring phenological transitions decreased or even reversed from cold climates to warm climates. This result suggests that the effect of the phenological regulatory mechanism on coupled carbon-water cycles is not only determined by the PFT but also by the habitat climate of an ecosystem. These observed differences between the ENF and DBF ecosystems will likely influence future phenological shifts related to competition for water and other resources in mixed species stands.

Multiple mechanisms determine the order of APC/C substrate degradation in mitosis

PubMed Central

Lu, Dan; Hsiao, Jennifer Y.; Davey, Norman E.; Van Voorhis, Vanessa A.; Foster, Scott A.

2014-01-01

The ubiquitin protein ligase anaphase-promoting complex or cyclosome (APC/C) controls mitosis by promoting ordered degradation of securin, cyclins, and other proteins. The mechanisms underlying the timing of APC/C substrate degradation are poorly understood. We explored these mechanisms using quantitative fluorescence microscopy of GFP-tagged APC/CCdc20 substrates in living budding yeast cells. Degradation of the S cyclin, Clb5, begins early in mitosis, followed 6 min later by the degradation of securin and Dbf4. Anaphase begins when less than half of securin is degraded. The spindle assembly checkpoint delays the onset of Clb5 degradation but does not influence securin degradation. Early Clb5 degradation depends on its interaction with the Cdk1–Cks1 complex and the presence of a Cdc20-binding “ABBA motif” in its N-terminal region. The degradation of securin and Dbf4 is delayed by Cdk1-dependent phosphorylation near their Cdc20-binding sites. Thus, a remarkably diverse array of mechanisms generates robust ordering of APC/CCdc20 substrate destruction. PMID:25287299

[Extraction of temperate vegetation phenology thresholds in North America based on flux tower observation data].

PubMed

Zhao, Jing-Jing; Liu, Liang-Yun

2013-02-01

Flux tower method can effectively monitor the vegetation seasonal and phenological variation processes. At present, the differences in the detection and quantitative evaluation of various phenology extraction methods were not well validated and quantified. Based on the gross primary productivity (GPP) and net ecosystem productivity (NEP) data of temperate forests from 9 forest FLUXNET sites in North America, and by using the start dates (SOS) and end dates (EOS) of the temperate forest growth seasons extracted by different phenology threshold extraction methods, in combining with the forest ecosystem carbon source/sink functions, this paper analyzed the effects of different threshold standards on the extraction results of the vegetations phenology. The results showed that the effects of different threshold standards on the stability of the extracted results of deciduous broadleaved forest (DBF) phenology were smaller than those on the stability of the extracted results of evergreen needleleaved forest (ENF) phenology. Among the extracted absolute and relative thresholds of the forests GPP, the extracted threshold of the DBF daily GPP= 2 g C.m-2.d-1 had the best agreement with the DBF daily GPP = 20% maximum GPP (GPPmax) , the phenological metrics with a threshold of daily GPP = 4 g C.m-2.d-1 was close to that between daily GPP = 20% GPPmax and daily GPP = 50% GPPmax, and the start date of ecosystem carbon sink function was close to the SOS metrics between daily GPP = 4 g C.m-2.d-1 and daily GPP= 20% GPPmax. For ENF, the phenological metrics with a threshold of daily GPP = 2 g C.m-2.d-1 and daily GPP = 4 g C.m-2.d-1 had the best agreement with the daily GPP = 20% GPPmax and daily GPP = 50% GPPmax, respectively, and the start date of the ecosystem carbon sink function was close to the SOS metrics between daily GPP = 2 g C.m-2.d-1 and daily GPP= 10% GPPmax.

External Validity of a Risk Stratification Score Predicting Early Distant Brain Failure and Salvage Whole Brain Radiation Therapy After Stereotactic Radiosurgery for Brain Metastases.

PubMed

Press, Robert H; Boselli, Danielle M; Symanowski, James T; Lankford, Scott P; McCammon, Robert J; Moeller, Benjamin J; Heinzerling, John H; Fasola, Carolina E; Burri, Stuart H; Patel, Kirtesh R; Asher, Anthony L; Sumrall, Ashley L; Curran, Walter J; Shu, Hui-Kuo G; Crocker, Ian R; Prabhu, Roshan S

2017-07-01

A scoring system using pretreatment factors was recently published for predicting the risk of early (≤6 months) distant brain failure (DBF) and salvage whole brain radiation therapy (WBRT) after stereotactic radiosurgery (SRS) alone. Four risk factors were identified: (1) lack of prior WBRT; (2) melanoma or breast histologic features; (3) multiple brain metastases; and (4) total volume of brain metastases <1.3 cm 3 , with each factor assigned 1 point. The purpose of this study was to assess the validity of this scoring system and its appropriateness for clinical use in an independent external patient population. We reviewed the records of 247 patients with 388 brain metastases treated with SRS between 2010 at 2013 at Levine Cancer Institute. The Press (Emory) risk score was calculated and applied to the validation cohort population, and subsequent risk groups were analyzed using cumulative incidence. The low-risk (LR) group had a significantly lower risk of early DBF than did the high-risk (HR) group (22.6% vs 44%, P=.004), but there was no difference between the HR and intermediate-risk (IR) groups (41.2% vs 44%, P=.79). Total lesion volume <1.3 cm 3  (P=.004), malignant melanoma (P=.007), and multiple metastases (P<.001) were validated as predictors for early DBF. Prior WBRT and breast cancer histologic features did not retain prognostic significance. Risk stratification for risk of early salvage WBRT were similar, with a trend toward an increased risk for HR compared with LR (P=.09) but no difference between IR and HR (P=.53). The 3-level Emory risk score was shown to not be externally valid, but the model was able to stratify between 2 levels (LR and not-LR [combined IR and HR]) for early (≤6 months) DBF. These results reinforce the importance of validating predictive models in independent cohorts. Further refinement of this scoring system with molecular information and in additional contemporary patient populations is warranted. Copyright © 2017 Elsevier Inc. All rights reserved.

The role of spring and autumn phenological switches on spatiotemporal variation in temperate and boreal forest C balance: A FLUXNET synthesis

NASA Astrophysics Data System (ADS)

Richardson, A. D.; Reichstein, M.; Piao, S.; Ciais, P.; Luyssaert, S.; Stockli, R.; Friedl, M.; Gobron, N.; Fluxnet Site Pis, 21

2009-04-01

In temperate and boreal ecosystems, phenological transitions (particularly the timing of spring onset and autumn senescence) are thought to represent a major control on spatial and temporal variation in forest carbon sequestration. To investigate these patterns, we analyzed 153 site-years of data from the FLUXNET ‘La Thuile' database. Eddy covariance measurements of surface-atmosphere exchanges of carbon and water from 21 research sites at latitudes from 36°N to 67°N were used in the synthesis. We defined a range of phenological indicators based on the first (spring) and last (autumn) dates of (1) C source/sink transitions (‘carbon uptake period'); (2) measurable photosynthetic uptake (‘physiologically active period'); (3) relative thresholds for latent heat (evapotranspiration) flux; (4) phenological thresholds derived from a range of remote sensing products (JRC fAPAR, MOD12Q2, and the PROGNOSTIC model with MODIS data assimilation); and (5) a climatological metric based on the date where soil temperature equals mean annual air temperature. We then tested whether site-level flux anomalies were significantly correlated with phenological anomalies across these metrics, and whether the slopes of these relationships (representing the sensitivity to phenological variation) differed between deciduous broadleaf (DBF) and evergreen needleleaf (ENF) forests. Within sites, interannual variation in most phenological metrics was about 5-10 d, compared to 10-30 d across sites. Both spatial and temporal phenological variation were consistently larger at ENF, compared to DBF, sites. Averaged across metrics, phenological variability was roughly comparable in spring and autumn, both across (17 d) and within (9 d) sites. However, patterns of interannual variation in fluxes were less well explained by the derived phenological metrics than were patterns of spatial variation in fluxes. Also, the observed pattern strongly depended on the metric used, with flux-derived metrics generally explaining more, and remote sensing-derived metrics generally explaining less, of the variation in flux anomalies. We found that GPP (gross primary productivity) was consistently more sensitive (both in terms of magnitude and statistical significance; ≈3 g C m-2 d-1 for DBF and ≈2 g C m-2 d-1 for ENF) to phenology than was Reco (ecosystem respiration), which meant that NEP (net ecosystem productivity) tended to be increased both by earlier springs and later autumns. Without exception, when the difference between DBF and ENF in the sensitivity to phenological anomalies was statistically significant, DBF sensitivity was always larger in absolute magnitude than ENF sensitivity. Phenology explained a much larger fraction of the variation in fluxes across sites compared to within sites. Across sites, the rate of increase in GPP with an "exta" day in spring (≈10 g C m-2 d-1) was much larger than in autumn (≈3 g C m-2 d-1). Furthermore, a one-day increase in growing season length across sites increased annual NEP by just ≈2 g C m-2 d-1; this resulted from an increase in GPP of ≈6 g C m-2 d-1 being offset by an increase in RE of ≈4 g C m-2 d-1. In general, there was no statistically significant difference between DBF and ENF in the sensitivity to spatial variation in phenology for either NEP or the component fluxes GPP and Reco. In relation to both within- and across-site variation in phenology and fluxes, the results obtained tended to depend on the phenological metric used, i.e. definition of "start" and "end" of growing season, emphasizing the need for improved understanding of the relationships between these different metrics and ecosystem processes. Furthermore, the differences in flux-phenology relationships in the context of spatial and temporal variation in phenology raise questions about using results from either short-term or space-for-time studies to anticipate responses to future climate change.

Roles of CDK and DDK in Genome Duplication and Maintenance: Meiotic Singularities.

PubMed

Gómez-Escoda, Blanca; Wu, Pei-Yun Jenny

2017-03-20

Cells reproduce using two types of divisions: mitosis, which generates two daughter cells each with the same genomic content as the mother cell, and meiosis, which reduces the number of chromosomes of the parent cell by half and gives rise to four gametes. The mechanisms that promote the proper progression of the mitotic and meiotic cycles are highly conserved and controlled. They require the activities of two types of serine-threonine kinases, the cyclin-dependent kinases (CDKs) and the Dbf4-dependent kinase (DDK). CDK and DDK are essential for genome duplication and maintenance in both mitotic and meiotic divisions. In this review, we aim to highlight how these kinases cooperate to orchestrate diverse processes during cellular reproduction, focusing on meiosis-specific adaptions of their regulation and functions in DNA metabolism.

Gibberellin Application at Pre-Bloom in Grapevines Down-Regulates the Expressions of VvIAA9 and VvARF7, Negative Regulators of Fruit Set Initiation, during Parthenocarpic Fruit Development

PubMed Central

Jung, Chan Jin; Hur, Youn Young; Yu, Hee-Ju; Noh, Jung-Ho; Park, Kyo-Sun; Lee, Hee Jae

2014-01-01

Fruit set is initiated only after fertilization and is tightly regulated primarily by gibberellins (GAs) and auxins. The application of either of these hormones induces parthenocarpy, fruit set without fertilization, but the molecular mechanism underlying this induction is poorly understood. In the present study, we have shown that the parthenocarpic fruits induced by GA application at pre-bloom result from the interaction of GA with auxin signaling. The transcriptional levels of the putative negative regulators of fruit set initiation, including Vitis auxin/indole-3-acetic acid transcription factor 9 (VvIAA9), Vitis auxin response factor 7 (VvARF7), and VvARF8 were monitored during inflorescence development in seeded diploid ‘Tamnara’ grapevines with or without GA application. Without GA application, VvIAA9, VvARF7, and VvARF8 were expressed at a relatively high level before full bloom, but decreased thereafter following pollination. After GA application at 14 days before full bloom (DBF); however, the expression levels of VvIAA9 and VvARF7 declined at 5 DBF prior to pollination. The effects of GA application on auxin levels or auxin signaling were also analyzed by monitoring the expression patterns of auxin biosynthesis genes and auxin-responsive genes with or without GA application. Transcription levels of the auxin biosynthesis genes Vitis anthranilate synthase β subunit (VvASB1-like), Vitis YUCCA2 (VvYUC2), and VvYUC6 were not significantly changed by GA application. However, the expressions of Vitis Gretchen Hagen3.2 (VvGH3.2) and VvGH3.3, auxin-responsive genes, were up-regulated from 2 DBF to full bloom with GA application. Furthermore, the Vitis GA signaling gene, VvDELLA was up-regulated by GA application during 12 DBF to 7 DBF, prior to down-regulation of VvIAA9 and VvARF7. These results suggest that VvIAA9 and VvARF7 are negative regulators of fruit set initiation in grapevines, and GA signaling is integrated with auxin signaling via VvDELLA during parthenocarpic fruit development in grapevines. PMID:24743886

Gibberellin application at pre-bloom in grapevines down-regulates the expressions of VvIAA9 and VvARF7, negative regulators of fruit set initiation, during parthenocarpic fruit development.

PubMed

Jung, Chan Jin; Hur, Youn Young; Yu, Hee-Ju; Noh, Jung-Ho; Park, Kyo-Sun; Lee, Hee Jae

2014-01-01

Fruit set is initiated only after fertilization and is tightly regulated primarily by gibberellins (GAs) and auxins. The application of either of these hormones induces parthenocarpy, fruit set without fertilization, but the molecular mechanism underlying this induction is poorly understood. In the present study, we have shown that the parthenocarpic fruits induced by GA application at pre-bloom result from the interaction of GA with auxin signaling. The transcriptional levels of the putative negative regulators of fruit set initiation, including Vitis auxin/indole-3-acetic acid transcription factor 9 (VvIAA9), Vitis auxin response factor 7 (VvARF7), and VvARF8 were monitored during inflorescence development in seeded diploid 'Tamnara' grapevines with or without GA application. Without GA application, VvIAA9, VvARF7, and VvARF8 were expressed at a relatively high level before full bloom, but decreased thereafter following pollination. After GA application at 14 days before full bloom (DBF); however, the expression levels of VvIAA9 and VvARF7 declined at 5 DBF prior to pollination. The effects of GA application on auxin levels or auxin signaling were also analyzed by monitoring the expression patterns of auxin biosynthesis genes and auxin-responsive genes with or without GA application. Transcription levels of the auxin biosynthesis genes Vitis anthranilate synthase β subunit (VvASB1-like), Vitis YUCCA2 (VvYUC2), and VvYUC6 were not significantly changed by GA application. However, the expressions of Vitis Gretchen Hagen3.2 (VvGH3.2) and VvGH3.3, auxin-responsive genes, were up-regulated from 2 DBF to full bloom with GA application. Furthermore, the Vitis GA signaling gene, VvDELLA was up-regulated by GA application during 12 DBF to 7 DBF, prior to down-regulation of VvIAA9 and VvARF7. These results suggest that VvIAA9 and VvARF7 are negative regulators of fruit set initiation in grapevines, and GA signaling is integrated with auxin signaling via VvDELLA during parthenocarpic fruit development in grapevines.

Dynamics of glass-forming di-n-butyl phthalate as studied by NMR.

PubMed

Szcześniak, E; Głowinkowski, S; Suchański, W; Jurga, S

1997-04-01

Spin-lattice relaxation times T1 and nuclear Overhauser effect (NOE) enhancement factors for the individual ring carbons in di-n-butyl phthalate (DBF) show that the reorientational correlation function corresponding to the global dynamics in supercooled liquid can be described by a Davidson-Cole distribution. Measurements of proton spin-lattice relaxation times T1 and T1p, as well as 1H NMR spectra at temperatures below the glass transition temperature, Tg, reveal that the same distribution holds also for description of local dynamics in glassy DBF. The activation parameters of the motions detected are derived.

Prototype Parts of a Digital Beam-Forming Wide-Band Receiver

NASA Technical Reports Server (NTRS)

Kaplan, Steven B.; Pylov, Sergey V.; Pambianchi, Michael

2003-01-01

Some prototype parts of a digital beamforming (DBF) receiver that would operate at multigigahertz carrier frequencies have been developed. The beam-forming algorithm in a DBF receiver processes signals from multiple antenna elements with appropriate time delays and weighting factors chosen to enhance the reception of signals from a specific direction while suppressing signals from other directions. Such a receiver would be used in the directional reception of weak wideband signals -- for example, spread-spectrum signals from a low-power transmitter on an Earth-orbiting spacecraft or other distant source. The prototype parts include superconducting components on integrated-circuit chips, and a multichip module (MCM), within which the chips are to be packaged and connected via special inter-chip-communication circuits. The design and the underlying principle of operation are based on the use of the rapid single-flux quantum (RSFQ) family of logic circuits to obtain the required processing speed and signal-to-noise ratio. RSFQ circuits are superconducting circuits that exploit the Josephson effect. They are well suited for this application, having been proven to perform well in some circuits at frequencies above 100 GHz. In order to maintain the superconductivity needed for proper functioning of the RSFQ circuits, the MCM must be kept in a cryogenic environment during operation.

Cdc7-Dbf4 Regulates NDT80 Transcription as Well as Reductional Segregation during Budding Yeast Meiosis

PubMed Central

Lo, Hsiao-Chi; Wan, Lihong; Rosebrock, Adam; Futcher, Bruce

2008-01-01

In budding yeast, as in other eukaryotes, the Cdc7 protein kinase is important for initiation of DNA synthesis in vegetative cells. In addition, Cdc7 has crucial meiotic functions: it facilitates premeiotic DNA replication, and it is essential for the initiation of recombination. This work uses a chemical genetic approach to demonstrate that Cdc7 kinase has additional roles in meiosis. First, Cdc7 allows expression of NDT80, a meiosis-specific transcriptional activator required for the induction of genes involved in exit from pachytene, meiotic progression, and spore formation. Second, Cdc7 is necessary for recruitment of monopolin to sister kinetochores, and it is necessary for the reductional segregation occurring at meiosis I. The use of the same kinase to regulate several distinct meiosis-specific processes may be important for the coordination of these processes during meiosis. PMID:18768747

Regulated Eukaryotic DNA Replication Origin Firing with Purified Proteins

PubMed Central

Yeeles, Joseph T.P.; Deegan, Tom D.; Janska, Agnieszka; Early, Anne; Diffley, John F. X.

2016-01-01

Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric MCM complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45, MCM, GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4 dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication. PMID:25739503

Regulated eukaryotic DNA replication origin firing with purified proteins.

PubMed

Yeeles, Joseph T P; Deegan, Tom D; Janska, Agnieszka; Early, Anne; Diffley, John F X

2015-03-26

Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.

Prereplicative complexes assembled in vitro support origin-dependent and independent DNA replication

PubMed Central

On, Kin Fan; Beuron, Fabienne; Frith, David; Snijders, Ambrosius P; Morris, Edward P; Diffley, John F X

2014-01-01

Eukaryotic DNA replication initiates from multiple replication origins. To ensure each origin fires just once per cell cycle, initiation is divided into two biochemically discrete steps: the Mcm2-7 helicase is first loaded into prereplicative complexes (pre-RCs) as an inactive double hexamer by the origin recognition complex (ORC), Cdt1 and Cdc6; the helicase is then activated by a set of “firing factors.” Here, we show that plasmids containing pre-RCs assembled with purified proteins support complete and semi-conservative replication in extracts from budding yeast cells overexpressing firing factors. Replication requires cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK). DDK phosphorylation of Mcm2-7 does not by itself promote separation of the double hexamer, but is required for the recruitment of firing factors and replisome components in the extract. Plasmid replication does not require a functional replication origin; however, in the presence of competitor DNA and limiting ORC concentrations, replication becomes origin-dependent in this system. These experiments indicate that Mcm2-7 double hexamers can be precursors of replication and provide insight into the nature of eukaryotic DNA replication origins. PMID:24566989

Validation of a mobility item bank for older patients in primary care.

PubMed

Cabrero-García, Julio; Ramos-Pichardo, Juan Diego; Muñoz-Mendoza, Carmen Luz; Cabañero-Martínez, María José; González-Llopis, Lorena; Reig-Ferrer, Abilio

2012-12-05

To develop and validate an item bank to measure mobility in older people in primary care and to analyse differential item functioning (DIF) and differential bundle functioning (DBF) by sex. A pool of 48 mobility items was administered by interview to 593 older people attending primary health care practices. The pool contained four domains based on the International Classification of Functioning: changing and maintaining body position, carrying, lifting and pushing, walking and going up and down stairs. The Late Life Mobility item bank consisted of 35 items, and measured with a reliability of 0.90 or more across the full spectrum of mobility, except at the higher end of better functioning. No evidence was found of non-uniform DIF but uniform DIF was observed, mainly for items in the changing and maintaining body position and carrying, lifting and pushing domains. The walking domain did not display DBF, but the other three domains did, principally the carrying, lifting and pushing items. During the design and validation of an item bank to measure mobility in older people, we found that strength (carrying, lifting and pushing) items formed a secondary dimension that produced DBF. More research is needed to determine how best to include strength items in a mobility measure, or whether it would be more appropriate to design separate measures for each construct.

The use of biochemical responses to assess ecotoxicological effects of Pharmaceutical and Personal Care Products (PPCPs) after injection in the mussel Elliptio complanata.

PubMed

Martín-Díaz, M Laura; Gagné, François; Blaise, Christian

2009-09-01

A biomarker approach was undertaken using the mussel Elliptio complanata to assess the ecotoxicological effects after injection of a range concentration (0-10mM) of three different PPCPs: carbamazepine, caffeine, methotrexate; and an effluent extract (C8) from St. Lawrence wastewaters treatment plant (Montreal, Canada). A battery of biomarkers, involving oxidative stress and genotoxicity responses: glutation-S-transferase (GST), ethoxyresorufin O-deethylase (EROD), dibenzylflourescein dealkylase (DBF), xanthine oxidoreductase (XOR) activities, lipid peroxidation (LPO) and DNA damage were determined in gonad and digestive gland tissues after 48 h of injection. Results showed an induction of the oxidative metabolism with increasing pharmaceutical concentration in those mussels injected with the PPCPs and the effluent extract. Phase I detoxification enzymes were significantly induced (p<0.05), concretely DBF activity was significantly induced after caffeine, carbamazipine and C8 injection; and EROD activity after C8 and methotrexate injection. Oxidative stress induction only lead to lipid peroxidation (p<0.05) in organisms injected with carbamazepine and caffeine and DNA damage in organisms injected with methotrexate (p<0.05). EROD and DBF enzymatic activities have been found to be suitable biomarkers to determine bioavailability of pharmaceuticals. LPO and DNA damage to determine possible associated adverse effects. Nevertheless, their validation in realistic exposure scenarios and under exposure conditions should be performed in future research.

Enhanced methodology of focus control and monitoring on scanner tool

NASA Astrophysics Data System (ADS)

Chen, Yen-Jen; Kim, Young Ki; Hao, Xueli; Gomez, Juan-Manuel; Tian, Ye; Kamalizadeh, Ferhad; Hanson, Justin K.

2017-03-01

As the demand of the technology node shrinks from 14nm to 7nm, the reliability of tool monitoring techniques in advanced semiconductor fabs to achieve high yield and quality becomes more critical. Tool health monitoring methods involve periodic sampling of moderately processed test wafers to detect for particles, defects, and tool stability in order to ensure proper tool health. For lithography TWINSCAN scanner tools, the requirements for overlay stability and focus control are very strict. Current scanner tool health monitoring methods include running BaseLiner to ensure proper tool stability on a periodic basis. The focus measurement on YIELDSTAR by real-time or library-based reconstruction of critical dimensions (CD) and side wall angle (SWA) has been demonstrated as an accurate metrology input to the control loop. The high accuracy and repeatability of the YIELDSTAR focus measurement provides a common reference of scanner setup and user process. In order to further improve the metrology and matching performance, Diffraction Based Focus (DBF) metrology enabling accurate, fast, and non-destructive focus acquisition, has been successfully utilized for focus monitoring/control of TWINSCAN NXT immersion scanners. The optimal DBF target was determined to have minimized dose crosstalk, dynamic precision, set-get residual, and lens aberration sensitivity. By exploiting this new measurement target design, 80% improvement in tool-to-tool matching, >16% improvement in run-to-run mean focus stability, and >32% improvement in focus uniformity have been demonstrated compared to the previous BaseLiner methodology. Matching <2.4 nm across multiple NXT immersion scanners has been achieved with the new methodology of set baseline reference. This baseline technique, with either conventional BaseLiner low numerical aperture (NA=1.20) mode or advanced illumination high NA mode (NA=1.35), has also been evaluated to have consistent performance. This enhanced methodology of focus control and monitoring on multiple illumination conditions, opens an avenue to significantly reduce Focus-Exposure Matrix (FEM) wafer exposure for new product/layer best focus (BF) setup.

Upgrade of deep bed filtration with activated carbon dosage for compact micropollutant removal from wastewater in technical scale.

PubMed

Löwenberg, Jonas; Zenker, Armin; Krahnstöver, Thérèse; Boehler, Marc; Baggenstos, Martin; Koch, Gerhard; Wintgens, Thomas

2016-05-01

The removal of micropollutants from drinking and wastewater by powdered activated carbon (PAC) adsorption has received considerable attention in research over the past decade with various separation options having been investigated. With Switzerland as the first country in the world having adopted a new legislation, which forces about 100 wastewater treatment plants to be upgraded for the removal of organic micropollutants from municipal wastewater, the topic has reached practical relevance. In this study, the process combination of powdered activated carbon (PAC) adsorption and deep bed filtration (DBF) for advanced municipal wastewater treatment was investigated over an extended period exceeding one year of operation in technical scale. The study aimed to determine optimum process conditions to achieve sufficient micropollutant removal in agreement with the new Swiss Water Ordinance under most economic process design. It was shown that the addition of PAC and Fe(3+) as combined coagulation and flocculation agent improved effluent water quality with respect to dissolved organic pollutants as well as total suspended solids (TSS), turbidity and PO4-P concentration in comparison to a DBF operated without the addition of PAC and Fe(3+). Sufficient micropollutant (MP) removal of around 80% was achieved at PAC dosages of 10 mg/L revealing that PAC retained in the filter bed maintained considerable adsorption capacity. In the investigated process combination the contact reactor serves for adsorption as well as for flocculation and allowed for small hydraulic retention times of minimum 10 min while maintaining sufficient MP removal. The flocculation of two different PAC types was shown to be fully concluded after 10-15 min, which determined the flocculation reactor size while both PAC types proved suitable for the application in combination with DBF and showed no significant differences in MP removal. Finally, the capping of PAC dosage during rain water periods, which resulted in lower dosage concentrations, was efficient in limiting PAC consumption during these events without suffering from negative effects on process performance or effluent quality. Copyright © 2016 Elsevier Ltd. All rights reserved.

Interactions of the Human MCM-BP Protein with MCM Complex Components and Dbf4

PubMed Central

Nguyen, Tin; Jagannathan, Madhav; Shire, Kathy; Frappier, Lori

2012-01-01

MCM-BP was discovered as a protein that co-purified from human cells with MCM proteins 3 through 7; results which were recapitulated in frogs, yeast and plants. Evidence in all of these organisms supports an important role for MCM-BP in DNA replication, including contributions to MCM complex unloading. However the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood. Here we show that human MCM-BP is capable of interacting with individual MCM proteins 2 through 7 when co-expressed in insect cells and can greatly increase the recovery of some recombinant MCM proteins. Glycerol gradient sedimentation analysis indicated that MCM-BP interacts most strongly with MCM4 and MCM7. Similar gradient analyses of human cell lysates showed that only a small amount of MCM-BP overlapped with the migration of MCM complexes and that MCM complexes were disrupted by exogenous MCM-BP. In addition, large complexes containing MCM-BP and MCM proteins were detected at mid to late S phase, suggesting that the formation of specific MCM-BP complexes is cell cycle regulated. We also identified an interaction between MCM-BP and the Dbf4 regulatory component of the DDK kinase in both yeast 2-hybrid and insect cell co-expression assays, and this interaction was verified by co-immunoprecipitation of endogenous proteins from human cells. In vitro kinase assays showed that MCM-BP was not a substrate for DDK but could inhibit DDK phosphorylation of MCM4,6,7 within MCM4,6,7 or MCM2-7 complexes, with little effect on DDK phosphorylation of MCM2. Since DDK is known to activate DNA replication through phosphorylation of these MCM proteins, our results suggest that MCM-BP may affect DNA replication in part by regulating MCM phosphorylation by DDK. PMID:22540012

Interactions of the human MCM-BP protein with MCM complex components and Dbf4.

PubMed

Nguyen, Tin; Jagannathan, Madhav; Shire, Kathy; Frappier, Lori

2012-01-01

MCM-BP was discovered as a protein that co-purified from human cells with MCM proteins 3 through 7; results which were recapitulated in frogs, yeast and plants. Evidence in all of these organisms supports an important role for MCM-BP in DNA replication, including contributions to MCM complex unloading. However the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood. Here we show that human MCM-BP is capable of interacting with individual MCM proteins 2 through 7 when co-expressed in insect cells and can greatly increase the recovery of some recombinant MCM proteins. Glycerol gradient sedimentation analysis indicated that MCM-BP interacts most strongly with MCM4 and MCM7. Similar gradient analyses of human cell lysates showed that only a small amount of MCM-BP overlapped with the migration of MCM complexes and that MCM complexes were disrupted by exogenous MCM-BP. In addition, large complexes containing MCM-BP and MCM proteins were detected at mid to late S phase, suggesting that the formation of specific MCM-BP complexes is cell cycle regulated. We also identified an interaction between MCM-BP and the Dbf4 regulatory component of the DDK kinase in both yeast 2-hybrid and insect cell co-expression assays, and this interaction was verified by co-immunoprecipitation of endogenous proteins from human cells. In vitro kinase assays showed that MCM-BP was not a substrate for DDK but could inhibit DDK phosphorylation of MCM4,6,7 within MCM4,6,7 or MCM2-7 complexes, with little effect on DDK phosphorylation of MCM2. Since DDK is known to activate DNA replication through phosphorylation of these MCM proteins, our results suggest that MCM-BP may affect DNA replication in part by regulating MCM phosphorylation by DDK.

Histological evaluation of direct pulp capping with all-in-one adhesives in rat teeth.

PubMed

Shinkai, Koichi; Taira, Yoshihisa; Kawashima, Satoki; Suzuki, Shiro; Suzuki, Masaya

2017-05-31

The aim of this study was to histologically evaluate direct pulp capping using different all-in-one adhesives in rat teeth. Five all-in-one adhesives and a control material (MTA) were used. Each material was applied on the exposed pulp, and each cavity was subsequently restored with the resin composite. Rats were sacrificed 14 days after the surgical procedure. Serial stained sections were histologically evaluated for examining pulp tissue disorganization (PTD), inflammatory cell infiltration (ICI), dentin bridge formation (DBF), and bacterial penetration (BP). We found that rat pulps, which were direct capped with all-in-one adhesives, showed various degrees of PTD, ICI, and DBF depending on the material, and that there were no complete dentin bridges. In contrast, rat pulps capped with MTA showed no PTD and ICI, and there were complete dentin bridges in all, but one specimen. No BP was observed in any specimen.

Reversal of DDK-Mediated MCM Phosphorylation by Rif1-PP1 Regulates Replication Initiation and Replisome Stability Independently of ATR/Chk1.

PubMed

Alver, Robert C; Chadha, Gaganmeet Singh; Gillespie, Peter J; Blow, J Julian

2017-03-07

Dbf4-dependent kinases (DDKs) are required for the initiation of DNA replication, their essential targets being the MCM2-7 proteins. We show that, in Xenopus laevis egg extracts and human cells, hyper-phosphorylation of DNA-bound Mcm4, but not phosphorylation of Mcm2, correlates with DNA replication. These phosphorylations are differentially affected by the DDK inhibitors PHA-767491 and XL413. We show that DDK-dependent MCM phosphorylation is reversed by protein phosphatase 1 (PP1) targeted to chromatin by Rif1. Loss of Rif1 increased MCM phosphorylation and the rate of replication initiation and also compromised the ability of cells to block initiation when challenged with replication inhibitors. We also provide evidence that Rif1 can mediate MCM dephosphorylation at replication forks and that the stability of dephosphorylated replisomes strongly depends on Chk1 activity. We propose that both replication initiation and replisome stability depend on MCM phosphorylation, which is maintained by a balance of DDK-dependent phosphorylation and Rif1-mediated dephosphorylation. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Distal Insertions of the Biceps Femoris

PubMed Central

Branch, Eric A.; Anz, Adam W.

2015-01-01

Background: Avulsion of the biceps femoris from the fibula and proximal tibia is encountered in clinical practice. While the anatomy of the primary posterolateral corner structures has been qualitatively and quantitatively described, a quantitative analysis regarding the insertions of the biceps femoris on the fibula and proximal tibia is lacking. Purpose: To quantitatively assess the insertions of the biceps femoris, fibular collateral ligament (FCL), and anterolateral ligament (ALL) on the fibula and proximal tibia as well as establish relationships among these structures and to pertinent surgical anatomy. Study Design: Descriptive laboratory study. Methods: Dissections were performed on 12 nonpaired, fresh-frozen cadaveric specimens identifying the biceps femoris, FCL, and ALL, and their insertions on the proximal tibia and fibula. The footprint areas, orientations, and distances from relevant osseous landmarks were measured using a 3-dimensional coordinate measurement device. Results: Dissection produced 6 easily identifiable and reproducible anatomic footprints. Tibial footprints included the insertion of the ALL and an insertion of the biceps femoris (TBF). Fibular footprints included the insertion of the FCL, a distal insertion of the biceps femoris (DBF), a medial footprint of the biceps femoris (MBF), and a proximal footprint of the biceps femoris (PBF). The mean area of these footprints (95% CI) was as follows: ALL, 53.0 mm2 (38.4-67.6); TBF, 93.9 mm2 (72.0-115.8); FCL, 86.8 mm2 (72.3-101.2); DBF, 119 mm2 (91.1-146.9); MBF, 46.8 mm2 (29.0-64.5); and PBF, 215 mm2 (192.4-237.5). The mean distance (95% CI) from the Gerdy tubercle to the center of the ALL footprint was 24.3 mm (21.6-27.0) and to the center of the TBF was 22.5 mm (21.0-24.0). The center of the DBF was 8.68 mm (7.0-10.3) from the anterior border of the fibula, the center of the FCL was 14.6 mm (12.5-16.7) from the anterior border of the fibula and 20.7 mm (19.0-22.4) from the tip of the fibular styloid, and the center of the PBF was 8.96 mm (8.2-9.7) from the tip of the fibular styloid. Conclusion: A tibial footprint, distal fibular footprint, medial fibular footprint, and proximal fibular footprint were all consistent components of the insertion of the biceps femoris. Consistent relationships existed between the biceps femoris and insertions of the ALL and FCL. Clinical Relevance: The size of these footprints and distances from pertinent surgical landmarks will guide repairs of biceps femoris avulsion injuries. PMID:26535398

High-resolution record of the deglaciation of the British-Irish Ice Sheet from North Atlantic deep-sea sediments.

NASA Astrophysics Data System (ADS)

Tarlati, S.; Benetti, S.; Callard, L.; O'Cofaigh, C.; Dunlop, P.; Chiverrell, R. C.; Fabel, D.; Moreton, S.; Clark, C.

2016-12-01

During the last glacial maximum the British-Irish Ice Sheet (BIIS) covered the majority of Ireland and Britain. Recent studies have described the BIIS as largely marine-based and highly dynamic with several advances and retreats recorded on the continental shelf. The focus of this study is the more recent sediment record from the Donegal Barra Fan (DBF), the largest sediment depocentre formed by the ice streaming of the western BIIS onto the North Atlantic continental margin. In this project, well-preserved, glacially-derived, deep-water sediments from 3 cores, up to 6.7 m long and retrieved from the DBF, are used to investigate and chronologically constrain the pattern of deglaciation of the BIIS. Deep-water sediments can record continuous sedimentation through time, avoiding hiatuses and erosional surfaces characteristic of a glacial environment and allow a detailed reconstruction of deglacial processes. Five lithofacies have been identified using sedimentology, x-rays, physical properties and grain size analysis. They include bioturbated foraminifera-bearing muds, interpreted as hemipelagic and contouritic deposits from interglacial periods. Chaotic and laminated muds, ice-rafted debris (IRD)-rich layers and laminated mud to sand couplets are characteristic of the glacial period including ice-sheet maximum extent and the beginning of retreat. These represent downslope mass movements, plumites from meltwater alongside melting icebergs and turbidites. Radiocarbon dates from foraminifera suggest that the deglacial sedimentary sequence is up to 5m thick. The IRD concentration and abundance of the foraminifera Neogloboquadrina pachyderma sinistral indicate a minimum of 3 different calving events during deglaciation and a marked Younger Dryas cooling and ice calving period. Additionally the δ 18O record will be used to investigate the record of climatic changes in the region and x-ray fluorescence will be used to assess sediment provenance during deglaciation.

1km Global Terrestrial Carbon Flux: Estimations and Evaluations

NASA Astrophysics Data System (ADS)

Murakami, K.; Sasai, T.; Kato, S.; Saito, M.; Matsunaga, T.; Hiraki, K.; Maksyutov, S. S.

2017-12-01

Estimating global scale of the terrestrial carbon flux change with high accuracy and high resolution is important to understand global environmental changes. Furthermore the estimations of the global spatiotemporal distribution may contribute to the political and social activities such as REDD+. In order to reveal the current state of terrestrial carbon fluxes covering all over the world and a decadal scale. The satellite-based diagnostic biosphere model is suitable for achieving this purpose owing to observing on the present global land surface condition uniformly at some time interval. In this study, we estimated the global terrestrial carbon fluxes with 1km grids by using the terrestrial biosphere model (BEAMS). And we evaluated our new carbon flux estimations on various spatial scales and showed the transition of forest carbon stocks in some regions. Because BEAMS required high resolution meteorological data and satellite data as input data, we made 1km interpolated data using a kriging method. The data used in this study were JRA-55, GPCP, GOSAT L4B atmospheric CO2 data as meteorological data, and MODIS land product as land surface satellite data. Interpolating process was performed on the meteorological data because of insufficient resolution, but not on MODIS data. We evaluated our new carbon flux estimations using the flux tower measurement (FLUXNET2015 Datasets) in a point scale. We used 166 sites data for evaluating our model results. These flux sites are classified following vegetation type (DBF, EBF, ENF, mixed forests, grass lands, croplands, shrub lands, Savannas, wetlands). In global scale, the BEAMS estimations was underestimated compared to the flux measurements in the case of carbon uptake and release. The monthly variations of NEP showed relatively high correlations in DBF and mixed forests, but the correlation coefficients of EBF, ENF, and grass lands were less than 0.5. In the meteorological factors, air temperature and solar radiation showed very high correlations, and slight variations were showed in precipitation data. LAI data that was another large driving factor of terrestrial carbon cycle was not included in FLUXNET2015 datasets and it could not be evaluated.

Study of LEO-SAT microwave link for broad-band mobile satellite communication system

NASA Technical Reports Server (NTRS)

Fujise, Masayuki; Chujo, Wataru; Chiba, Isamu; Furuhama, Yoji; Kawabata, Kazuaki; Konishi, Yoshihiko

1993-01-01

In the field of mobile satellite communications, a system based on low-earth-orbit satellites (LEO-SAT's) such as the Iridium system has been proposed. The LEO-SAT system is able to offer mobile telecommunication services in high-latitude areas. Rain degradation, fading and shadowing are also expected to be decreased when the system is operated at a high elevation angle. Furthermore, the propagation delay generated in the LEO-SAT system is less pronounced than that in the geostationary orbit satellite (GEO-SAT) system and, in voice services, the effect of the delay is almost negligible. We proposed a concept of a broad-band mobile satellite communication system with LEO-SAT's and Optical ISL. In that system, a fixed L-band (1.6/1.5 GHz) multibeam is used to offer narrow band service to the mobile terminals in the entire area covered by a LEO-SAT and steerable Ka-band (30/20 GHz) spot beams are used for the wide band service. In this paper, we present results of a study of LEO-SAT microwave link between a satellite and a mobile terminal for a broad-band mobile satellite communication system. First, the results of link budget calculations are presented and the antennas mounted on satellites are shown. For a future mobile antenna technology, we also show digital beamforming (DBF) techniques. DBF, together with modulation and/or demodulation, is becoming a key technique for mobile antennas with advanced functions such as antenna pattern calibration, correction, and radio interference suppression. In this paper, efficient DBF techniques for transmitting and receiving are presented. Furthermore, an adaptive array antenna system suitable for this LEO-SAT is presented.

ENU mutagenesis screening for dominant behavioral mutations based on normal control data obtained in home-cage activity, open-field, and passive avoidance tests.

PubMed

Wada, Yumiko; Furuse, Tamio; Yamada, Ikuko; Masuya, Hiroshi; Kushida, Tomoko; Shibukawa, Yoko; Nakai, Yuji; Kobayashi, Kimio; Kaneda, Hideki; Gondo, Yoichi; Noda, Tetsuo; Shiroishi, Toshihiko; Wakana, Shigeharu

2010-01-01

To establish the cutoff values for screening ENU-induced behavioral mutations, normal variations in mouse behavioral data were examined in home-cage activity (HA), open-field (OF), and passive-avoidance (PA) tests. We defined the normal range as one that included more than 95% of the normal control values. The cutoffs were defined to identify outliers yielding values that deviated from the normal by less than 5% for C57BL/6J, DBA/2J, DBF(1), and N(2) (DXDB) progenies. Cutoff values for G1-phenodeviant (DBF(1)) identification were defined based on values over +/- 3.0 SD from the mean of DBF(1) for all parameters assessed in the HA and OF tests. For the PA test, the cutoff values were defined based on whether the mice met the learning criterion during the 2nd (at a shock intensity of 0.3 mA) or the 3rd (at a shock intensity of 0.15 mA) retention test. For several parameters, the lower outliers were undetectable as the calculated cutoffs were negative values. Based on the cutoff criteria, we identified 275 behavioral phenodeviants among 2,646 G1 progeny. Of these, 64 were crossed with wild-type DBA/2J individuals, and the phenotype transmission was examined in the G2 progeny using the cutoffs defined for N(2) mice. In the G2 mice, we identified 15 novel dominant mutants exhibiting behavioral abnormalities, including hyperactivity in the HA or OF tests, hypoactivity in the OF test, and PA deficits. Genetic and detailed behavioral analysis of these ENU-induced mutants will provide novel insights into the molecular mechanisms underlying behavior.

DNA repair decline during mouse spermiogenesis results in the accumulation of heritable DNA damage

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

Marchetti, Francesco; Marchetti, Francesco; Wryobek, Andrew J

The post-meiotic phase of mouse spermatogenesis (spermiogenesis) is very sensitive to the genomic effects of environmental mutagens because as male germ cells form mature sperm they progressively lose the ability to repair DNA damage. We hypothesized that repeated exposures to mutagens during this repair-deficient phase result in the accumulation of heritable genomic damage in mouse sperm that leads to chromosomal aberrations in zygotes after fertilization. We used a combination of single or fractionated exposures to diepoxybutane (DEB), a component of tobacco smoke, to investigate how differential DNA repair efficiencies during the three weeks of spermiogenesis affected the accumulation of DEB-inducedmore » heritable damage in early spermatids (21-15 days before fertilization, dbf), late spermatids (14-8 dbf) and sperm (7- 1 dbf). Analysis of chromosomalaberrations in zygotic metaphases using PAINT/DAPI showed that late spermatids and sperm are unable to repair DEB-induced DNA damage as demonstrated by significant increases (P<0.001) in the frequencies of zygotes with chromosomal aberrations. Comparisons between single and fractionated exposures suggested that the DNA repair-deficient window during late spermiogenesis may be less than two weeks in the mouse and that during this repair-deficient window there is accumulation of DNA damage in sperm. Finally, the dose-response study in sperm indicated a linear response for both single and repeated exposures. These findings show that the differential DNA repair capacity of post-meioitic male germ cells has a major impact on the risk of paternally transmitted heritable damage and suggest that chronic exposures that may occur in the weeks prior to fertilization because of occupational or lifestyle factors (i.e, smoking) can lead to an accumulation of genetic damage in sperm and result in heritable chromosomal aberrations of paternal origin.« less

DNA Repair Decline During Mouse Spermiogenesis Results in the Accumulation of Heritable DNA Damage

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

Marchetti, Francesco; Marchetti, Francesco; Wyrobek, Andrew J.

The post-meiotic phase of mouse spermatogenesis (spermiogenesis) is very sensitive to the genomic effects of environmental mutagens because as male germ cells form mature sperm they progressively lose the ability to repair DNA damage. We hypothesized that repeated exposures to mutagens during this repair-deficient phase result in the accumulation of heritable genomic damage in mouse sperm that leads to chromosomal aberrations in zygotes after fertilization. We used a combination of single or fractionated exposures to diepoxybutane (DEB), a component of tobacco smoke, to investigate how differential DNA repair efficiencies during the three weeks of spermiogenesis affected the accumulation of DEB-inducedmore » heritable damage in early spermatids (21-15 days before fertilization, dbf), late spermatids (14-8 dbf) and sperm (7-1 dbf). Analysis of chromosomal aberrations in zygotic metaphases using PAINT/DAPI showed that late spermatids and sperm are unable to repair DEB-induced DNA damage as demonstrated by significant increases (P<0.001) in the frequencies of zygotes with chromosomal aberrations. Comparisons between single and fractionated exposures suggested that the DNA repair-deficient window during late spermiogenesis may be less than two weeks in the mouse and that during this repair-deficient window there is accumulation of DNA damage in sperm. Finally, the dose-response study in sperm indicated a linear response for both single and repeated exposures. These findings show that the differential DNA repair capacity of post-meioitic male germ cells has a major impact on the risk of paternally transmitted heritable damage and suggest that chronic exposures that may occur in the weeks prior to fertilization because of occupational or lifestyle factors (i.e, smoking) can lead to an accumulation of genetic damage in sperm and result in heritable chromosomal aberrations of paternal origin.« less

Increased Cdc7 expression is a marker of oral squamous cell carcinoma and overexpression of Cdc7 contributes to the resistance to DNA-damaging agents.

PubMed

Cheng, An Ning; Jiang, Shih Sheng; Fan, Chi-Chen; Lo, Yu-Kang; Kuo, Chan-Yen; Chen, Chung-Hsing; Liu, Ying-Lan; Lee, Chun-Chung; Chen, Wei-Shone; Huang, Tze-Sing; Wang, Tao-Yeuan; Lee, Alan Yueh-Luen

2013-09-01

Cdc7-Dbf4 kinase (Dbf4-dependent kinase, DDK) is an essential factor of DNA replication and DNA damage response (DDR), which is associated with tumorigenesis. However, Cdc7 expression has never been associated to the outcome of oral squamous cell carcinoma (OSCC) patients, and the mechanism underlying cancer cell survival mediated by Cdc7 remains unclear. The Cdc7 protein expression of 105 OSCC tumor and 30 benign tissues was examined by immunohistochemistry assay. Overall survival rates of 80 OSCC patients were measured using Kaplan-Meier estimates and the log-rank tests. Cdc7 overexpression by adenovirus system was used to scrutinize the underlying mechanism contributed to cancer cell survival upon DDR. In silico analysis showed that increased Cdc7 is a common feature of cancer. Cdc7 overexpression was found in 96 of 105 (91.4%) studied cases of OSCC patients. Patients with higher Cdc7 expression, either categorized into two groups: Cdc7 high expression (2+ to 3+) versus Cdc7 low expression (0 to 1+) [hazard ratios (HR)=2.6; 95% confidence interval (CI)=1.28-5.43; P=0.0087] or four groups (0 to 3+) [HR=1.71; 95% CI=1.20-2.44; P=0.0032], exhibited a poorer outcome. Multivariate analysis showed that Cdc7 is an independent marker for survival prediction. Overexpressed Cdc7 inhibits genotoxin-induced apoptosis to increase the survival of cancer cells. In summary, Cdc7 expression, which is universally upregulated in cancer, is an independent prognostic marker of OSCC. Cdc7 inhibits genotoxin-induced apoptosis and increases survival in cancer cells upon DDR, suggesting that high expression of Cdc7 enhances the resistance to chemotherapy. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

The MIMIC Model as a Tool for Differential Bundle Functioning Detection

ERIC Educational Resources Information Center

Finch, W. Holmes

2012-01-01

Increasingly, researchers interested in identifying potentially biased test items are encouraged to use a confirmatory, rather than exploratory, approach. One such method for confirmatory testing is rooted in differential bundle functioning (DBF), where hypotheses regarding potential differential item functioning (DIF) for sets of items (bundles)…

Repellency to ticks (Acari: Ixodidae) of extracts of nigella sativa L.(Ranunculaceae) and the anti-inflammatory DogsBestFriend™

USDA-ARS?s Scientific Manuscript database

Motivated by observations that the canine anti-inflammatory cream DogsBestFriend™ (DBF) appeared to deter flies, mosquitoes, and ticks from treated animals, repellent efficacy bioassays using four species of ticks were conducted with three extracts of Nigella sativa L. (Ranunculaceae), a constituent...

Exploring Differential Bundle Functioning in Mathematics by Gender: The Effect of Hierarchical Modelling

ERIC Educational Resources Information Center

Ong, Yoke Mooi; Williams, Julian; Lamprianou, Iasonas

2013-01-01

Researchers interested in exploring substantive group differences are increasingly attending to bundles of items (or testlets): the aim is to understand how gender differences, for instance, are explained by differential performances on different types or bundles of items, hence differential bundle functioning (DBF). Some previous work has…

Relationships between net primary productivity and stand age for several forest types and their influence on China's carbon balance.

PubMed

Wang, Shaoqiang; Zhou, Lei; Chen, Jingming; Ju, Weimin; Feng, Xianfeng; Wu, Weixing

2011-06-01

Affected by natural and anthropogenic disturbances such as forest fires, insect-induced mortality and harvesting, forest stand age plays an important role in determining the distribution of carbon pools and fluxes in a variety of forest ecosystems. An improved understanding of the relationship between net primary productivity (NPP) and stand age (i.e., age-related increase and decline in forest productivity) is essential for the simulation and prediction of the global carbon cycle at annual, decadal, centurial, or even longer temporal scales. In this paper, we developed functions describing the relationship between national mean NPP and stand age using stand age information derived from forest inventory data and NPP simulated by the BEPS (Boreal Ecosystem Productivity Simulator) model in 2001. Due to differences in ecobiophysical characteristics of different forest types, NPP-age equations were developed for five typical forest ecosystems in China (deciduous needleleaf forest (DNF), evergreen needleleaf forest in tropic and subtropical zones (ENF-S), deciduous broadleaf forest (DBF), evergreen broadleaf forest (EBF), and mixed broadleaf forest (MBF)). For DNF, ENF-S, EBF, and MBF, changes in NPP with age were well fitted with a common non-linear function, with R(2) values equal to 0.90, 0.75, 0.66, and 0.67, respectively. In contrast, a second order polynomial was best suitable for simulating the change of NPP for DBF, with an R(2) value of 0.79. The timing and magnitude of the maximum NPP varied with forest types. DNF, EBF, and MBF reached the peak NPP at the age of 54, 40, and 32 years, respectively, while the NPP of ENF-S maximizes at the age of 13 years. The highest NPP of DBF appeared at 122 years. NPP was generally lower in older stands with the exception of DBF, and this particular finding runs counter to the paradigm of age-related decline in forest growth. Evaluation based on measurements of NPP and stand age at the plot-level demonstrates the reliability and applicability of the fitted NPP-age relationships. These relationships were used to replace the normalized NPP-age relationship used in the original InTEC (Integrated Terrestrial Ecosystem Carbon) model, to improve the accuracy of estimated carbon balance for China's forest ecosystems. With the revised NPP-age relationship, the InTEC model simulated a larger carbon source from 1950-1980 and a larger carbon sink from 1985-2001 for China's forests than the original InTEC model did because of the modification to the age-related carbon dynamics in forests. This finding confirms the importance of considering the dynamics of NPP related to forest age in estimating regional and global terrestrial carbon budgets. Copyright © 2011 Elsevier Ltd. All rights reserved.

Excess electron is trapped in a large single molecular cage C60F60.

PubMed

Wang, Yin-Feng; Li, Zhi-Ru; Wu, Di; Sun, Chia-Chung; Gu, Feng-Long

2010-01-15

A new kind of solvated electron systems, sphere-shaped e(-)@C60F60 (I(h)) and capsule-shaped e(-)@C60F60 (D6h), in contrast to the endohedral complex M@C60, is represented at the B3LYP/6-31G(d) + dBF (diffusive basis functions) density functional theory. It is proven, by examining the singly occupied molecular orbital (SOMO) and the spin density map of e(-)@C60F60, that the excess electron is indeed encapsulated inside the C60F60 cage. The shape of the electron cloud in SOMO matches with the shape of C60F60 cage. These cage-like single molecular solvated electrons have considerably large vertical electron detachment energies VDE of 4.95 (I(h)) and 4.67 eV (D6h) at B3LYP/6-31+G(3df) + dBF level compared to the VDE of 3.2 eV for an electron in bulk water (Coe et al., Int Rev Phys Chem 2001, 20, 33) and that of 3.66 eV for e(-)@C20F20 (Irikura, J Phys Chem A 2008, 112, 983), which shows their higher stability. The VDE of the sphere-shaped e(-)@C60F60 (I(h)) is greater than that of the capsule-shaped e(-)@C60F60 (D6h), indicating that the excess electron prefers to reside in the cage with the higher symmetry to form the more stable solvated electron. It is also noticed that the cage size [7.994 (I(h)), 5.714 and 9.978 A (D6h) in diameter] is much larger than that (2.826 A) of (H2O)20- dodecahedral cluster (Khan, Chem Phys Lett 2005, 401, 85). Copyright 2009 Wiley Periodicals, Inc.

Assessing the Effect of Language Demand in Bundles of Math Word Problems

ERIC Educational Resources Information Center

Banks, Kathleen; Jeddeeni, Ahmad; Walker, Cindy M.

2016-01-01

Differential bundle functioning (DBF) analyses were conducted to determine whether seventh and eighth grade second language learners (SLLs) had lower probabilities of answering bundles of math word problems correctly that had heavy language demands, when compared to non-SLLs of equal math proficiency. Math word problems on each of four test forms…

The Nuclear Dbf2-Related Kinase COT1 and the Mitogen-Activated Protein Kinases MAK1 and MAK2 Genetically Interact to Regulate Filamentous Growth, Hyphal Fusion and Sexual Development in Neurospora crassa

PubMed Central

Maerz, Sabine; Ziv, Carmit; Vogt, Nico; Helmstaedt, Kerstin; Cohen, Nourit; Gorovits, Rena; Yarden, Oded; Seiler, Stephan

2008-01-01

Ndr kinases, such as Neurospora crassa COT1, are important for cell differentiation and polar morphogenesis, yet their input signals as well as their integration into a cellular signaling context are still elusive. Here, we identify the cot-1 suppressor gul-4 as mak-2 and show that mutants of the gul-4/mak-2 mitogen-activated protein (MAP) kinase pathway suppress cot-1 phenotypes along with a concomitant reduction in protein kinase A (PKA) activity. Furthermore, mak-2 pathway defects are partially overcome in a cot-1 background and are associated with increased MAK1 MAPK signaling. A comparative characterization of N. crassa MAPKs revealed that they act as three distinct modules during vegetative growth and asexual development. In addition, common functions of MAK1 and MAK2 signaling during maintenance of cell-wall integrity distinguished the two ERK-type pathways from the p38-type OS2 osmosensing pathway. In contrast to separate functions during vegetative growth, the concerted activity of the three MAPK pathways is essential for cell fusion and for the subsequent formation of multicellular structures that are required for sexual development. Taken together, our data indicate a functional link between COT1 and MAPK signaling in regulating filamentous growth, hyphal fusion, and sexual development. PMID:18562669

Behavioral indicators of sublethal toxicity in rainbow trout

USGS Publications Warehouse

Little, Edward E.; Archeski, Richard D.; Flerov, Boris A.; Kozlovskaya, Vera I.

1990-01-01

Four measures of behavior-spontaneous swimming activity, swimming capacity, feeding behavior, and vulnerability to predation-were assessed as indicators of sublethal toxicity in rainbow trout (Oncorhynchus mykiss) in 96-hr exposures to sublethal concentrations of six agricultural chemicals: carbaryl, chlordane, dimethylamine salt of 2,4-dichlorophenoxyacetic acid (2,4-DMA), tributyl phosphorotrithioate (DBF 1), methyl parathion, and pentachlorophenol. After exposures, behavioral changes consistently demonstrated sublethal toxicity, but effects on specific behaviors varied with contaminants and their concentrations were altered by the water quality criterion concentration for chlordane (2 μg/L), and at a concentration of DEF (5 μg/L) that had previously been shown to inhibit growth and survival after a 90-day exposure. Feeding behavior was inhibited most by exposure to DEF, 2,4-DMA, and methyl parathion. Vulnerability to predation was heightened most by exposure to carbaryl and pentachlorophenol. Although all chemicals inhibited spontaneous swimming activity, only carbaryl, DEF, and 2,4-DMA influenced swimming capacity.

D/B/F 98: Final Report Of the AIAA Student Aircraft Design, Build & Fly Competition

DTIC Science & Technology

1998-01-17

Jason Nichol Configuration, Materials (Leader) Greg Mondeau Aerodynamics (Leader) April Register Configuration Sung-LiehLin Aerodynamics Jefferson...and Astronautics Team Members: Aruni Athuada Lashan Athuada Jason Bachelor Sebastian Echinique Shelly Ellis Wayne Fulford Benjamin Goff...hierarchy of our design team: AIAA OFFICERS Jennifer Huddle - President Benjamin Goff- Vice President Cheree Kiernan - Secretary Jason Bachelor

Fire performance and decay resistance of solid wood and plywood treated with quaternary ammonia compounds and common fire retardants

Treesearch

Evren Terzi; S. Nami Kartal; Robert White; Katsumi Shinoda; Yuji Imamura

2010-01-01

In this study, the fire performance and decay resistance of solid wood and plywood treated with quaternary ammonia compounds (didecyl dimethyl ammonium chloride (DDAC) and didecyl dimethyl ammonium tetrafluoroborate (DBF)) were compared with the performance of untreated control specimens and specimens treated with common fire retardants ((monoammonium phosphate (MAP),...

Interaction between clay-based sealing components and crystalline host rock

NASA Astrophysics Data System (ADS)

Priyanto, D. G.; Dixon, D. A.; Man, A. G.

The results of hydraulic-mechanical (H-M) numerical simulation of a shaft seal installed at a fracture zone (FZ) in a crystalline host rock using the finite element method are presented. The primary function of a shaft seal is to limit short-circuiting of the groundwater flow regime via the shaft in a deep geological repository. Two different stages of system evolution were considered in this numerical modelling. Stage 1 simulates the groundwater flow into an open shaft, prior to seal installation. Stage 2 simulates the groundwater flow into the shaft seal after seal installation. Four different cases were completed to: (i) evaluate H-M response due to the interaction between clay-based sealing material and crystalline host rock in the shaft seal structure; (ii) quantify the effect of the different times between the completion of the shaft excavation and the completion of shaft seal installation on the H-M response; and (iii) define the potential effects of different sealing material configurations. Shaft sealing materials include the bentonite-sand mixture (BSM), dense backfill (DBF), and concrete plug (CP). The BSM has greater swelling capacity and lower hydraulic conductivity ( K) than the DBF. The results of these analyses show that the decrease of the pore water pressure is concentrated along the fracture zone (FZ), which has the greatest K. As the time increases, the greatest decrease in pore water pressure is found around the FZ. Following FZ isolation and the subsequent filling of the shaft with water as it floods, the pore water pressure profile tends to recover back to the initial conditions prior to shaft excavation. The majority of the fluids that ultimately saturate the centre of the shaft seal flow radially inwards from the FZ. The time between the completion of the shaft excavation and the completion of shaft seal installation has a significant effect on the saturation time. A shorter time can reduce the saturation time. Since most of the inflow comes from the FZ, application of the BSM for extended distances above and below the FZ does not significantly affect the saturation time of the volume adjacent to the FZ. The application of BSM near the FZ rather than a low swelling capacity, more permeable filling material is very significant. This study assumed a perfect contact between seal materials and host rock. Limited to the assumptions used in this study, use of BSM near the FZ was found to increase the time before the centre of the shaft seal became fully saturated from between 4 and 30 years (when the DBF is used) to between 90 and 100 years (when the BSM is used).

mer, fac, and Bidentate Coordination of an Alkyl-POP Ligand in the Chemistry of Nonclassical Osmium Hydrides.

PubMed

Esteruelas, Miguel A; García-Yebra, Cristina; Martín, Jaime; Oñate, Enrique

2017-01-03

Nonclassical and classical osmium polyhydrides containing the diphosphine 9,9-dimethyl-4,5-bis(diisopropylphosphino)xanthene (xant(P i Pr 2 ) 2 ), coordinated in κ 3 -mer, κ 3 -fac, and κ 2 -P,P fashions, have been isolated during the cyclic formation of H 2 by means of the sequential addition of H + and H - or H - and H + to the classical trihydride OsH 3 Cl{xant(P i Pr 2 ) 2 } (1). This complex adds H + to form the compressed dihydride dihydrogen complex [OsCl(H···H)(η 2 -H 2 ){xant(P i Pr 2 ) 2 }] + (2). Under argon, cation 2 loses H 2 and the resulting unsaturated fragment dimerizes to give [(Os(H···H){xant(P i Pr 2 ) 2 }) 2 (μ-Cl) 2 ] 2+ (3). During the transformation the phosphine changes its coordination mode from mer to fac. The benzofuran counterpart of 1, OsH 3 Cl{dbf(P i Pr 2 ) 2 } (4; dbf(P i Pr 2 ) 2 = 4,6-bis(diisopropylphosphino)dibenzofuran), also adds H + to afford the benzofuran counterpart of 2, [OsCl(H···H)(η 2 -H 2 ){xant(P i Pr 2 ) 2 }] + (5), which in contrast to the latter is stable and does not dimerize. Acetonitrile breaks the chloride bridge of 3 to form the dihydrogen [OsCl(η 2 -H 2 )(CH 3 CN){xant(P i Pr 2 ) 2 }] + (6), regenerating the mer coordination of the diphosphine. The hydride ion also breaks the chloride bridge of 3. The addition of KH to 3 leads to 1, closing a cycle for the formation of H 2 . Complex 1 reacts with a second hydride ion to give OsH 4 {xant(P i Pr 2 ) 2 } (7) as consequence of the displacement of the chloride. Similarly to the latter, the oxygen atom of the mer-coordinated diphosphine of 7 has a tendency to be displaced by the hydride ion. Thus, the addition of KH to 7 yields [OsH 5 {xant(P i Pr 2 ) 2 }] - (8), containing a κ 2 -P,P-diphosphine. Complex 8 is easily protonated to afford OsH 6 {xant(P i Pr 2 ) 2 } (9), which releases H 2 to regenerate 7, closing a second cycle for the formation of molecular hydrogen.

Focus control enhancement and on-product focus response analysis methodology

NASA Astrophysics Data System (ADS)

Kim, Young Ki; Chen, Yen-Jen; Hao, Xueli; Samudrala, Pavan; Gomez, Juan-Manuel; Mahoney, Mark O.; Kamalizadeh, Ferhad; Hanson, Justin K.; Lee, Shawn; Tian, Ye

2016-03-01

With decreasing CDOF (Critical Depth Of Focus) for 20/14nm technology and beyond, focus errors are becoming increasingly critical for on-product performance. Current on product focus control techniques in high volume manufacturing are limited; It is difficult to define measurable focus error and optimize focus response on product with existing methods due to lack of credible focus measurement methodologies. Next to developments in imaging and focus control capability of scanners and general tool stability maintenance, on-product focus control improvements are also required to meet on-product imaging specifications. In this paper, we discuss focus monitoring, wafer (edge) fingerprint correction and on-product focus budget analysis through diffraction based focus (DBF) measurement methodology. Several examples will be presented showing better focus response and control on product wafers. Also, a method will be discussed for a focus interlock automation system on product for a high volume manufacturing (HVM) environment.

The wheat AGC kinase TaAGC1 is a positive contributor to host resistance to the necrotrophic pathogen Rhizoctonia cerealis

PubMed Central

Zhu, Xiuliang; Yang, Kun; Wei, Xuening; Zhang, Qiaofeng; Rong, Wei; Du, Lipu; Ye, Xingguo; Qi, Lin; Zhang, Zengyan

2015-01-01

Considerable progress has been made in understanding the roles of AGC kinases in mammalian systems. However, very little is known about the roles of AGC kinases in wheat (Triticum aestivum). The necrotrophic fungus Rhizoctonia cerealis is the major pathogen of the destructive disease sharp eyespot of wheat. In this study, the wheat AGC kinase gene TaAGC1, responding to R. cerealis infection, was isolated, and its properties and role in wheat defence were characterized. R. cerealis-resistant wheat lines expressed TaAGC1 at higher levels than susceptible wheat lines. Sequence and phylogenetic analyses showed that the TaAGC1 protein is a serine/threonine kinase belonging to the NDR (nuclear Dbf2-related) subgroup of AGC kinases. Kinase activity assays proved that TaAGC1 is a functional kinase and the Asp-239 residue located in the conserved serine/threonine kinase domain of TaAGC1 is required for the kinase activity. Subcellular localization assays indicated that TaAGC1 localized in the cytoplasm and nucleus. Virus-induced TaAGC1 silencing revealed that the down-regulation of TaAGC1 transcripts significantly impaired wheat resistance to R. cerealis. The molecular characterization and responses of TaAGC1 overexpressing transgenic wheat plants indicated that TaAGC1 overexpression significantly enhanced resistance to sharp eyespot and reduced the accumulation of reactive oxygen species (ROS) in wheat plants challenged with R. cerealis. Furthermore, ROS-scavenging and certain defence-associated genes were up-regulated in resistant plants overexpressing TaAGC1 but down-regulated in susceptible knock-down plants. These results suggested that the kinase TaAGC1 positively contributes to wheat immunity to R. cerealis through regulating expression of ROS-related and defence-associated genes. PMID:26220083

Phosphorylation of CMG helicase and Tof1 is required for programmed fork arrest

PubMed Central

Bastia, Deepak; Srivastava, Pankaj; Zaman, Shamsu; Choudhury, Malay; Mohanty, Bidyut K.; Bacal, Julien; Langston, Lance D.; Pasero, Philippe; O’Donnell, Michael E.

2016-01-01

Several important physiological transactions, including control of replicative life span (RLS), prevention of collision between replication and transcription, and cellular differentiation, require programmed replication fork arrest (PFA). However, a general mechanism of PFA has remained elusive. We previously showed that the Tof1–Csm3 fork protection complex is essential for PFA by antagonizing the Rrm3 helicase that displaces nonhistone protein barriers that impede fork progression. Here we show that mutations of Dbf4-dependent kinase (DDK) of Saccharomyces cerevisiae, but not other DNA replication factors, greatly reduced PFA at replication fork barriers in the spacer regions of the ribosomal DNA array. A key target of DDK is the mini chromosome maintenance (Mcm) 2–7 complex, which is known to require phosphorylation by DDK to form an active CMG [Cdc45 (cell division cycle gene 45), Mcm2–7, GINS (Go, Ichi, Ni, and San)] helicase. In vivo experiments showed that mutational inactivation of DDK caused release of Tof1 from the chromatin fractions. In vitro binding experiments confirmed that CMG and/or Mcm2–7 had to be phosphorylated for binding to phospho-Tof1–Csm3 but not to its dephosphorylated form. Suppressor mutations that bypass the requirement for Mcm2–7 phosphorylation by DDK restored PFA in the absence of the kinase. Retention of Tof1 in the chromatin fraction and PFA in vivo was promoted by the suppressor mcm5-bob1, which bypassed DDK requirement, indicating that under this condition a kinase other than DDK catalyzed the phosphorylation of Tof1. We propose that phosphorylation regulates the recruitment and retention of Tof1–Csm3 by the replisome and that this complex antagonizes the Rrm3 helicase, thereby promoting PFA, by preserving the integrity of the Fob1–Ter complex. PMID:27298353

DDT Contamination of Migrating Birds Using White-faced Ibis as an Indicator Species

DTIC Science & Technology

2005-02-01

en Alimentación y Desarrollo A.C. (Dr. Jaqueline Garcia) and the Universidad de Guadalajara (Dr. Eduardo Santana and Rodrigo Esparza). Mary Gustafson...Argos were processed by Earthspan and files created (.xls, .dbf, and shapefiles) for use in ArcGIS (ESRI GIS and Mapping Software ). Locations were...the latter. The critical task of collecting invertebrates and detailing avian associates in Mexico was ably performed by the Centro de Investigación

TIGER 2010 Boundaries

EPA Pesticide Factsheets

This EnviroAtlas web service supports research and online mapping activities related to EnviroAtlas (https://www.epa.gov/enviroatlas). This web service includes the State and County boundaries from the TIGER shapefiles compiled into a single national coverage for each layer. The TIGER/Line Files are shapefiles and related database files (.dbf) that are an extract of selected geographic and cartographic information from the U.S. Census Bureau's Master Address File / Topologically Integrated Geographic Encoding and Referencing (MAF/TIGER) Database (MTDB).

TIGER 2010 Boundaries

EPA Pesticide Factsheets

This EnviroAtlas web service supports research and online mapping activities related to EnviroAtlas (https://www.epa.gov/enviroatlas). This web service includes the State, County, and Census Block Groups boundaries from the TIGER shapefiles compiled into a single national coverage for each layer. The TIGER/Line Files are shapefiles and related database files (.dbf) that are an extract of selected geographic and cartographic information from the U.S. Census Bureau's Master Address File / Topologically Integrated Geographic Encoding and Referencing (MAF/TIGER) Database (MTDB).

Wld S protein requires Nmnat activity and a short N-terminal sequence to protect axons in mice.

PubMed

Conforti, Laura; Wilbrey, Anna; Morreale, Giacomo; Janeckova, Lucie; Beirowski, Bogdan; Adalbert, Robert; Mazzola, Francesca; Di Stefano, Michele; Hartley, Robert; Babetto, Elisabetta; Smith, Trevor; Gilley, Jonathan; Billington, Richard A; Genazzani, Armando A; Ribchester, Richard R; Magni, Giulio; Coleman, Michael

2009-02-23

The slow Wallerian degeneration (Wld(S)) protein protects injured axons from degeneration. This unusual chimeric protein fuses a 70-amino acid N-terminal sequence from the Ube4b multiubiquitination factor with the nicotinamide adenine dinucleotide-synthesizing enzyme nicotinamide mononucleotide adenylyl transferase 1. The requirement for these components and the mechanism of Wld(S)-mediated neuroprotection remain highly controversial. The Ube4b domain is necessary for the protective phenotype in mice, but precisely which sequence is essential and why are unclear. Binding to the AAA adenosine triphosphatase valosin-containing protein (VCP)/p97 is the only known biochemical property of the Ube4b domain. Using an in vivo approach, we show that removing the VCP-binding sequence abolishes axon protection. Replacing the Wld(S) VCP-binding domain with an alternative ataxin-3-derived VCP-binding sequence restores its protective function. Enzyme-dead Wld(S) is unable to delay Wallerian degeneration in mice. Thus, neither domain is effective without the function of the other. Wld(S) requires both of its components to protect axons from degeneration.

Familial discoid lupus erythematosus associated with heterozygote C2 deficiency.

PubMed

Belin, D C; Bordwell, B J; Einarson, M E; McLean, R H; Weinstein, A; Yunis, E J; Rothfield, N F

1980-08-01

Two siblings with chronic discoid lupus erythematosus and several family members were found with heterozygous C2 deficiency. An association with histocompatibility markers HLA-B18 and HLA-Dw2 was demonstrated, and the slow allotype of factor B was present. Linkage studies in this family suggested a close linkage between the C2 deficiency gene and genes coding for B18, Dw2, and BfS antigens. One HLA-ACB/DBf recombinant was observed showing closer linkage between HLA-D and Bf than between HLA-B and Bf.

The wheat AGC kinase TaAGC1 is a positive contributor to host resistance to the necrotrophic pathogen Rhizoctonia cerealis.

PubMed

Zhu, Xiuliang; Yang, Kun; Wei, Xuening; Zhang, Qiaofeng; Rong, Wei; Du, Lipu; Ye, Xingguo; Qi, Lin; Zhang, Zengyan

2015-11-01

Considerable progress has been made in understanding the roles of AGC kinases in mammalian systems. However, very little is known about the roles of AGC kinases in wheat (Triticum aestivum). The necrotrophic fungus Rhizoctonia cerealis is the major pathogen of the destructive disease sharp eyespot of wheat. In this study, the wheat AGC kinase gene TaAGC1, responding to R. cerealis infection, was isolated, and its properties and role in wheat defence were characterized. R. cerealis-resistant wheat lines expressed TaAGC1 at higher levels than susceptible wheat lines. Sequence and phylogenetic analyses showed that the TaAGC1 protein is a serine/threonine kinase belonging to the NDR (nuclear Dbf2-related) subgroup of AGC kinases. Kinase activity assays proved that TaAGC1 is a functional kinase and the Asp-239 residue located in the conserved serine/threonine kinase domain of TaAGC1 is required for the kinase activity. Subcellular localization assays indicated that TaAGC1 localized in the cytoplasm and nucleus. Virus-induced TaAGC1 silencing revealed that the down-regulation of TaAGC1 transcripts significantly impaired wheat resistance to R. cerealis. The molecular characterization and responses of TaAGC1 overexpressing transgenic wheat plants indicated that TaAGC1 overexpression significantly enhanced resistance to sharp eyespot and reduced the accumulation of reactive oxygen species (ROS) in wheat plants challenged with R. cerealis. Furthermore, ROS-scavenging and certain defence-associated genes were up-regulated in resistant plants overexpressing TaAGC1 but down-regulated in susceptible knock-down plants. These results suggested that the kinase TaAGC1 positively contributes to wheat immunity to R. cerealis through regulating expression of ROS-related and defence-associated genes. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Control of the mitotic exit network during meiosis

PubMed Central

Attner, Michelle A.; Amon, Angelika

2012-01-01

The mitotic exit network (MEN) is an essential GTPase signaling pathway that triggers exit from mitosis in budding yeast. We show here that during meiosis, the MEN is dispensable for exit from meiosis I but contributes to the timely exit from meiosis II. Consistent with a role for the MEN during meiosis II, we find that the signaling pathway is active only during meiosis II. Our analysis further shows that MEN signaling is modulated during meiosis in several key ways. Whereas binding of MEN components to spindle pole bodies (SPBs) is necessary for MEN signaling during mitosis, during meiosis MEN signaling occurs off SPBs and does not require the SPB recruitment factor Nud1. Furthermore, unlike during mitosis, MEN signaling is controlled through the regulated interaction between the MEN kinase Dbf20 and its activating subunit Mob1. Our data lead to the conclusion that a pathway essential for vegetative growth is largely dispensable for the specialized meiotic divisions and provide insights into how cell cycle regulatory pathways are modulated to accommodate different modes of cell division. PMID:22718910

Novel probabilistic and distributed algorithms for guidance, control, and nonlinear estimation of large-scale multi-agent systems

NASA Astrophysics Data System (ADS)

Bandyopadhyay, Saptarshi

Multi-agent systems are widely used for constructing a desired formation shape, exploring an area, surveillance, coverage, and other cooperative tasks. This dissertation introduces novel algorithms in the three main areas of shape formation, distributed estimation, and attitude control of large-scale multi-agent systems. In the first part of this dissertation, we address the problem of shape formation for thousands to millions of agents. Here, we present two novel algorithms for guiding a large-scale swarm of robotic systems into a desired formation shape in a distributed and scalable manner. These probabilistic swarm guidance algorithms adopt an Eulerian framework, where the physical space is partitioned into bins and the swarm's density distribution over each bin is controlled using tunable Markov chains. In the first algorithm - Probabilistic Swarm Guidance using Inhomogeneous Markov Chains (PSG-IMC) - each agent determines its bin transition probabilities using a time-inhomogeneous Markov chain that is constructed in real-time using feedback from the current swarm distribution. This PSG-IMC algorithm minimizes the expected cost of the transitions required to achieve and maintain the desired formation shape, even when agents are added to or removed from the swarm. The algorithm scales well with a large number of agents and complex formation shapes, and can also be adapted for area exploration applications. In the second algorithm - Probabilistic Swarm Guidance using Optimal Transport (PSG-OT) - each agent determines its bin transition probabilities by solving an optimal transport problem, which is recast as a linear program. In the presence of perfect feedback of the current swarm distribution, this algorithm minimizes the given cost function, guarantees faster convergence, reduces the number of transitions for achieving the desired formation, and is robust to disturbances or damages to the formation. We demonstrate the effectiveness of these two proposed swarm guidance algorithms using results from numerical simulations and closed-loop hardware experiments on multiple quadrotors. In the second part of this dissertation, we present two novel discrete-time algorithms for distributed estimation, which track a single target using a network of heterogeneous sensing agents. The Distributed Bayesian Filtering (DBF) algorithm, the sensing agents combine their normalized likelihood functions using the logarithmic opinion pool and the discrete-time dynamic average consensus algorithm. Each agent's estimated likelihood function converges to an error ball centered on the joint likelihood function of the centralized multi-sensor Bayesian filtering algorithm. Using a new proof technique, the convergence, stability, and robustness properties of the DBF algorithm are rigorously characterized. The explicit bounds on the time step of the robust DBF algorithm are shown to depend on the time-scale of the target dynamics. Furthermore, the DBF algorithm for linear-Gaussian models can be cast into a modified form of the Kalman information filter. In the Bayesian Consensus Filtering (BCF) algorithm, the agents combine their estimated posterior pdfs multiple times within each time step using the logarithmic opinion pool scheme. Thus, each agent's consensual pdf minimizes the sum of Kullback-Leibler divergences with the local posterior pdfs. The performance and robust properties of these algorithms are validated using numerical simulations. In the third part of this dissertation, we present an attitude control strategy and a new nonlinear tracking controller for a spacecraft carrying a large object, such as an asteroid or a boulder. If the captured object is larger or comparable in size to the spacecraft and has significant modeling uncertainties, conventional nonlinear control laws that use exact feed-forward cancellation are not suitable because they exhibit a large resultant disturbance torque. The proposed nonlinear tracking control law guarantees global exponential convergence of tracking errors with finite-gain Lp stability in the presence of modeling uncertainties and disturbances, and reduces the resultant disturbance torque. Further, this control law permits the use of any attitude representation and its integral control formulation eliminates any constant disturbance. Under small uncertainties, the best strategy for stabilizing the combined system is to track a fuel-optimal reference trajectory using this nonlinear control law, because it consumes the least amount of fuel. In the presence of large uncertainties, the most effective strategy is to track the derivative plus proportional-derivative based reference trajectory, because it reduces the resultant disturbance torque. The effectiveness of the proposed attitude control law is demonstrated by using results of numerical simulation based on an Asteroid Redirect Mission concept. The new algorithms proposed in this dissertation will facilitate the development of versatile autonomous multi-agent systems that are capable of performing a variety of complex tasks in a robust and scalable manner.

Phenological and physiological mechanisms underlying interannual variability of terrestrial net ecosystem production

NASA Astrophysics Data System (ADS)

Niu, S.; Luo, Y.; Hui, D.; Chen, J.

2013-12-01

The interannual variability (IAV) of atmospheric CO2 concentration varies substantial and is largely ascribed to IAV of terrestrial ecosystem carbon fluxes. However, we have limited understanding on the mechanisms that control the IAV on the carbon flux of terrestrial ecosystems. Here, we hypothesized that physiological and phonological processes regulate IAV significantly in terrestrial carbon uptake (i.e., net ecosystem production, NEP). To test this hypothesis, we analyzed eddy-covariance data from 24 sites with more than 8 years data in deciduous broadleaf forests (DBF), evergreen forests (EF), and grasslands (GRA) in the northern hemisphere. Ecosystem physiology is represented by the maximum carbon uptake capacity (NEPmax) in one year whereas phonology is represented by carbon uptake period (CUP). We found that yearly anomalies of CUP and NEPmax accounted for 40% and 60% separately, and 73% in combination, of the anomalies in annual NEP across all the 253 site-years, with their relative contributions varying among the sites. The IAV of CUP was determined by the anomalies of spring and autumn carbon uptake phenology, both of which were sensitive to climate changes but controlled by different environmental factors in different biomes. IAV of NEPmax was determined by summer precipitation anomalies in DBF and GRA. The results suggest that IAV of NEP is consistently co-determined by CUP and NEPmax anomalies among sites in the northern hemisphere. Overall, the mechanisms revealed by our study on NEP anomalies through changing in phenology and physiology contribute to predictive understanding of temporal dynamics of terrestrial carbon uptake.

42 CFR 84.100 - Man tests 1, 2, 3, and 4; requirements.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 1 2011-10-01 2011-10-01 false Man tests 1, 2, 3, and 4; requirements. 84.100...-Contained Breathing Apparatus § 84.100 Man tests 1, 2, 3, and 4; requirements. Man tests 1, 2, 3, and 4, set forth in Tables 1, 2, 3, and 4 of this subpart, respectively, prescribe the duration and sequence of...

Functional relevance of “seed” and “non-seed” sequences in microRNA-mediated promotion of C. elegans developmental progression

PubMed Central

Zhang, Huibin; Artiles, Karen L.; Fire, Andrew Z.

2015-01-01

The founding heterochronic microRNAs, lin-4 and let-7, together with their validated targets and well-characterized phenotypes in C. elegans, offer an opportunity to test functionality of microRNAs in a developmental context. In this study, we defined sequence requirements at the microRNA level for these two microRNAs, evaluating lin-4 and let-7 mutant microRNAs for their ability to support temporal development under conditions where the wild-type lin-4 and let-7 gene products are absent. For lin-4, we found a strong requirement for seed sequences, with function drastically affected by several central mutations in the seed sequence, while rescue was retained by a set of mutations peripheral to the seed. let-7 rescuing activity was retained to a surprising degree by a variety of central seed mutations, while several non-seed mutant effects support potential noncanonical contributions to let-7 function. Taken together, this work illustrates both the functional partnership between seed and non-seed sequences in mediating C. elegans temporal development and a diversity among microRNA effectors in the contributions of seed and non-seed regions to activity. PMID:26385508

Spliced leader RNA of trypanosomes: in vivo mutational analysis reveals extensive and distinct requirements for trans splicing and cap4 formation.

PubMed Central

Lücke, S; Xu, G L; Palfi, Z; Cross, M; Bellofatto, V; Bindereif, A

1996-01-01

In trypanosomes mRNAs are generated through trans splicing. The spliced leader (SL) RNA, which donates the 5'-terminal mini-exon to each of the protein coding exons, plays a central role in the trans splicing process. We have established in vivo assays to study in detail trans splicing, cap4 modification, and RNP assembly of the SL RNA in the trypanosomatid species Leptomonas seymouri. First, we found that extensive sequences within the mini-exon are required for SL RNA function in vivo, although a conserved length of 39 nt is not essential. In contrast, the intron sequence appears to be surprisingly tolerant to mutation; only the stem-loop II structure is indispensable. The asymmetry of the sequence requirements in the stem I region suggests that this domain may exist in different functional conformations. Second, distinct mini-exon sequences outside the modification site are important for efficient cap4 formation. Third, all SL RNA mutations tested allowed core RNP assembly, suggesting flexible requirements for core protein binding. In sum, the results of our mutational analysis provide evidence for a discrete domain structure of the SL RNA and help to explain the strong phylogenetic conservation of the mini-exon sequence and of the overall SL RNA secondary structure; they also suggest that there may be certain differences between trans splicing in nematodes and trypanosomes. This approach provides a basis for studying RNA-RNA interactions in the trans spliceosome. Images PMID:8861965

Structural Plasticity and Rapid Evolution in a Viral RNA Revealed by In Vivo Genetic Selection▿ †

PubMed Central

Guo, Rong; Lin, Wai; Zhang, Jiuchun; Simon, Anne E.; Kushner, David B.

2009-01-01

Satellite RNAs usually lack substantial homology with their helper viruses. The 356-nucleotide satC of Turnip crinkle virus (TCV) is unusual in that its 3′-half shares high sequence similarity with the TCV 3′ end. Computer modeling, structure probing, and/or compensatory mutagenesis identified four hairpins and three pseudoknots in this TCV region that participate in replication and/or translation. Two hairpins and two pseudoknots have been confirmed as important for satC replication. One portion of the related 3′ end of satC that remains poorly characterized corresponds to juxtaposed TCV hairpins H4a and H4b and pseudoknot ψ3, which are required for the TCV-specific requirement of translation (V. A. Stupina et al., RNA 14:2379-2393, 2008). Replacement of satC H4a with randomized sequence and scoring for fitness in plants by in vivo genetic selection (SELEX) resulted in winning sequences that contain an H4a-like stem-loop, which can have additional upstream sequence composing a portion of the stem. SELEX of the combined H4a and H4b region in satC generated three distinct groups of winning sequences. One group models into two stem-loops similar to H4a and H4b of TCV. However, the selected sequences in the other two groups model into single hairpins. Evolution of these single-hairpin SELEX winners in plants resulted in satC that can accumulate to wild-type (wt) levels in protoplasts but remain less fit in planta when competed against wt satC. These data indicate that two highly distinct RNA conformations in the H4a and H4b region can mediate satC fitness in protoplasts. PMID:19004956

Preliminary surficial geologic map database of the Amboy 30 x 60 minute quadrangle, California

USGS Publications Warehouse

Bedford, David R.; Miller, David M.; Phelps, Geoffrey A.

2006-01-01

The surficial geologic map database of the Amboy 30x60 minute quadrangle presents characteristics of surficial materials for an area approximately 5,000 km2 in the eastern Mojave Desert of California. This map consists of new surficial mapping conducted between 2000 and 2005, as well as compilations of previous surficial mapping. Surficial geology units are mapped and described based on depositional process and age categories that reflect the mode of deposition, pedogenic effects occurring post-deposition, and, where appropriate, the lithologic nature of the material. The physical properties recorded in the database focus on those that drive hydrologic, biologic, and physical processes such as particle size distribution (PSD) and bulk density. This version of the database is distributed with point data representing locations of samples for both laboratory determined physical properties and semi-quantitative field-based information. Future publications will include the field and laboratory data as well as maps of distributed physical properties across the landscape tied to physical process models where appropriate. The database is distributed in three parts: documentation, spatial map-based data, and printable map graphics of the database. Documentation includes this file, which provides a discussion of the surficial geology and describes the format and content of the map data, a database 'readme' file, which describes the database contents, and FGDC metadata for the spatial map information. Spatial data are distributed as Arc/Info coverage in ESRI interchange (e00) format, or as tabular data in the form of DBF3-file (.DBF) file formats. Map graphics files are distributed as Postscript and Adobe Portable Document Format (PDF) files, and are appropriate for representing a view of the spatial database at the mapped scale.

Cdc7 kinase - a new target for drug development.

PubMed

Swords, Ronan; Mahalingam, Devalingam; O'Dwyer, Michael; Santocanale, Corrado; Kelly, Kevin; Carew, Jennifer; Giles, Francis

2010-01-01

The cell division cycle 7 (Cdc7) is a serine threonine kinase that is of critical importance in the regulation of normal cell cycle progression. Cdc7 kinase is highly conserved during evolution and much has been learned about its biological roles in humans through the study of lower eukaryotes, particularly yeasts. Two important regulator proteins, Dbf4 and Drf1, bind to and modulate the kinase activity of human Cdc7 which phosphorylates several sites on Mcm2 (minichromosome maintenance protein 2), one of the six subunits of the replicative DNA helicase needed for duplication of the genome. Through regulation of both DNA synthesis and DNA damage response, both key functions in the survival of tumour cells, Cdc7 becomes an attractive target for pharmacological inhibition. There are much data available on the pre-clinical anti-cancer effects of Cdc7 depletion and although there are no available Cdc7 inhibitors in clinical trials as yet, several lead compounds are being optimised for this purpose. In this review, we will address the current status of Cdc7 as an important target for new drug development.

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.

Xenopus origin recognition complex (ORC) initiates DNA replication preferentially at sequences targeted by Schizosaccharomyces pombe ORC

PubMed Central

Kong, Daochun; Coleman, Thomas R.; DePamphilis, Melvin L.

2003-01-01

Budding yeast (Saccharomyces cerevisiae) origin recognition complex (ORC) requires ATP to bind specific DNA sequences, whereas fission yeast (Schizosaccharomyces pombe) ORC binds to specific, asymmetric A:T-rich sites within replication origins, independently of ATP, and frog (Xenopus laevis) ORC seems to bind DNA non-specifically. Here we show that despite these differences, ORCs are functionally conserved. Firstly, SpOrc1, SpOrc4 and SpOrc5, like those from other eukaryotes, bound ATP and exhibited ATPase activity, suggesting that ATP is required for pre-replication complex (pre-RC) assembly rather than origin specificity. Secondly, SpOrc4, which is solely responsible for binding SpORC to DNA, inhibited up to 70% of XlORC-dependent DNA replication in Xenopus egg extract by preventing XlORC from binding to chromatin and assembling pre-RCs. Chromatin-bound SpOrc4 was located at AT-rich sequences. XlORC in egg extract bound preferentially to asymmetric A:T-sequences in either bare DNA or in sperm chromatin, and it recruited XlCdc6 and XlMcm proteins to these sequences. These results reveal that XlORC initiates DNA replication preferentially at the same or similar sites to those targeted in S.pombe. PMID:12840006

Increasing time to postoperative stereotactic radiation therapy for patients with resected brain metastases: investigating clinical outcomes and identifying predictors associated with time to initiation.

PubMed

Yusuf, Mehran B; Amsbaugh, Mark J; Burton, Eric; Nelson, Megan; Williams, Brian; Koutourousiou, Maria; Nauta, Haring; Woo, Shiao

2018-02-01

We sought to determine the impact of time to initiation (TTI) of post-operative radiosurgery on clinical outcomes for patients with resected brain metastases and to identify predictors associated with TTI. All patients with resected brain metastases treated with postoperative SRS or fractionated stereotactic radiation therapy (fSRT) from 2012 to 2016 at a single institution were reviewed. TTI was defined as the interval from resection to first day of radiosurgery. Receiver operating characteristic (ROC) curves were used to identify an optimal threshold for TTI with respect to local failure (LF). Survival outcomes were estimated using the Kaplan-Meier method and analyzed using the log-rank test and Cox proportional hazards models. Logistic regression models were used to identify factors associated with ROC-determined TTI covariates. A total of 79 resected lesions from 73 patients were evaluated. An ROC curve of LF and TTI identified an optimal threshold for TTI of 30.5 days, with an area under the curve of 0.637. TTI > 30 days was associated with an increased hazard of LF (HR 4.525, CI 1.239-16.527) but was not significantly associated with survival (HR 1.002, CI 0.547-1.823) or distant brain failure (DBF, HR 1.943, CI 0.989-3.816). Fifteen patients (20.5%) required post-operative inpatient rehabilitation. Post-operative rehabilitation was associated with TTI > 30 days (OR 1.48, CI 1.142-1.922). In our study of resected brain metastases, longer time to initiation of post-operative radiosurgery was associated with increased local failure. Ideally, post-op SRS should be initiated within 30 days of resection if feasible.

Nucleotide sequence of the Saccharomyces cerevisiae PUT4 proline-permease-encoding gene: similarities between CAN1, HIP1 and PUT4 permeases.

PubMed

Vandenbol, M; Jauniaux, J C; Grenson, M

1989-11-15

The complete nucleotide (nt) sequence of the PUT4 gene, whose product is required for high-affinity proline active transport in the yeast Saccharomyces cerevisiae, is presented. The sequence contains a single long open reading frame of 1881 nt, encoding a polypeptide with a calculated Mr of 68,795. The predicted protein is strongly hydrophobic and exhibits six potential glycosylation sites. Its hydropathy profile suggests the presence of twelve membrane-spanning regions flanked by hydrophilic N- and C-terminal domains. The N terminus does not resemble signal sequences found in secreted proteins. These features are characteristic of integral membrane proteins catalyzing translocation of ligands across cellular membranes. Protein sequence comparisons indicate strong resemblance to the arginine and histidine permeases of S. cerevisiae, but no marked sequence similarity to the proline permease of Escherichia coli or to other known prokaryotic or eukaryotic transport proteins. The strong similarity between the three yeast amino acid permeases suggests a common ancestor for the three proteins.

Activity Enhancement of G-Quadruplex/Hemin DNAzyme by Flanking d(CCC).

PubMed

Chang, Tianjun; Gong, Hongmei; Ding, Pi; Liu, Xiangjun; Li, Weiguo; Bing, Tao; Cao, Zehui; Shangguan, Dihua

2016-03-14

G-quadruplex (G4)/hemin DNAzymes have been extensively applied in bioanalysis and molecular devices. However, their catalytic activity is still much lower than that of proteinous enzymes. The G4/hemin DNAzyme activity is correlated with the G4 conformations and the solution conditions. However, little is known about the effect of the flanking sequences on the activity, though they are important parts of G4s. Here, we report sequences containing d(CCC), flanked on both ends of the G4-core sequences remarkably enhance their DNAzyme activity. By using circular dichroism and UV-visible spectroscopy, the d(CCC) flanking sequences were demonstrated to improve the hemin binding affinity to G4s instead of increasing the parallel G4 formation, which might explain the enhanced DNAzyme activity. Meanwhile, the increased hemin binding ability promoted the degradation of hemin within the DNAzyme by H2O2. Furthermore, the DNAzyme with d(CCC) flanking sequences showed strong tolerance to pH value changes, which makes it more suitable for applications requiring wide pH conditions. The results highlight the influence of the flanking sequences on the DNAzyme activity and provide insightful information for the design of highly active DNAzymes. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Sequence search on a supercomputer.

PubMed

Gotoh, O; Tagashira, Y

1986-01-10

A set of programs was developed for searching nucleic acid and protein sequence data bases for sequences similar to a given sequence. The programs, written in FORTRAN 77, were optimized for vector processing on a Hitachi S810-20 supercomputer. A search of a 500-residue protein sequence against the entire PIR data base Ver. 1.0 (1) (0.5 M residues) is carried out in a CPU time of 45 sec. About 4 min is required for an exhaustive search of a 1500-base nucleotide sequence against all mammalian sequences (1.2M bases) in Genbank Ver. 29.0. The CPU time is reduced to about a quarter with a faster version.

Complete nucleotide sequences of the coat protein messenger RNAs of brome mosaic virus and cowpea chlorotic mottle virus.

PubMed Central

Dasgupta, R; Kaesberg, P

1982-01-01

The nucleotide sequences of the subgenomic coat protein messengers (RNA4's) of two related bromoviruses, brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV), have been determined by direct RNA and CDNA sequencing without cloning. BMV RNA4 is 876 b long including a 5' noncoding region of nine nucleotides and a 3' noncoding region of 300 nucleotides. CCMV RNA 4 is 824 b long, including a 5' noncoding region of 10 nucleotides and a 3' noncoding region of 244 nucleotides. The encoded coat proteins are similar in length (188 amino acids for BMV and 189 amino acids for CCMV) and display about 70% homology in their amino acid sequences. Length difference between the two RNAs is due mostly to a single deletion, in CCMV with respect to BMV, of about 57 b immediately following the coding region. Allowing for this deletion the RNAs are indicate that mutations leading to divergence were constrained in the coding region primarily by the requirement of maintaining a favorable coat protein structure and in the 3' noncoding region primarily by the requirement of maintaining a favorable RNA spatial configuration. PMID:6895941

Digital Beamforming Synthetic Aperture Radar Developments at NASA Goddard Space Flight Center

NASA Technical Reports Server (NTRS)

Rincon, Rafael; Fatoyinbo, Temilola; Osmanoglu, Batuhan; Lee, Seung Kuk; Du Toit, Cornelis F.; Perrine, Martin; Ranson, K. Jon; Sun, Guoqing; Deshpande, Manohar; Beck, Jaclyn;

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

PubMed Central

Mandal, Bijoy Kumar; Kim, Tai-hoon

2013-01-01

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

Sequences required for induction of neurotensin receptor gene expression during neuronal differentiation of N1E-115 neuroblastoma cells.

PubMed

Tavares, D; Tully, K; Dobner, P R

1999-10-15

The promoter region of the mouse high affinity neurotensin receptor (Ntr-1) gene was characterized, and sequences required for expression in neuroblastoma cell lines that express high affinity NT-binding sites were characterized. Me(2)SO-induced neuronal differentiation of N1E-115 neuroblastoma cells increased both the expression of the endogenous Ntr-1 gene and reporter genes driven by NTR-1 promoter sequences by 3-4-fold. Deletion analysis revealed that an 83-base pair promoter region containing the transcriptional start site is required for Me(2)SO activation. Detailed mutational analysis of this region revealed that a CACCC box and the central region of a large GC-rich palindrome are the crucial cis-regulatory elements required for Me(2)SO induction. The CACCC box is bound by at least one factor that is induced upon Me(2)SO treatment of N1E-115 cells. The Me(2)SO effect was found to be both selective and cell type-restricted. Basal expression in the neuroblastoma cell lines required a distinct set of sequences, including an Sp1-like sequence, and a sequence resembling an NGFI-A-binding site; however, a more distal 5' sequence was found to repress basal activity in N1E-115 cells. These results provide evidence that Ntr-1 gene regulation involves both positive and negative regulatory elements located in the 5'-flanking region and that Ntr-1 gene activation involves the coordinate activation or induction of several factors, including a CACCC box binding complex.

EEG and ECG changes during simulator operation reflect mental workload and vigilance.

PubMed

Dussault, Caroline; Jouanin, Jean-Claude; Philippe, Matthieu; Guezennec, Charles-Yannick

2005-04-01

Performing mission tasks in a simulator influences many neurophysiological measures. Quantitative assessments of electroencephalography (EEG) and electrocardiography (ECG) have made it possible to develop indicators of mental workload and to estimate relative physiological responses to cognitive requirements. To evaluate the effects of mental workload without actual physical risk, we studied the cortical and cardiovascular changes that occurred during simulated flight. There were 12 pilots (8 novices and 4 experts) who simulated a flight composed of 10 sequences that induced several different mental workload levels. EEG was recorded at 12 electrode sites during rest and flight sequences; ECG activity was also recorded. Subjective tests were used to evaluate anxiety and vigilance levels. Theta band activity was lower during the two simulated flight rest sequences than during visual and instrument flight sequences at central, parietal, and occipital sites (p < 0.05). On the other hand, rest sequences resulted in higher beta (at the C4 site; p < 0.05) and gamma (at the central, parietal, and occipital sites; p < 0.05) power than active segments. The mean heart rate (HR) was not significantly different during any simulated flight sequence, but HR was lower for expert subjects than for novices. The subjective tests revealed no significant anxiety and high values for vigilance levels before and during flight. The different flight sequences performed on the simulator resulted in electrophysiological changes that expressed variations in mental workload. These results corroborate those found during study of real flights, particularly during sequences requiring the heaviest mental workload.

An automated genotyping tool for enteroviruses and noroviruses.

PubMed

Kroneman, A; Vennema, H; Deforche, K; v d Avoort, H; Peñaranda, S; Oberste, M S; Vinjé, J; Koopmans, M

2011-06-01

Molecular techniques are established as routine in virological laboratories and virus typing through (partial) sequence analysis is increasingly common. Quality assurance for the use of typing data requires harmonization of genotype nomenclature, and agreement on target genes, depending on the level of resolution required, and robustness of methods. To develop and validate web-based open-access typing-tools for enteroviruses and noroviruses. An automated web-based typing algorithm was developed, starting with BLAST analysis of the query sequence against a reference set of sequences from viruses in the family Picornaviridae or Caliciviridae. The second step is phylogenetic analysis of the query sequence and a sub-set of the reference sequences, to assign the enterovirus type or norovirus genotype and/or variant, with profile alignment, construction of phylogenetic trees and bootstrap validation. Typing is performed on VP1 sequences of Human enterovirus A to D, and ORF1 and ORF2 sequences of genogroup I and II noroviruses. For validation, we used the tools to automatically type sequences in the RIVM and CDC enterovirus databases and the FBVE norovirus database. Using the typing-tools, 785(99%) of 795 Enterovirus VP1 sequences, and 8154(98.5%) of 8342 norovirus sequences were typed in accordance with previously used methods. Subtyping into variants was achieved for 4439(78.4%) of 5838 NoV GII.4 sequences. The online typing-tools reliably assign genotypes for enteroviruses and noroviruses. The use of phylogenetic methods makes these tools robust to ongoing evolution. This should facilitate standardized genotyping and nomenclature in clinical and public health laboratories, thus supporting inter-laboratory comparisons. Copyright © 2011 Elsevier B.V. All rights reserved.

Early responses measured in the brachyuran crab Carcinus maenas exposed to carbamazepine and novobiocin: application of a 2-tier approach.

PubMed

Aguirre-Martínez, G V; Del Valls, T A; Martín-Díaz, M L

2013-11-01

One of the main consequences of the constant input of pharmaceuticals to the aquatic environment is that biota might develop unknown chronic effects, thus affecting their health even at low concentrations. The aim of this study is to evaluate the health status of Carcinus maenas employing a 2-tier approach, after 28 days of exposure to carbamazepine (CBZ) and novobiocin (NOV) at 0.1, 1, 10 and 50µgL(-1). Lysosomal membrane stability (LMS) is employed in tier 1. In tier 2 was applied a battery of biomarkers of exposure and effect (ethoxyresorufin O-deethylase (EROD), dibenzyl flourescein dealkylase (DBF), glutathione S-transferase (GST), glutathione peroxidase (GPx), lipid peroxidation (LPO) and DNA adducts) measured in gill, hepatopancreas, muscle and gonad tissues. Results show a dose-dependent effect. LMS in crabs exposed to environmental concentrations of pharmaceuticals was significantly lower compared to controls (p<0.05), indicating their stressed status. EROD activity was induced significantly (p<0.05) in all tissues by NOV (10-50µgL(-1)). DBF activity was induced significantly (p<0.05) in gill and hepatopancreas tissues by CBZ (10-50µgL(-1)). GST activity was activated in all tissues of crabs exposed to the highest concentrations tested (p<0.05). All tissues showed induction of GPX activity after exposure to selected drugs (p<0.05). LPO was activated in gill and hepatopancreas tissues by the pharmaceuticals at 50µgL(-1) (p<0.05). Crabs exposed to NOV (50µgL(-1)) presented DNA damage in gill and hepatopancreas tissues (p<0.05). Environmental concentrations of these pharmaceuticals have a measurable effect on the biomarkers studied. The 2-tier approach applied might be a suitable tool for the assessment of sublethal responses in crabs exposed to pharmaceuticals in the marine environment. Copyright © 2013 Elsevier Inc. All rights reserved.

Pharmacokinetic-Pharmacodynamic Relationship of Erenumab (AMG 334) and Capsaicin-Induced Dermal Blood Flow in Healthy and Migraine Subjects.

PubMed

Vu, Thuy; Ma, Peiming; Chen, Jiyun Sunny; de Hoon, Jan; Van Hecken, Anne; Yan, Lucy; Wu, Liviawati Sutjandra; Hamilton, Lisa; Vargas, Gabriel

2017-09-01

Capsaicin-induced dermal blood flow (CIDBF) is a validated biomarker used to evaluate the target engagement of potential calcitonin gene-related peptide-blocking therapeutics for migraine. To characterize the pharmacokinetics (PK) and quantify the inhibitory effects of erenumab (AMG 334) on CIDBF, CIDBF data were pooled from a single- and a multiple-dose study in healthy and migraine subjects. Repeated capsaicin challenges and DBF measurements were performed and serum erenumab concentrations determined. A population analysis was conducted using a nonlinear mixed-effects modeling approach. Effects of body weight, gender, and age on model parameters were evaluated. Two-compartment target-mediated drug disposition (TMDD) model assuming binding of erenumab in the central compartment best described the nonlinear PK of erenumab. Subcutaneous absorption half-life was 1.6 days and bioavailability was 74%. Erenumab produced a maximum inhibition of 89% (95% confidence interval: 87-91%). Erenumab concentrations required for 50% and 99% of maximum inhibition were 255 ng/mL and 1134 ng/mL, respectively. Increased body weight was associated with increased erenumab clearance but had no effect on the inhibitory effect on CIDBF. Our results show that erenumab pharmacokinetics was best characterized by a TMDD model and resulted in potent inhibition of CIDBF.

Alternation blindness in the representation of binary sequences.

PubMed

Yu, Ru Qi; Osherson, Daniel; Zhao, Jiaying

2018-03-01

Binary information is prevalent in the environment and contains 2 distinct outcomes. Binary sequences consist of a mixture of alternation and repetition. Understanding how people perceive such sequences would contribute to a general theory of information processing. In this study, we examined how people process alternation and repetition in binary sequences. Across 4 paradigms involving estimation, working memory, change detection, and visual search, we found that the number of alternations is underestimated compared with repetitions (Experiment 1). Moreover, recall for binary sequences deteriorates as the sequence alternates more (Experiment 2). Changes in bits are also harder to detect as the sequence alternates more (Experiment 3). Finally, visual targets superimposed on bits of a binary sequence take longer to process as alternation increases (Experiment 4). Overall, our results indicate that compared with repetition, alternation in a binary sequence is less salient in the sense of requiring more attention for successful encoding. The current study thus reveals the cognitive constraints in the representation of alternation and provides a new explanation for the overalternation bias in randomness perception. (PsycINFO Database Record (c) 2018 APA, all rights reserved).

An Mcm10 Mutant Defective in ssDNA Binding Shows Defects in DNA Replication Initiation.

PubMed

Perez-Arnaiz, Patricia; Kaplan, Daniel L

2016-11-20

Mcm10 is an essential protein that functions to initiate DNA replication after the formation of the replication fork helicase. In this manuscript, we identified a budding yeast Mcm10 mutant (Mcm10-m2,3,4) that is defective in DNA binding in vitro. Moreover, this Mcm10-m2,3,4 mutant does not stimulate the phosphorylation of Mcm2 by Dbf4-dependent kinase (DDK) in vitro. When we expressed wild-type levels of mcm10-m2,3,4 in budding yeast cells, we observed a severe growth defect and a substantially decreased DNA replication. We also observed a substantially reduced replication protein A- chromatin immunoprecipitation signal at origins of replication, reduced levels of DDK-phosphorylated Mcm2, and diminished Go, Ichi, Ni, and San (GINS) association with Mcm2-7 in vivo. mcm5-bob1 bypasses the growth defect conferred by DDK-phosphodead Mcm2 in budding yeast. However, the growth defect observed by expressing mcm10-m2,3,4 is not bypassed by the mcm5-bob1 mutation. Furthermore, origin melting and GINS association with Mcm2-7 are substantially decreased for cells expressing mcm10-m2,3,4 in the mcm5-bob1 background. Thus, the origin melting and GINS-Mcm2-7 interaction defects we observed for mcm10-m2,3,4 are not explained by decreased Mcm2 phosphorylation by DDK, since the defects persist in an mcm5-bob1 background. These data suggest that DNA binding by Mcm10 is essential for the initiation of DNA replication. Copyright © 2016 Elsevier Ltd. All rights reserved.

SEED 2: a user-friendly platform for amplicon high-throughput sequencing data analyses.

PubMed

Vetrovský, Tomáš; Baldrian, Petr; Morais, Daniel; Berger, Bonnie

2018-02-14

Modern molecular methods have increased our ability to describe microbial communities. Along with the advances brought by new sequencing technologies, we now require intensive computational resources to make sense of the large numbers of sequences continuously produced. The software developed by the scientific community to address this demand, although very useful, require experience of the command-line environment, extensive training and have steep learning curves, limiting their use. We created SEED 2, a graphical user interface for handling high-throughput amplicon-sequencing data under Windows operating systems. SEED 2 is the only sequence visualizer that empowers users with tools to handle amplicon-sequencing data of microbial community markers. It is suitable for any marker genes sequences obtained through Illumina, IonTorrent or Sanger sequencing. SEED 2 allows the user to process raw sequencing data, identify specific taxa, produce of OTU-tables, create sequence alignments and construct phylogenetic trees. Standard dual core laptops with 8 GB of RAM can handle ca. 8 million of Illumina PE 300 bp sequences, ca. 4GB of data. SEED 2 was implemented in Object Pascal and uses internal functions and external software for amplicon data processing. SEED 2 is a freeware software, available at http://www.biomed.cas.cz/mbu/lbwrf/seed/ as a self-contained file, including all the dependencies, and does not require installation. Supplementary data contain a comprehensive list of supported functions. daniel.morais@biomed.cas.cz. Supplementary data are available at Bioinformatics online. © The Author(s) 2018. Published by Oxford University Press.

Cooperative heteroassembly of the adenoviral L4-22K and IVa2 proteins onto the viral packaging sequence DNA.

PubMed

Yang, Teng-Chieh; Maluf, Nasib Karl

2012-02-21

Human adenovirus (Ad) is an icosahedral, double-stranded DNA virus. Viral DNA packaging refers to the process whereby the viral genome becomes encapsulated by the viral particle. In Ad, activation of the DNA packaging reaction requires at least three viral components: the IVa2 and L4-22K proteins and a section of DNA within the viral genome, called the packaging sequence. Previous studies have shown that the IVa2 and L4-22K proteins specifically bind to conserved elements within the packaging sequence and that these interactions are absolutely required for the observation of DNA packaging. However, the equilibrium mechanism for assembly of IVa2 and L4-22K onto the packaging sequence has not been determined. Here we characterize the assembly of the IVa2 and L4-22K proteins onto truncated packaging sequence DNA by analytical sedimentation velocity and equilibrium methods. At limiting concentrations of L4-22K, we observe a species with two IVa2 monomers and one L4-22K monomer bound to the DNA. In this species, the L4-22K monomer is promoting positive cooperative interactions between the two bound IVa2 monomers. As L4-22K levels are increased, we observe a species with one IVa2 monomer and three L4-22K monomers bound to the DNA. To explain this result, we propose a model in which L4-22K self-assembly on the DNA competes with IVa2 for positive heterocooperative interactions, destabilizing binding of the second IVa2 monomer. Thus, we propose that L4-22K levels control the extent of cooperativity observed between adjacently bound IVa2 monomers. We have also determined the hydrodynamic properties of all observed stoichiometric species; we observe that species with three L4-22K monomers bound have more extended conformations than species with a single L4-22K bound. We suggest this might reflect a molecular switch that controls insertion of the viral DNA into the capsid.

Regional centromeres in the yeast Candida lusitaniae lack pericentromeric heterochromatin

PubMed Central

Kapoor, Shivali; Zhu, Lisha; Froyd, Cara; Liu, Tao; Rusche, Laura N.

2015-01-01

Point centromeres are specified by a short consensus sequence that seeds kinetochore formation, whereas regional centromeres lack a conserved sequence and instead are epigenetically inherited. Regional centromeres are generally flanked by heterochromatin that ensures high levels of cohesin and promotes faithful chromosome segregation. However, it is not known whether regional centromeres require pericentromeric heterochromatin. In the yeast Candida lusitaniae, we identified a distinct type of regional centromere that lacks pericentromeric heterochromatin. Centromere locations were determined by ChIP-sequencing of two key centromere proteins, Cse4 and Mif2, and are consistent with bioinformatic predictions. The centromeric DNA sequence was unique for each chromosome and spanned 4–4.5 kbp, consistent with regional epigenetically inherited centromeres. However, unlike other regional centromeres, there was no evidence of pericentromeric heterochromatin in C. lusitaniae. In particular, flanking genes were expressed at a similar level to the rest of the genome, and a URA3 reporter inserted adjacent to a centromere was not repressed. In addition, regions flanking the centromeric core were not associated with hypoacetylated histones or a sirtuin deacetylase that generates heterochromatin in other yeast. Interestingly, the centromeric chromatin had a distinct pattern of histone modifications, being enriched for methylated H3K79 and H3R2 but lacking methylation of H3K4, which is found at other regional centromeres. Thus, not all regional centromeres require flanking heterochromatin. PMID:26371315

miRNA-embedded shRNAs for Lineage-specific BCL11A Knockdown and Hemoglobin F Induction

PubMed Central

Guda, Swaroopa; Brendel, Christian; Renella, Raffaele; Du, Peng; Bauer, Daniel E; Canver, Matthew C; Grenier, Jennifer K; Grimson, Andrew W; Kamran, Sophia C; Thornton, James; de Boer, Helen; Root, David E; Milsom, Michael D; Orkin, Stuart H; Gregory, Richard I; Williams, David A

2015-01-01

RNA interference (RNAi) technology using short hairpin RNAs (shRNAs) expressed via RNA polymerase (pol) III promoters has been widely exploited to modulate gene expression in a variety of mammalian cell types. For certain applications, such as lineage-specific knockdown, embedding targeting sequences into pol II-driven microRNA (miRNA) architecture is required. Here, using the potential therapeutic target BCL11A, we demonstrate that pol III-driven shRNAs lead to significantly increased knockdown but also increased cytotoxcity in comparison to pol II-driven miRNA adapted shRNAs (shRNAmiR) in multiple hematopoietic cell lines. We show that the two expression systems yield mature guide strand sequences that differ by a 4 bp shift. This results in alternate seed sequences and consequently influences the efficacy of target gene knockdown. Incorporating a corresponding 4 bp shift into the guide strand of shRNAmiRs resulted in improved knockdown efficiency of BCL11A. This was associated with a significant de-repression of the hemoglobin target of BCL11A, human γ-globin or the murine homolog Hbb-y. Our results suggest the requirement for optimization of shRNA sequences upon incorporation into a miRNA backbone. These findings have important implications in future design of shRNAmiRs for RNAi-based therapy in hemoglobinopathies and other diseases requiring lineage-specific expression of gene silencing sequences. PMID:26080908

Infant auditory short-term memory for non-linguistic sounds.

PubMed

Ross-Sheehy, Shannon; Newman, Rochelle S

2015-04-01

This research explores auditory short-term memory (STM) capacity for non-linguistic sounds in 10-month-old infants. Infants were presented with auditory streams composed of repeating sequences of either 2 or 4 unique instruments (e.g., flute, piano, cello; 350 or 700 ms in duration) followed by a 500-ms retention interval. These instrument sequences either stayed the same for every repetition (Constant) or changed by 1 instrument per sequence (Varying). Using the head-turn preference procedure, infant listening durations were recorded for each stream type (2- or 4-instrument sequences composed of 350- or 700-ms notes). Preference for the Varying stream was taken as evidence of auditory STM because detection of the novel instrument required memory for all of the instruments in a given sequence. Results demonstrate that infants listened longer to Varying streams for 2-instrument sequences, but not 4-instrument sequences, composed of 350-ms notes (Experiment 1), although this effect did not hold when note durations were increased to 700 ms (Experiment 2). Experiment 3 replicates and extends results from Experiments 1 and 2 and provides support for a duration account of capacity limits in infant auditory STM. Copyright © 2014 Elsevier Inc. All rights reserved.

The Hsk1(Cdc7) Replication Kinase Regulates Origin Efficiency

PubMed Central

Patel, Prasanta K.; Kommajosyula, Naveen; Rosebrock, Adam; Bensimon, Aaron; Leatherwood, Janet; Bechhoefer, John

2008-01-01

Origins of DNA replication are generally inefficient, with most firing in fewer than half of cell cycles. However, neither the mechanism nor the importance of the regulation of origin efficiency is clear. In fission yeast, origin firing is stochastic, leading us to hypothesize that origin inefficiency and stochasticity are the result of a diffusible, rate-limiting activator. We show that the Hsk1-Dfp1 replication kinase (the fission yeast Cdc7-Dbf4 homologue) plays such a role. Increasing or decreasing Hsk1-Dfp1 levels correspondingly increases or decreases origin efficiency. Furthermore, tethering Hsk1-Dfp1 near an origin increases the efficiency of that origin, suggesting that the effective local concentration of Hsk1-Dfp1 regulates origin firing. Using photobleaching, we show that Hsk1-Dfp1 is freely diffusible in the nucleus. These results support a model in which the accessibility of replication origins to Hsk1-Dfp1 regulates origin efficiency and provides a potential mechanistic link between chromatin structure and replication timing. By manipulating Hsk1-Dfp1 levels, we show that increasing or decreasing origin firing rates leads to an increase in genomic instability, demonstrating the biological importance of appropriate origin efficiency. PMID:18799612

The geochemical landscape of northwestern Wisconsin and adjacent parts of northern Michigan and Minnesota (geochemical data files)

USGS Publications Warehouse

Cannon, William F.; Woodruff, Laurel G.

2003-01-01

This data set consists of nine files of geochemical information on various types of surficial deposits in northwestern Wisconsin and immediately adjacent parts of Michigan and Minnesota. The files are presented in two formats: as dbase files in dbaseIV form and Microsoft Excel form. The data present multi-element chemical analyses of soils, stream sediments, and lake sediments. Latitude and longitude values are provided in each file so that the dbf files can be readily imported to GIS applications. Metadata files are provided in outline form, question and answer form and text form. The metadata includes information on procedures for sample collection, sample preparation, and chemical analyses including sensitivity and precision.

Robust adaptive multichannel SAR processing based on covariance matrix reconstruction

NASA Astrophysics Data System (ADS)

Tan, Zhen-ya; He, Feng

2018-04-01

With the combination of digital beamforming (DBF) processing, multichannel synthetic aperture radar(SAR) systems in azimuth promise well in high-resolution and wide-swath imaging, whereas conventional processing methods don't take the nonuniformity of scattering coefficient into consideration. This paper brings up a robust adaptive Multichannel SAR processing method which utilizes the Capon spatial spectrum estimator to obtain the spatial spectrum distribution over all ambiguous directions first, and then the interference-plus-noise covariance Matrix is reconstructed based on definition to acquire the Multichannel SAR processing filter. The performance of processing under nonuniform scattering coefficient is promoted by this novel method and it is robust again array errors. The experiments with real measured data demonstrate the effectiveness and robustness of the proposed method.

Quick and clean cloning.

PubMed

Thieme, Frank; Marillonnet, Sylvestre

2014-01-01

Identification of unknown sequences that flank known sequences of interest requires PCR amplification of DNA fragments that contain the junction between the known and unknown flanking sequences. Since amplified products often contain a mixture of specific and nonspecific products, the quick and clean (QC) cloning procedure was developed to clone specific products only. QC cloning is a ligation-independent cloning procedure that relies on the exonuclease activity of T4 DNA polymerase to generate single-stranded extensions at the ends of the vector and insert. A specific feature of QC cloning is the use of vectors that contain a sequence called catching sequence that allows cloning specific products only. QC cloning is performed by a one-pot incubation of insert and vector in the presence of T4 DNA polymerase at room temperature for 10 min followed by direct transformation of the incubation mix in chemo-competent Escherichia coli cells.

Adaptation of the Haloarcula hispanica CRISPR-Cas system to a purified virus strictly requires a priming process

PubMed Central

Li, Ming; Wang, Rui; Zhao, Dahe; Xiang, Hua

2014-01-01

The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system mediates adaptive immunity against foreign nucleic acids in prokaryotes. However, efficient adaptation of a native CRISPR to purified viruses has only been observed for the type II-A system from a Streptococcus thermophilus industry strain, and rarely reported for laboratory strains. Here, we provide a second native system showing efficient adaptation. Infected by a newly isolated virus HHPV-2, Haloarcula hispanica type I-B CRISPR system acquired spacers discriminatively from viral sequences. Unexpectedly, in addition to Cas1, Cas2 and Cas4, this process also requires Cas3 and at least partial Cascade proteins, which are involved in interference and/or CRISPR RNA maturation. Intriguingly, a preexisting spacer partially matching a viral sequence is also required, and spacer acquisition from upstream and downstream sequences of its target sequence (i.e. priming protospacer) shows different strand bias. These evidences strongly indicate that adaptation in this system strictly requires a priming process. This requirement, if validated also true for other CRISPR systems as implied by our bioinformatic analysis, may help to explain failures to observe efficient adaptation to purified viruses in many laboratory strains, and the discrimination mechanism at the adaptation level that has confused scientists for years. PMID:24265226

The Phosphatidic Acid Binding Site of the Arabidopsis Trigalactosyldiacylglycerol 4 (TGD4) Protein Required for Lipid Import into Chloroplasts*

PubMed Central

Wang, Zhen; Anderson, Nicholas Scott; Benning, Christoph

2013-01-01

Chloroplast membrane lipid synthesis relies on the import of glycerolipids from the ER. The TGD (TriGalactosylDiacylglycerol) proteins are required for this lipid transfer process. The TGD1, -2, and -3 proteins form a putative ABC (ATP-binding cassette) transporter transporting ER-derived lipids through the inner envelope membrane of the chloroplast, while TGD4 binds phosphatidic acid (PtdOH) and resides in the outer chloroplast envelope. We identified two sequences in TGD4, amino acids 1–80 and 110–145, which are necessary and sufficient for PtdOH binding. Deletion of both sequences abolished PtdOH binding activity. We also found that TGD4 from 18:3 plants bound specifically and with increased affinity PtdOH. TGD4 did not interact with other proteins and formed a homodimer both in vitro and in vivo. Our results suggest that TGD4 is an integral dimeric β-barrel lipid transfer protein that binds PtdOH with its N terminus and contains dimerization domains at its C terminus. PMID:23297418

Centrifuge: rapid and sensitive classification of metagenomic sequences

PubMed Central

Song, Li; Breitwieser, Florian P.

2016-01-01

Centrifuge is a novel microbial classification engine that enables rapid, accurate, and sensitive labeling of reads and quantification of species on desktop computers. The system uses an indexing scheme based on the Burrows-Wheeler transform (BWT) and the Ferragina-Manzini (FM) index, optimized specifically for the metagenomic classification problem. Centrifuge requires a relatively small index (4.2 GB for 4078 bacterial and 200 archaeal genomes) and classifies sequences at very high speed, allowing it to process the millions of reads from a typical high-throughput DNA sequencing run within a few minutes. Together, these advances enable timely and accurate analysis of large metagenomics data sets on conventional desktop computers. Because of its space-optimized indexing schemes, Centrifuge also makes it possible to index the entire NCBI nonredundant nucleotide sequence database (a total of 109 billion bases) with an index size of 69 GB, in contrast to k-mer-based indexing schemes, which require far more extensive space. PMID:27852649

Identification of Sequence Specificity of 5-Methylcytosine Oxidation by Tet1 Protein with High-Throughput Sequencing.

PubMed

Kizaki, Seiichiro; Chandran, Anandhakumar; Sugiyama, Hiroshi

2016-03-02

Tet (ten-eleven translocation) family proteins have the ability to oxidize 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxycytosine (caC). However, the oxidation reaction of Tet is not understood completely. Evaluation of genomic-level epigenetic changes by Tet protein requires unbiased identification of the highly selective oxidation sites. In this study, we used high-throughput sequencing to investigate the sequence specificity of mC oxidation by Tet1. A 6.6×10(4) -member mC-containing random DNA-sequence library was constructed. The library was subjected to Tet-reactive pulldown followed by high-throughput sequencing. Analysis of the obtained sequence data identified the Tet1-reactive sequences. We identified mCpG as a highly reactive sequence of Tet1 protein. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Epigenetic Instability due to Defective Replication of Structured DNA

PubMed Central

Sarkies, Peter; Reams, Charlie; Simpson, Laura J.; Sale, Julian E.

2010-01-01

Summary The accurate propagation of histone marks during chromosomal replication is proposed to rely on the tight coupling of replication with the recycling of parental histones to the daughter strands. Here, we show in the avian cell line DT40 that REV1, a key regulator of DNA translesion synthesis at the replication fork, is required for the maintenance of repressive chromatin marks and gene silencing in the vicinity of DNA capable of forming G-quadruplex (G4) structures. We demonstrate a previously unappreciated requirement for REV1 in replication of G4 forming sequences and show that transplanting a G4 forming sequence into a silent locus leads to its derepression in REV1-deficient cells. Together, our observations support a model in which failure to maintain processive DNA replication at G4 DNA in REV1-deficient cells leads to uncoupling of DNA synthesis from histone recycling, resulting in localized loss of repressive chromatin through biased incorporation of newly synthesized histones. PMID:21145480

GIS Methodic and New Database for Magmatic Rocks. Application for Atlantic Oceanic Magmatism.

NASA Astrophysics Data System (ADS)

Asavin, A. M.

2001-12-01

There are several geochemical Databases in INTERNET available now. There one of the main peculiarities of stored geochemical information is geographical coordinates of each samples in those Databases. As rule the software of this Database use spatial information only for users interface search procedures. In the other side, GIS-software (Geographical Information System software),for example ARC/INFO software which using for creation and analyzing special geological, geochemical and geophysical e-map, have been deeply involved with geographical coordinates for of samples. We join peculiarities GIS systems and relational geochemical Database from special software. Our geochemical information system created in Vernadsky Geological State Museum and institute of Geochemistry and Analytical Chemistry from Moscow. Now we tested system with data of geochemistry oceanic rock from Atlantic and Pacific oceans, about 10000 chemical analysis. GIS information content consist from e-map covers Wold Globes. Parts of these maps are Atlantic ocean covers gravica map (with grid 2''), oceanic bottom hot stream, altimeteric maps, seismic activity, tectonic map and geological map. Combination of this information content makes possible created new geochemical maps and combination of spatial analysis and numerical geochemical modeling of volcanic process in ocean segment. Now we tested information system on thick client technology. Interface between GIS system Arc/View and Database resides in special multiply SQL-queries sequence. The result of the above gueries were simple DBF-file with geographical coordinates. This file act at the instant of creation geochemical and other special e-map from oceanic region. We used more complex method for geophysical data. From ARC\\View we created grid cover for polygon spatial geophysical information.

The influence of viral coding sequences on pestivirus IRES activity reveals further parallels with translation initiation in prokaryotes.

PubMed Central

Fletcher, Simon P; Ali, Iraj K; Kaminski, Ann; Digard, Paul; Jackson, Richard J

2002-01-01

Classical swine fever virus (CSFV) is a member of the pestivirus family, which shares many features in common with hepatitis C virus (HCV). It is shown here that CSFV has an exceptionally efficient cis-acting internal ribosome entry segment (IRES), which, like that of HCV, is strongly influenced by the sequences immediately downstream of the initiation codon, and is optimal with viral coding sequences in this position. Constructs that retained 17 or more codons of viral coding sequence exhibited full IRES activity, but with only 12 codons, activity was approximately 66% of maximum in vitro (though close to maximum in transfected BHK cells), whereas with just 3 codons or fewer, the activity was only approximately 15% of maximum. The minimal coding region elements required for high activity were exchanged between HCV and CSFV. Although maximum activity was observed in each case with the homologous combination of coding region and 5' UTR, the heterologous combinations were sufficiently active to rule out a highly specific functional interplay between the 5' UTR and coding sequences. On the other hand, inversion of the coding sequences resulted in low IRES activity, particularly with the HCV coding sequences. RNA structure probing showed that the efficiency of internal initiation of these chimeric constructs correlated most closely with the degree of single-strandedness of the region around and immediately downstream of the initiation codon. The low activity IRESs could not be rescued by addition of supplementary eIF4A (the initiation factor with ATP-dependent RNA helicase activity). The extreme sensitivity to secondary structure around the initiation codon is likely to be due to the fact that the eIF4F complex (which has eIF4A as one of its subunits) is not required for and does not participate in initiation on these IRESs. PMID:12515388

Identification of optimum sequencing depth especially for de novo genome assembly of small genomes using next generation sequencing data.

PubMed

Desai, Aarti; Marwah, Veer Singh; Yadav, Akshay; Jha, Vineet; Dhaygude, Kishor; Bangar, Ujwala; Kulkarni, Vivek; Jere, Abhay

2013-01-01

Next Generation Sequencing (NGS) is a disruptive technology that has found widespread acceptance in the life sciences research community. The high throughput and low cost of sequencing has encouraged researchers to undertake ambitious genomic projects, especially in de novo genome sequencing. Currently, NGS systems generate sequence data as short reads and de novo genome assembly using these short reads is computationally very intensive. Due to lower cost of sequencing and higher throughput, NGS systems now provide the ability to sequence genomes at high depth. However, currently no report is available highlighting the impact of high sequence depth on genome assembly using real data sets and multiple assembly algorithms. Recently, some studies have evaluated the impact of sequence coverage, error rate and average read length on genome assembly using multiple assembly algorithms, however, these evaluations were performed using simulated datasets. One limitation of using simulated datasets is that variables such as error rates, read length and coverage which are known to impact genome assembly are carefully controlled. Hence, this study was undertaken to identify the minimum depth of sequencing required for de novo assembly for different sized genomes using graph based assembly algorithms and real datasets. Illumina reads for E.coli (4.6 MB) S.kudriavzevii (11.18 MB) and C.elegans (100 MB) were assembled using SOAPdenovo, Velvet, ABySS, Meraculous and IDBA-UD. Our analysis shows that 50X is the optimum read depth for assembling these genomes using all assemblers except Meraculous which requires 100X read depth. Moreover, our analysis shows that de novo assembly from 50X read data requires only 6-40 GB RAM depending on the genome size and assembly algorithm used. We believe that this information can be extremely valuable for researchers in designing experiments and multiplexing which will enable optimum utilization of sequencing as well as analysis resources.

Substituted 1H-1,2,3-Triazol-4-yl-1H-pyrrolo[2,3-b]pyridines by De Novo One-Pot Ring Forming Coupling-Cyclization-Desilylation-CuAAC-Sequence.

PubMed

Müller, Thomas J J; Lessing, Timo; van Mark, Hauke

2018-05-04

Substituted 1H-1,2,3-triazol-4-yl-pyrrolo[2,3-b]pyridines are efficiently prepared by a one-pot coupling-cyclization-desilylation-CuAAC-sequence in the sense of a consecutive three-component fashion. The key feature of this novel de novo formation of azole and triazole anellation is the sequentially Pd/Cu-catalyzed process employing tri(iso-propyl)silylbutadiyne (TIPS-butadiyne) as a four-carbon building block. In addition, the sequence can be expanded in a four-component fashion also employing the in situ formation of the require azides. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Relative performance of three stream bed stability indices as indicators of stream health

USGS Publications Warehouse

Kusnierz, Paul C; Holbrook, Christopher

2017-01-01

Bed stability is an important stream habitat attribute because it affects geomorphology and biotic communities. Natural resource managers desire indices of bed stability that can be used under a wide range of geomorphic conditions, are biologically meaningful, and are easily incorporated into sampling protocols. To eliminate potential bias due to presence of instream wood and increase precision of stability values, we modified a stream bed instability index (ISI) to include measurements of bankfull depth (dbf) and median particle diameter (D50) only in riffles and increased the pebble count to decrease variability (i.e., increase precision) in D50.The new riffle-based instability index (RISI) was compared to two established indices: ISI and the riffle stability index (RSI). RISI and ISI were strongly associated with each other but neither was closely associated with RSI. RISI and ISI were closely associated with both a diatom- and two macrovertebrate-based stream health indices, but RSI was only weakly associated with the macroinvertebrate indices. Unexpectedly, precision of D50 did not differ between RISI and ISI. Results suggest that RISI is a viable alternative to both ISI and RSI for evaluating bed stability in multiple stream types. With few data requirements and a simple protocol, RISI may also better conform to riffle-based sampling methods used by some water quality practitioners.

Intronic sequences are required for AINTEGUMENTA-LIKE6 expression in Arabidopsis flowers.

PubMed

Krizek, Beth A

2015-10-12

The AINTEGUMENTA-LIKE6/PLETHORA3 (AIL6/PLT3) gene of Arabidopsis thaliana is a key regulator of growth and patterning in both shoots and roots. AIL6 encodes an AINTEGUMENTA-LIKE/PLETHORA (AIL/PLT) transcription factor that is expressed in the root stem cell niche, the peripheral region of the shoot apical meristem and young lateral organ primordia. In flowers, AIL6 acts redundantly with AINTEGUMENTA (ANT) to regulate floral organ positioning, growth, identity and patterning. Experiments were undertaken to define the genomic regions required for AIL6 function and expression in flowers. Transgenic plants expressing a copy of the coding region of AIL6 in the context of 7.7 kb of 5' sequence and 919 bp of 3' sequence (AIL6:cAIL6-3') fail to fully complement AIL6 function when assayed in the ant-4 ail6-2 double mutant background. In contrast, a genomic copy of AIL6 with the same amount of 5' and 3' sequence (AIL6:gAIL6-3') can fully complement ant-4 ail6-2. In addition, a genomic copy of AIL6 with 590 bp of 5' sequence and 919 bp of 3' sequence (AIL6m:gAIL6-3') complements ant-4 ail6-2 and contains all regulatory elements needed to confer normal AIL6 expression in inflorescences. Efforts to map cis-regulatory elements reveal that the third intron of AIL6 contains enhancer elements that confer expression in young flowers but in a broader pattern than that of AIL6 mRNA in wild-type flowers. Some AIL6:gAIL6-3' and AIL6m:gAIL6-3' lines confer an over-rescue phenotype in the ant-4 ail6-2 background that is correlated with higher levels of AIL6 mRNA accumulation. The results presented here indicate that AIL6 intronic sequences serve as transcriptional enhancer elements. In addition, the results show that increased expression of AIL6 can partially compensate for loss of ANT function in flowers.

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

Polyomavirus BK non-coding control region rearrangements in health and disease.

PubMed

Sharma, Preety M; Gupta, Gaurav; Vats, Abhay; Shapiro, Ron; Randhawa, Parmjeet S

2007-08-01

BK virus is an increasingly recognized pathogen in transplanted patients. DNA sequencing of this virus shows considerable genomic variability. To understand the clinical significance of rearrangements in the non-coding control region (NCCR) of BK virus (BKV), we report a meta-analysis of 507 sequences, including 40 sequences generated in our own laboratory, for associations between rearrangements and disease, tissue tropism, geographic origin, and viral genotype. NCCR rearrangements were less frequent in (a) asymptomatic BKV viruria compared to patients viral nephropathy (1.7% vs. 22.5%), and (b) viral genotype 1 compared to other genotypes (2.4% vs. 11.2%). Rearrangements were commoner in malignancy (78.6%), and Norwegians (45.7%), and less common in East Indians (0%), and Japanese (4.3%). A surprising number of rearranged sequences were reported from mononuclear cells of healthy subjects, whereas most plasma sequences were archetypal. This difference could not be related to potential recombinase activity in lymphocytes, as consensus recombination signal sequences could not be found in the NCCR region. NCCR rearrangements are neither required nor a sufficient condition to produce clinical disease. BKV nephropathy and hemorrhagic cystitis are not associated with any unique NCCR configuration or nucleotide sequence.

Sequential steps in DNA replication are inhibited to ensure reduction of ploidy in meiosis

PubMed Central

Hua, Hui; Namdar, Mandana; Ganier, Olivier; Gregan, Juraj; Méchali, Marcel; Kearsey, Stephen E.

2013-01-01

Meiosis involves two successive rounds of chromosome segregation without an intervening S phase. Exit from meiosis I is distinct from mitotic exit, in that replication origins are not licensed by Mcm2-7 chromatin binding, but spindle disassembly occurs during a transient interphase-like state before meiosis II. The absence of licensing is assumed to explain the block to DNA replication, but this has not been formally tested. Here we attempt to subvert this block by expressing the licensing control factors Cdc18 and Cdt1 during the interval between meiotic nuclear divisions. Surprisingly, this leads only to a partial round of DNA replication, even when these factors are overexpressed and effect clear Mcm2-7 chromatin binding. Combining Cdc18 and Cdt1 expression with modulation of cyclin-dependent kinase activity, activation of Dbf4-dependent kinase, or deletion of the Spd1 inhibitor of ribonucleotide reductase has little additional effect on the extent of DNA replication. Single-molecule analysis indicates this partial round of replication results from inefficient progression of replication forks, and thus both initiation and elongation replication steps may be inhibited in late meiosis. In addition, DNA replication or damage during the meiosis I–II interval fails to arrest meiotic progress, suggesting absence of checkpoint regulation of meiosis II entry. PMID:23303250

Conserved mechanism for coordinating replication fork helicase assembly with phosphorylation of the helicase

PubMed Central

Bruck, Irina; Kaplan, Daniel L.

2015-01-01

Dbf4-dependent kinase (DDK) phosphorylates minichromosome maintenance 2 (Mcm2) during S phase in yeast, and Sld3 recruits cell division cycle 45 (Cdc45) to minichromosome maintenance 2-7 (Mcm2-7). We show here DDK-phosphoryled Mcm2 preferentially interacts with Cdc45 in vivo, and that Sld3 stimulates DDK phosphorylation of Mcm2 by 11-fold. We identified a mutation of the replication initiation factor Sld3, Sld3-m16, that is specifically defective in stimulating DDK phosphorylation of Mcm2. Wild-type expression levels of sld3-m16 result in severe growth and DNA replication defects. Cells expressing sld3-m16 exhibit no detectable Mcm2 phosphorylation in vivo, reduced replication protein A-ChIP signal at an origin, and diminished Go, Ichi, Ni, and San association with Mcm2-7. Treslin, the human homolog of Sld3, stimulates human DDK phosphorylation of human Mcm2 by 15-fold. DDK phosphorylation of human Mcm2 decreases the affinity of Mcm5 for Mcm2, suggesting a potential mechanism for helicase ring opening. These data suggest a conserved mechanism for replication initiation: Sld3/Treslin coordinates Cdc45 recruitment to Mcm2-7 with DDK phosphorylation of Mcm2 during S phase. PMID:26305950

Conserved mechanism for coordinating replication fork helicase assembly with phosphorylation of the helicase.

PubMed

Bruck, Irina; Kaplan, Daniel L

2015-09-08

Dbf4-dependent kinase (DDK) phosphorylates minichromosome maintenance 2 (Mcm2) during S phase in yeast, and Sld3 recruits cell division cycle 45 (Cdc45) to minichromosome maintenance 2-7 (Mcm2-7). We show here DDK-phosphoryled Mcm2 preferentially interacts with Cdc45 in vivo, and that Sld3 stimulates DDK phosphorylation of Mcm2 by 11-fold. We identified a mutation of the replication initiation factor Sld3, Sld3-m16, that is specifically defective in stimulating DDK phosphorylation of Mcm2. Wild-type expression levels of sld3-m16 result in severe growth and DNA replication defects. Cells expressing sld3-m16 exhibit no detectable Mcm2 phosphorylation in vivo, reduced replication protein A-ChIP signal at an origin, and diminished Go, Ichi, Ni, and San association with Mcm2-7. Treslin, the human homolog of Sld3, stimulates human DDK phosphorylation of human Mcm2 by 15-fold. DDK phosphorylation of human Mcm2 decreases the affinity of Mcm5 for Mcm2, suggesting a potential mechanism for helicase ring opening. These data suggest a conserved mechanism for replication initiation: Sld3/Treslin coordinates Cdc45 recruitment to Mcm2-7 with DDK phosphorylation of Mcm2 during S phase.

The glycan-specific sulfotransferase (R77W)GalNAc-4-ST1 putatively responsible for peeling skin syndrome has normal properties consistent with a simple sequence polymorphisim.

PubMed

Fiete, Dorothy; Mi, Yiling; Beranek, Mary; Baenziger, Nancy L; Baenziger, Jacques U

2017-05-01

Expanded access to DNA sequencing now fosters ready detection of site-specific human genome alterations whose actual significance requires in-depth functional study to rule in or out disease-causing mutations. This is a particular concern for genomic sequence differences in glycosyltransferases, whose implications are often difficult to assess. A recent whole-exome sequencing study identifies (c.229 C > T) in the GalNAc-4-ST1 glycosyltransferase (CHST8) as a disease-causing missense R77W mutation yielding the genodermatosis peeling skin syndrome (PSS) when homozygous. Cabral et al. (Genomics. 2012;99:202-208) cite this sequence change as reducing keratinocyte GalNAc-4-ST1 activity, thus decreasing glycosaminoglycan sulfation, as the mechanism for this blistering disorder. Such an identification could point toward potential clinical and/or prenatal diagnosis of a harmful medical condition. However, GalNAc-4-ST1 has minimal activity toward glycosaminoglycans, instead modifying terminal β1,4-linked GalNAc on N- and O-linked oligosaccharides on specific glycoproteins. We find expression, processing and catalytic activity of GalNAc-4-ST1 completely equivalent between wild type and (R77W) sulfotransferases. Moreover, keratinocytes have little or no GalNAc-4-ST1 mRNA, indicating that they do not express GalNAc-4-ST1. In addition, loss-of-function of GalNAc-4-ST1 primarily presents as reproductive system aberrations rather than skin effects. These findings, an allele frequency of 0.004357, and a 10-fold difference in prevalence of CHST8 (c.299 C > T, R77W) across different ethnic groups, suggest that this sequence represents a "passenger" distributed polymorphism, a simple sequence variant form of the enzyme having normal activity, rather than a "driver" disease-causing mutation that accounts for PSS. This study presents an example for guiding biomedical research initiatives, as well as medical and personal/family perspectives, regarding newly-identified genomic sequence differences. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex

PubMed Central

Wolle, Daniel; Cleard, Fabienne; Aoki, Tsutomu; Deshpande, Girish; Karch, Francois

2015-01-01

Chromatin boundaries are architectural elements that determine the three-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 boundary from the Drosophila bithorax complex (BX-C) is required for the parasegment-specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in the BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and we describe a novel ∼700-kDa complex, the late boundary complex (LBC), that binds to Fab-7 sequences that have insulator functions in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late, but not early, embryos and that it contains three insulator proteins, GAF, Mod(mdg4), and E(y)2. Its DNA binding properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display few sequence similarities. Finally, we show that mutations which abrogate LBC binding in vitro inactivate the Fab-7 boundary in the BX-C. PMID:26303531

Impact of CyberKnife Radiosurgery on Overall Survival and Various Parameters of Patients with 1-3 versus ≥ 4 Brain Metastases.

PubMed

Murovic, Judith; Ding, Victoria; Han, Summer S; Adler, John R; Chang, Steven D

2017-10-24

Introduction This study's objective is to compare the overall survivals (OSs) and various parameters of patients with 1-3 versus ≥ 4 brain metastases post-CyberKnife radiosurgery (CKRS) (Accuray, Sunnyvale, California) alone. Methods Charts of 150 patients, from 2009-2014, treated with only CKRS for brain metastases were reviewed retrospectively for overall survival (OS) and patient, tumor, and imaging characteristics. Parameters included demographics, Eastern Cooperative Oncology Group (ECOG) performance scores, number and control of extracranial disease (ECD) sites, cause of death (COD), histology, tumor volume (TV), and post-CKRS whole brain radiotherapy (WBRT). The imaging characteristics assessed were time of complete response (CR), partial response (PR), stable imaging or local failure (LF), and distal brain failure (DBF). Patients and their data were divided into those with 1-3 (group 1) versus ≥ 4 brain metastases (group 2). For each CR and LF patient, absolute neutrophil count (ANC), absolute lymphocyte count (ALC)), and ANC/ALC ratio (NLR) were obtained, when available, at the time of CKRS. Results Both group 1 and group 2 had a median OS of 13 months. The patient median age for the 115 group 1 patients versus the 35 group 2 patients was 62 versus 56 years. Group 1 had slightly more males and group 2, females. The predominant ECOG score for each group was 1 and the number of ECD sites was one and two, respectively. Uncontrolled ECD occurred in the majority of both group 1 and group 2 patients. The main COD was ECD in both groups. The prevalent tumor histology for groups 1 and 2 was non-small cell lung carcinoma. Median TVs were 1.08 cc versus 1.42 cc for groups 1 and 2, respectively. The majority of patients in both groups did not undergo post-CKRS WBRT. Imaging outcomes were LC (CR, PR, or stable imaging) in 93 (80.9%) and 26 (74.3%) group 1 and 2 patients, of whom 32 (27.8%) and six (17.1%) had CR; 38 (33.0%) and 18 (51.4%), PR and 23 (20.0%) and two (5.7%), stable imaging; LF was the outcome in 22 (19.1%) and nine (25.7%) patients, and DBF occurred in 62 (53.9%) and 21 (60.0%), respectively. Uni- and multivariable analyses showed the independent parameters of a lower ECOG score, a greater number of ECD sites and uncontrolled ECD were significantly associated with greater mortality risk with and without accounting for other covariates. At CKRS, 19 group 1 and 2 CR patients had a mean ANC of 5.88 K/µL and a mean ALC of 1.31 K/µL and 13 (68%) of 19 had NLRs ≤ five, while 11 with LFs had a mean ANC of 5.22 K/µL and a mean ALC of 0.93 K/µL and seven (64%) had NLRs > five. An NLR ≤ five and high ALC was associated with a CR and an NLR > five and a low ALC with an LF. Conclusions Median OS post-CKRS was 13 months for both patients with 1-3 brain metastases and with ≥ 4. This is the only study in the literature to evaluate OS in patients with 1-3 and ≥ 4 brain metastases who were treated with CKRS alone. For groups 1 and 2 patients combined, 119 (79.3%) had LC and 38 (25.3%) had CR. The ANC, ALC, and NLR values are likely predictive of CR and LF outcomes.

Centrifuge: rapid and sensitive classification of metagenomic sequences.

PubMed

Kim, Daehwan; Song, Li; Breitwieser, Florian P; Salzberg, Steven L

2016-12-01

Centrifuge is a novel microbial classification engine that enables rapid, accurate, and sensitive labeling of reads and quantification of species on desktop computers. The system uses an indexing scheme based on the Burrows-Wheeler transform (BWT) and the Ferragina-Manzini (FM) index, optimized specifically for the metagenomic classification problem. Centrifuge requires a relatively small index (4.2 GB for 4078 bacterial and 200 archaeal genomes) and classifies sequences at very high speed, allowing it to process the millions of reads from a typical high-throughput DNA sequencing run within a few minutes. Together, these advances enable timely and accurate analysis of large metagenomics data sets on conventional desktop computers. Because of its space-optimized indexing schemes, Centrifuge also makes it possible to index the entire NCBI nonredundant nucleotide sequence database (a total of 109 billion bases) with an index size of 69 GB, in contrast to k-mer-based indexing schemes, which require far more extensive space. © 2016 Kim et al.; Published by Cold Spring Harbor Laboratory Press.

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

A clone-free, single molecule map of the domestic cow (Bos taurus) genome.

PubMed

Zhou, Shiguo; Goldstein, Steve; Place, Michael; Bechner, Michael; Patino, Diego; Potamousis, Konstantinos; Ravindran, Prabu; Pape, Louise; Rincon, Gonzalo; Hernandez-Ortiz, Juan; Medrano, Juan F; Schwartz, David C

2015-08-28

The cattle (Bos taurus) genome was originally selected for sequencing due to its economic importance and unique biology as a model organism for understanding other ruminants, or mammals. Currently, there are two cattle genome sequence assemblies (UMD3.1 and Btau4.6) from groups using dissimilar assembly algorithms, which were complemented by genetic and physical map resources. However, past comparisons between these assemblies revealed substantial differences. Consequently, such discordances have engendered ambiguities when using reference sequence data, impacting genomic studies in cattle and motivating construction of a new optical map resource--BtOM1.0--to guide comparisons and improvements to the current sequence builds. Accordingly, our comprehensive comparisons of BtOM1.0 against the UMD3.1 and Btau4.6 sequence builds tabulate large-to-immediate scale discordances requiring mediation. The optical map, BtOM1.0, spanning the B. taurus genome (Hereford breed, L1 Dominette 01449) was assembled from an optical map dataset consisting of 2,973,315 (439 X; raw dataset size before assembly) single molecule optical maps (Rmaps; 1 Rmap = 1 restriction mapped DNA molecule) generated by the Optical Mapping System. The BamHI map spans 2,575.30 Mb and comprises 78 optical contigs assembled by a combination of iterative (using the reference sequence: UMD3.1) and de novo assembly techniques. BtOM1.0 is a high-resolution physical map featuring an average restriction fragment size of 8.91 Kb. Comparisons of BtOM1.0 vs. UMD3.1, or Btau4.6, revealed that Btau4.6 presented far more discordances (7,463) vs. UMD3.1 (4,754). Overall, we found that Btau4.6 presented almost double the number of discordances than UMD3.1 across most of the 6 categories of sequence vs. map discrepancies, which are: COMPLEX (misassembly), DELs (extraneous sequences), INSs (missing sequences), ITs (Inverted/Translocated sequences), ECs (extra restriction cuts) and MCs (missing restriction cuts). Alignments of UMD3.1 and Btau4.6 to BtOM1.0 reveal discordances commensurate with previous reports, and affirm the NCBI's current designation of UMD3.1 sequence assembly as the "reference assembly" and the Btau4.6 as the "alternate assembly." The cattle genome optical map, BtOM1.0, when used as a comprehensive and largely independent guide, will greatly assist improvements to existing sequence builds, and later serve as an accurate physical scaffold for studies concerning the comparative genomics of cattle breeds.

Targeted RNA-Sequencing with Competitive Multiplex-PCR Amplicon Libraries

PubMed Central

Blomquist, Thomas M.; Crawford, Erin L.; Lovett, Jennie L.; Yeo, Jiyoun; Stanoszek, Lauren M.; Levin, Albert; Li, Jia; Lu, Mei; Shi, Leming; Muldrew, Kenneth; Willey, James C.

2013-01-01

Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation. We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression. We demonstrate excellent reproducibility (R2 = 0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R2 = 0.97–0.99) using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R2 = 0.96) and whole transcriptome RNA-sequencing following “traditional” library preparation using Illumina NGS kits (R2 = 0.94). Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 107-fold range was reduced more than 10,000-fold, from 2.3×109 to 1.4×105 sequencing reads. These studies demonstrate that the competitive multiplex-PCR amplicon library preparation method presented here provides the quality control, reproducibility, and reduced sequencing reads necessary for development and implementation of targeted quantitative RNA-sequencing biomarkers in molecular diagnostic testing. PMID:24236095

A Novel Model System to Examine Agents Used in Breast Cancer Therapy.

DTIC Science & Technology

1995-07-01

We have recently characterized a multiprotein DNA replication complex (MRC) that was purified from NODA NIB 468 human breast cancer cells by a series...proliferating cell nuclear antigen (PCNA), RE-C RP-A and DNA topoisomerase I. Based upon its requirements for DNA replication activity and its...SV4O) origin sequences, the MRC executes all of the steps required for the in vitro, bidirectional replication of the SV4O genome. Several of the DNA

Activation of the yeast Hippo pathway by phosphorylation-dependent assembly of signaling complexes.

PubMed

Rock, Jeremy M; Lim, Daniel; Stach, Lasse; Ogrodowicz, Roksana W; Keck, Jamie M; Jones, Michele H; Wong, Catherine C L; Yates, John R; Winey, Mark; Smerdon, Stephen J; Yaffe, Michael B; Amon, Angelika

2013-05-17

Scaffold-assisted signaling cascades guide cellular decision-making. In budding yeast, one such signal transduction pathway called the mitotic exit network (MEN) governs the transition from mitosis to the G1 phase of the cell cycle. The MEN is conserved and in metazoans is known as the Hippo tumor-suppressor pathway. We found that signaling through the MEN kinase cascade was mediated by an unusual two-step process. The MEN kinase Cdc15 first phosphorylated the scaffold Nud1. This created a phospho-docking site on Nud1, to which the effector kinase complex Dbf2-Mob1 bound through a phosphoserine-threonine binding domain, in order to be activated by Cdc15. This mechanism of pathway activation has implications for signal transmission through other kinase cascades and might represent a general principle in scaffold-assisted signaling.

Evaluation of the Abbott realtime HCV genotype II RUO (GT II) assay with reference to 5'UTR, core and NS5B sequencing.

PubMed

Mallory, Melanie A; Lucic, Danijela X; Sears, Mitchell T; Cloherty, Gavin A; Hillyard, David R

2014-05-01

HCV genotyping is a critical tool for guiding initiation of therapy and selecting the most appropriate treatment regimen. To evaluate the concordance between the Abbott GT II assay and genotyping by sequencing subregions of the HCV 5'UTR, core and NS5B. The Abbott assay was used to genotype 127 routine patient specimens and 35 patient specimens with unusual subtypes and mixed infection. Abbott results were compared to genotyping by 5'UTR, core and NS5B sequencing. Sequences were genotyped using the NCBI non-redundant database and the online genotyping tool COMET. Among routine specimens, core/NS5B sequencing identified 93 genotype 1s, 13 genotype 2s, 15 genotype 3s, three genotype 4s, two genotype 6s and one recombinant specimen. Genotype calls by 5'UTR, core, NS5B sequencing and the Abbott assay were 97.6% concordant. Core/NS5B sequencing identified two discrepant samples as genotype 6 (subtypes 6l and 6u) while Abbott and 5'UTR sequencing identified these samples as genotype 1 with no subtype. The Abbott assay subtyped 91.4% of genotype 1 specimens. Among the 35 rare specimens, the Abbott assay inaccurately genotyped 3k, 6e, 6o, 6q and one genotype 4 variant; gave indeterminate results for 3g, 3h, 4r, 6m, 6n, and 6q specimens; and agreed with core/NS5B sequencing for mixed specimens. The Abbott assay is an automated HCV genotyping method with improved accuracy over 5'UTR sequencing. Samples identified by the Abbott assay as genotype 1 with no subtype may be rare subtypes of other genotypes and thus require confirmation by another method. Copyright © 2014 Elsevier B.V. All rights reserved.

Industrial approach to piezoelectric damping of large fighter aircraft components

NASA Astrophysics Data System (ADS)

Simpson, John; Schweiger, Johannes

1998-06-01

Different concepts to damp structural vibrations of the vertical tail of fighter aircraft are reported. The various requirements for a vertical tail bias an integrated approach for the design. Several active vibrations suppression concepts had been investigated during the preparatory phase of a research program shared by Daimler-Benz Aerospace Military Aircraft (Dasa), Daimler-Benz Forschung (DBF) and Deutsche Forschungsandstalt fuer Luftund Raumfahrt (DLR). Now in the main phase of the programme, four concepts were finally chosen: two concepts with aerodynamic control surfaces and two concepts with piezoelectric components. One piezo concept approach will be described rigorously, the other concepts are briefly addressed. In the Dasa concept, thin surface piezo actuators are set out carefully to flatten the dynamic portion of the combined static and dynamic maximum bending moment loading case directly in the shell structure. The second piezo concept by DLR involves pre-loaded lead zirconate titanate (PZT)-block actuators at host structure fixtures. To this end a research apparatus was designed and built as a full scale simplified fin box with carbon fiber reinformed plastic skins and an aluminium stringer-rib substructure restrained by relevant aircraft fixtures. It constitutes a benchmark 3D-structural impedance. The engineering design incorporates 7kg of PZT surface actuators. The structural system then should be excited to more than 15mm tip displacement amplitude. This prepares the final step to total A/C integration. Typical analysis methods using cyclic thermal analogies adapted to induced load levels are compared. Commercial approaches leading onto basic state space model interpretation wrt. actuator sizing and positioning, structural integrity constraints, FE-validation and testing are described. Both piezoelectric strategies are aimed at straight open-loop performance related to concept weight penalty and input electric power. The required actuators, power and integration are then enhanced to specification standards. An adapted qualification program plan is used to improve analytical read across, specifications, manufacturing decisions, handling requirements. The next research goals are outlined.

Saccharomyces cerevisiae Mob1p Is Required for Cytokinesis and Mitotic Exit

PubMed Central

Luca, Francis C.; Mody, Manali; Kurischko, Cornelia; Roof, David M.; Giddings, Thomas H.; Winey, Mark

2001-01-01

The Saccharomyces cerevisiae mitotic exit network (MEN) is a conserved set of genes that mediate the transition from mitosis to G1 by regulating mitotic cyclin degradation and the inactivation of cyclin-dependent kinase (CDK). Here, we demonstrate that, in addition to mitotic exit, S. cerevisiae MEN gene MOB1 is required for cytokinesis and cell separation. The cytokinesis defect was evident in mob1 mutants under conditions in which there was no mitotic-exit defect. Observation of live cells showed that yeast myosin II, Myo1p, was present in the contractile ring at the bud neck but that the ring failed to contract and disassemble. The cytokinesis defect persisted for several mitotic cycles, resulting in chains of cells with correctly segregated nuclei but with uncontracted actomyosin rings. The cytokinesis proteins Cdc3p (a septin), actin, and Iqg1p/ Cyk1p (an IQGAP-like protein) appeared to correctly localize in mob1 mutants, suggesting that MOB1 functions subsequent to actomyosin ring assembly. We also examined the subcellular distribution of Mob1p during the cell cycle and found that Mob1p first localized to the spindle pole bodies during mid-anaphase and then localized to a ring at the bud neck just before and during cytokinesis. Localization of Mob1p to the bud neck required CDC3, MEN genes CDC5, CDC14, CDC15, and DBF2, and spindle pole body gene NUD1 but was independent of MYO1. The localization of Mob1p to both spindle poles was abolished in cdc15 and nud1 mutants and was perturbed in cdc5 and cdc14 mutants. These results suggest that the MEN functions during the mitosis-to-G1 transition to control cyclin-CDK inactivation and cytokinesis. PMID:11564880

A machine learning model to determine the accuracy of variant calls in capture-based next generation sequencing.

PubMed

van den Akker, Jeroen; Mishne, Gilad; Zimmer, Anjali D; Zhou, Alicia Y

2018-04-17

Next generation sequencing (NGS) has become a common technology for clinical genetic tests. The quality of NGS calls varies widely and is influenced by features like reference sequence characteristics, read depth, and mapping accuracy. With recent advances in NGS technology and software tools, the majority of variants called using NGS alone are in fact accurate and reliable. However, a small subset of difficult-to-call variants that still do require orthogonal confirmation exist. For this reason, many clinical laboratories confirm NGS results using orthogonal technologies such as Sanger sequencing. Here, we report the development of a deterministic machine-learning-based model to differentiate between these two types of variant calls: those that do not require confirmation using an orthogonal technology (high confidence), and those that require additional quality testing (low confidence). This approach allows reliable NGS-based calling in a clinical setting by identifying the few important variant calls that require orthogonal confirmation. We developed and tested the model using a set of 7179 variants identified by a targeted NGS panel and re-tested by Sanger sequencing. The model incorporated several signals of sequence characteristics and call quality to determine if a variant was identified at high or low confidence. The model was tuned to eliminate false positives, defined as variants that were called by NGS but not confirmed by Sanger sequencing. The model achieved very high accuracy: 99.4% (95% confidence interval: +/- 0.03%). It categorized 92.2% (6622/7179) of the variants as high confidence, and 100% of these were confirmed to be present by Sanger sequencing. Among the variants that were categorized as low confidence, defined as NGS calls of low quality that are likely to be artifacts, 92.1% (513/557) were found to be not present by Sanger sequencing. This work shows that NGS data contains sufficient characteristics for a machine-learning-based model to differentiate low from high confidence variants. Additionally, it reveals the importance of incorporating site-specific features as well as variant call features in such a model.

HIV-1 transmission linkage in an HIV-1 prevention clinical trial

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

Leitner, Thomas; Campbell, Mary S; Mullins, James I

2009-01-01

HIV-1 sequencing has been used extensively in epidemiologic and forensic studies to investigate patterns of HIV-1 transmission. However, the criteria for establishing genetic linkage between HIV-1 strains in HIV-1 prevention trials have not been formalized. The Partners in Prevention HSV/HIV Transmission Study (ClinicaITrials.gov NCT00194519) enrolled 3408 HIV-1 serodiscordant heterosexual African couples to determine the efficacy of genital herpes suppression with acyclovir in reducing HIV-1 transmission. The trial analysis required laboratory confirmation of HIV-1 linkage between enrolled partners in couples in which seroconversion occurred. Here we describe the process and results from HIV-1 sequencing studies used to perform transmission linkage determinationmore » in this clinical trial. Consensus Sanger sequencing of env (C2-V3-C3) and gag (p17-p24) genes was performed on plasma HIV-1 RNA from both partners within 3 months of seroconversion; env single molecule or pyrosequencing was also performed in some cases. For linkage, we required monophyletic clustering between HIV-1 sequences in the transmitting and seroconverting partners, and developed a Bayesian algorithm using genetic distances to evaluate the posterior probability of linkage of participants sequences. Adjudicators classified transmissions as linked, unlinked, or indeterminate. Among 151 seroconversion events, we found 108 (71.5%) linked, 40 (26.5%) unlinked, and 3 (2.0%) to have indeterminate transmissions. Nine (8.3%) were linked by consensus gag sequencing only and 8 (7.4%) required deep sequencing of env. In this first use of HIV-1 sequencing to establish endpoints in a large clinical trial, more than one-fourth of transmissions were unlinked to the enrolled partner, illustrating the relevance of these methods in the design of future HIV-1 prevention trials in serodiscordant couples. A hierarchy of sequencing techniques, analysis methods, and expert adjudication contributed to the linkage determination process.« less

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

The SRL peptide of Rhesus Rotavirus VP4 protein governs cholangiocyte infection and the murine model of biliary atresia

PubMed Central

Mohanty, Sujit K.; Donnelly, Bryan; Lobeck, Inna; Walther, Ashley; Dupree, Phylicia; Coots, Abigail; Meller, Jaroslaw; McNeal, Monica; Sestak, Karol; Tiao, Greg

2016-01-01

Biliary atresia (BA) is a neonatal obstructive cholangiopathy which progresses to end stage liver disease, often requiring transplantation. The murine model of BA, employing rhesus rotavirus (RRV), parallels human disease and has been used to elucidate mechanistic aspects of a virus induced biliary cholangiopathy. We previously reported that RRV VP4 gene plays an integral role in activating the immune system and induction of BA. Utilizing rotavirus binding and blocking assays, this study elucidated how RRV VP4 protein governs cholangiocyte susceptibility to infection both in vitro and in vivo in the murine model of BA. We identified the amino acid sequence on VP4 and its cholangiocyte binding protein, finding that the sequence is specific to those rotavirus strains which cause an obstructive cholangiopathy. Pretreatment of murine and human cholangiocytes with this VP4 derived peptide (TRTRVSRLY), significantly reduced RRV’s ability to bind and infect the cells. However, the peptide did not block cholangiocyte binding of TUCH and Ro1845, strains which do not induce murine BA. The SRL sequence within TRTRVSRLY is required for cholangiocyte binding and viral replication. The cholangiocyte membrane protein bound by SRL was found to be Hsc70. Inhibition of Hsc70 by siRNAs reduced RRV’s ability to infect cholangiocytes. This virus-cholangiocyte interaction is also seen in vivo in the murine model of BA, where inoculation of mice with TRTRVSRLY peptide significantly reduced symptoms and mortality in RRV-injected mice. Conclusion The tri-peptide SRL on RRV VP4 binds to the cholangiocyte membrane protein Hsc70 defining a novel binding site governing VP4 attachment. Investigations are underway to determine the cellular response following this interaction to understand how it contributes to the pathogenesis of BA. PMID:27859498

DNA sequence requirements for the accurate transcription of a protein-coding plastid gene in a plastid in vitro system from mustard (Sinapis alba L.)

PubMed Central

Link, Gerhard

1984-01-01

A nuclease-treated plastid extract from mustard (Sinapis alba L.) allows efficient transcription of cloned plastid DNA templates. In this in vitro system, the major runoff transcript of the truncated gene for the 32 000 mol. wt. photosystem II protein was accurately initiated from a site close to or identical with the in vivo start site. By using plasmids with deletions in the 5'-flanking region of this gene as templates, a DNA region required for efficient and selective initiation was detected ˜28-35 nucleotides upstream of the transcription start site. This region contains the sequence element TTGACA, which matches the consensus sequence for prokaryotic `−35' promoter elements. In the absence of this region, a region ˜13-27 nucleotides upstream of the start site still enables a basic level of specific transcription. This second region contains the sequence element TATATAA, which matches the consensus sequence for the `TATA' box of genes transcribed by RNA polymerase II (or B). The region between the `TATA'-like element and the transcription start site is not sufficient but may be required for specific transcription of the plastid gene. This latter region contains the sequence element TATACT, which resembles the prokaryotic `−10' (Pribnow) box. Based on the structural and transcriptional features of the 5' upstream region, a `promoter switch' mechanism is proposed, which may account for the developmentally regulated expression of this plastid gene. ImagesFig. 1.Fig. 2.Fig. 3.Fig. 4.Figure 5. PMID:16453540

Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex.

PubMed

Wolle, Daniel; Cleard, Fabienne; Aoki, Tsutomu; Deshpande, Girish; Schedl, Paul; Karch, Francois

2015-11-01

Chromatin boundaries are architectural elements that determine the three-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 boundary from the Drosophila bithorax complex (BX-C) is required for the parasegment-specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in the BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and we describe a novel ∼700-kDa complex, the late boundary complex (LBC), that binds to Fab-7 sequences that have insulator functions in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late, but not early, embryos and that it contains three insulator proteins, GAF, Mod(mdg4), and E(y)2. Its DNA binding properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display few sequence similarities. Finally, we show that mutations which abrogate LBC binding in vitro inactivate the Fab-7 boundary in the BX-C. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

37 CFR 1.823 - Requirements for nucleotide and/or amino acid sequences as part of the application.

Code of Federal Regulations, 2011 CFR

2011-07-01

... and/or amino acid sequences as part of the application. 1.823 Section 1.823 Patents, Trademarks, and... Amino Acid Sequences § 1.823 Requirements for nucleotide and/or amino acid sequences as part of the... incorporation-by-reference of the Sequence Listing as required by § 1.52(e)(5). The presentation of the...

37 CFR 1.823 - Requirements for nucleotide and/or amino acid sequences as part of the application.

Code of Federal Regulations, 2013 CFR

2013-07-01

... and/or amino acid sequences as part of the application. 1.823 Section 1.823 Patents, Trademarks, and... Amino Acid Sequences § 1.823 Requirements for nucleotide and/or amino acid sequences as part of the... incorporation-by-reference of the Sequence Listing as required by § 1.52(e)(5). The presentation of the...

37 CFR 1.823 - Requirements for nucleotide and/or amino acid sequences as part of the application.

Code of Federal Regulations, 2012 CFR

2012-07-01

... and/or amino acid sequences as part of the application. 1.823 Section 1.823 Patents, Trademarks, and... Amino Acid Sequences § 1.823 Requirements for nucleotide and/or amino acid sequences as part of the... incorporation-by-reference of the Sequence Listing as required by § 1.52(e)(5). The presentation of the...

37 CFR 1.823 - Requirements for nucleotide and/or amino acid sequences as part of the application.

Code of Federal Regulations, 2010 CFR

2010-07-01

... and/or amino acid sequences as part of the application. 1.823 Section 1.823 Patents, Trademarks, and... Amino Acid Sequences § 1.823 Requirements for nucleotide and/or amino acid sequences as part of the... incorporation-by-reference of the Sequence Listing as required by § 1.52(e)(5). The presentation of the...

37 CFR 1.823 - Requirements for nucleotide and/or amino acid sequences as part of the application.

Code of Federal Regulations, 2014 CFR

2014-07-01

... and/or amino acid sequences as part of the application. 1.823 Section 1.823 Patents, Trademarks, and... Amino Acid Sequences § 1.823 Requirements for nucleotide and/or amino acid sequences as part of the... incorporation-by-reference of the Sequence Listing as required by § 1.52(e)(5). The presentation of the...

Investigation of the role of TCF4 rare sequence variants in schizophrenia.

PubMed

Basmanav, F Buket; Forstner, Andreas J; Fier, Heide; Herms, Stefan; Meier, Sandra; Degenhardt, Franziska; Hoffmann, Per; Barth, Sandra; Fricker, Nadine; Strohmaier, Jana; Witt, Stephanie H; Ludwig, Michael; Schmael, Christine; Moebus, Susanne; Maier, Wolfgang; Mössner, Rainald; Rujescu, Dan; Rietschel, Marcella; Lange, Christoph; Nöthen, Markus M; Cichon, Sven

2015-07-01

Transcription factor 4 (TCF4) is one of the most robust of all reported schizophrenia risk loci and is supported by several genetic and functional lines of evidence. While numerous studies have implicated common genetic variation at TCF4 in schizophrenia risk, the role of rare, small-sized variants at this locus-such as single nucleotide variants and short indels which are below the resolution of chip-based arrays requires further exploration. The aim of the present study was to investigate the association between rare TCF4 sequence variants and schizophrenia. Exon-targeted resequencing was performed in 190 German schizophrenia patients. Six rare variants at the coding exons and flanking sequences of the TCF4 gene were identified, including two missense variants and one splice site variant. These six variants were then pooled with nine additional rare variants identified in 379 European participants of the 1000 Genomes Project, and all 15 variants were genotyped in an independent German sample (n = 1,808 patients; n = 2,261 controls). These data were then analyzed using six statistical methods developed for the association analysis of rare variants. No significant association (P < 0.05) was found. However, the results from our association and power analyses suggest that further research into the possible involvement of rare TCF4 sequence variants in schizophrenia risk is warranted by the assessment of larger cohorts with higher statistical power to identify rare variant associations. © 2015 Wiley Periodicals, Inc.

Aim44p regulates phosphorylation of Hof1p to promote contractile ring closure during cytokinesis in budding yeast

PubMed Central

Wolken, Dana M. Alessi; McInnes, Joseph; Pon, Liza A.

2014-01-01

Whereas actomyosin and septin ring organization and function in cytokinesis are thoroughly described, little is known regarding the mechanisms by which the actomyosin ring interacts with septins and associated proteins to coordinate cell division. Here we show that the protein product of YPL158C, Aim44p, undergoes septin-dependent recruitment to the site of cell division. Aim44p colocalizes with Myo1p, the type II myosin of the contractile ring, throughout most of the cell cycle. The Aim44p ring does not contract when the actomyosin ring closes. Instead, it forms a double ring that associates with septin rings on mother and daughter cells after cell separation. Deletion of AIM44 results in defects in contractile ring closure. Aim44p coimmunoprecipitates with Hof1p, a conserved F-BAR protein that binds both septins and type II myosins and promotes contractile ring closure. Deletion of AIM44 results in a delay in Hof1p phosphorylation and altered Hof1p localization. Finally, overexpression of Dbf2p, a kinase that phosphorylates Hof1p and is required for relocalization of Hof1p from septin rings to the contractile ring and for Hof1p-triggered contractile ring closure, rescues the cytokinesis defect observed in aim44∆ cells. Our studies reveal a novel role for Aim44p in regulating contractile ring closure through effects on Hof1p. PMID:24451263

Persistence of evolutionary memory: primordial six-transmembrane helical domain mu opiate receptors selectively linked to endogenous morphine signaling.

PubMed

Kream, Richard M; Sheehan, Melinda; Cadet, Patrick; Mantione, Kirk J; Zhu, Wei; Casares, Federico; Stefano, George B

2007-12-01

Biochemical, molecular and pharmacological evidence for two unique six-transmembrane helical (TMH) domain opiate receptors expressed from the micro opioid receptor (MOR) gene have been shown. Designated micro3 and micro4 receptors, both protein species are Class A rhodopsin-like members of the superfamily of G-protein coupled receptors but are selectively tailored to mediate the cellular regulatory effects of endogenous morphine and related morphinan alkaloids via stimulation of nitric oxide (NO) production and release. Both micro3 and micro4 receptors lack an amino acid sequence of approximately 90 amino acids that constitute the extracellular N-terminal and TMH1 domains and part of the first intracellular loop of the micro1 receptor, but retain the empirically defined ligand binding pocket distributed across conserved TMH2, TMH3, and TMH7 domains of the micro1 sequence. Additionally, the receptor proteins are terminated by unique intracellular C-terminal amino acid sequences that serve as putative coupling or docking domains required for constitutive NO synthase activation. Because the recognition profile of micro3 and micro4 receptors is restricted to rigid benzylisoquinoline alkaloids typified by morphine and its extended family of chemical congeners, it is hypothesized that conformational stabilization provided by interaction of extended extracellular N-terminal protein domains and the extracellular loops is required for binding of endogenous opioid peptides as well as synthetic flexible opiate alkaloids.

A double-labeling procedure for sequence analysis of picomole amounts of nonradioactive RNA fragments.

PubMed Central

Gupta, R C; Randerath, E; Randerath, K

1976-01-01

A double-labeling procedure for sequence analysis of nonradioactive polyribonucleotides is detailed, which is based on controlled endonucleolytic degradation of 3'-terminally (3H)-labeled oligonucleotide-(3') dialcohols and 5"-terminal analysis of the partial (3H)-labeled fragments following their separation according to chain length by polyethyleneimine- (PEI-)cellulose TLC and detection by fluorography. Undesired nonradioactive partial digestion products are eliminated by periodate oxidation. The 5'-termini are assayed by enzymic incorporation of (32p)-label into the isolated fragments, enzymic release of (32p)-labeled nucleoside-(5') monophosphates, two-dimensional PEI-cellulose chromatography, and autoradiography. Using this procedure, as little as 0.1 - 0.3 A260 unit of tRNA is needed to sequence all fragments in complete ribonuclease T1 and A digests, whereas radioactive derivative methods previously described by us1-4 required 4 - 6 A260 units. Images PMID:826884

Investigation of a Quadruplex-Forming Repeat Sequence Highly Enriched in Xanthomonas and Nostoc sp.

PubMed

Rehm, Charlotte; Wurmthaler, Lena A; Li, Yuanhao; Frickey, Tancred; Hartig, Jörg S

2015-01-01

In prokaryotes simple sequence repeats (SSRs) with unit sizes of 1-5 nucleotides (nt) are causative for phase and antigenic variation. Although an increased abundance of heptameric repeats was noticed in bacteria, reports about SSRs of 6-9 nt are rare. In particular G-rich repeat sequences with the propensity to fold into G-quadruplex (G4) structures have received little attention. In silico analysis of prokaryotic genomes show putative G4 forming sequences to be abundant. This report focuses on a surprisingly enriched G-rich repeat of the type GGGNATC in Xanthomonas and cyanobacteria such as Nostoc. We studied in detail the genomes of Xanthomonas campestris pv. campestris ATCC 33913 (Xcc), Xanthomonas axonopodis pv. citri str. 306 (Xac), and Nostoc sp. strain PCC7120 (Ana). In all three organisms repeats are spread all over the genome with an over-representation in non-coding regions. Extensive variation of the number of repetitive units was observed with repeat numbers ranging from two up to 26 units. However a clear preference for four units was detected. The strong bias for four units coincides with the requirement of four consecutive G-tracts for G4 formation. Evidence for G4 formation of the consensus repeat sequences was found in biophysical studies utilizing CD spectroscopy. The G-rich repeats are preferably located between aligned open reading frames (ORFs) and are under-represented in coding regions or between divergent ORFs. The G-rich repeats are preferentially located within a distance of 50 bp upstream of an ORF on the anti-sense strand or within 50 bp from the stop codon on the sense strand. Analysis of whole transcriptome sequence data showed that the majority of repeat sequences are transcribed. The genetic loci in the vicinity of repeat regions show increased genomic stability. In conclusion, we introduce and characterize a special class of highly abundant and wide-spread quadruplex-forming repeat sequences in bacteria.

Investigation of a Quadruplex-Forming Repeat Sequence Highly Enriched in Xanthomonas and Nostoc sp.

PubMed Central

Rehm, Charlotte; Wurmthaler, Lena A.; Li, Yuanhao; Frickey, Tancred; Hartig, Jörg S.

2015-01-01

In prokaryotes simple sequence repeats (SSRs) with unit sizes of 1–5 nucleotides (nt) are causative for phase and antigenic variation. Although an increased abundance of heptameric repeats was noticed in bacteria, reports about SSRs of 6–9 nt are rare. In particular G-rich repeat sequences with the propensity to fold into G-quadruplex (G4) structures have received little attention. In silico analysis of prokaryotic genomes show putative G4 forming sequences to be abundant. This report focuses on a surprisingly enriched G-rich repeat of the type GGGNATC in Xanthomonas and cyanobacteria such as Nostoc. We studied in detail the genomes of Xanthomonas campestris pv. campestris ATCC 33913 (Xcc), Xanthomonas axonopodis pv. citri str. 306 (Xac), and Nostoc sp. strain PCC7120 (Ana). In all three organisms repeats are spread all over the genome with an over-representation in non-coding regions. Extensive variation of the number of repetitive units was observed with repeat numbers ranging from two up to 26 units. However a clear preference for four units was detected. The strong bias for four units coincides with the requirement of four consecutive G-tracts for G4 formation. Evidence for G4 formation of the consensus repeat sequences was found in biophysical studies utilizing CD spectroscopy. The G-rich repeats are preferably located between aligned open reading frames (ORFs) and are under-represented in coding regions or between divergent ORFs. The G-rich repeats are preferentially located within a distance of 50 bp upstream of an ORF on the anti-sense strand or within 50 bp from the stop codon on the sense strand. Analysis of whole transcriptome sequence data showed that the majority of repeat sequences are transcribed. The genetic loci in the vicinity of repeat regions show increased genomic stability. In conclusion, we introduce and characterize a special class of highly abundant and wide-spread quadruplex-forming repeat sequences in bacteria. PMID:26695179

RNA synthesis is modulated by G-quadruplex formation in Hepatitis C virus negative RNA strand.

PubMed

Chloé, Jaubert; Amina, Bedrat; Laura, Bartolucci; Carmelo, Di Primo; Michel, Ventura; Jean-Louis, Mergny; Samir, Amrane; Marie-Line, Andreola

2018-05-25

DNA and RNA guanine-rich oligonucleotides can form non-canonical structures called G-quadruplexes or "G4" that are based on the stacking of G-quartets. The role of DNA and RNA G4 is documented in eukaryotic cells and in pathogens such as viruses. Yet, G4 have been identified only in a few RNA viruses, including the Flaviviridae family. In this study, we analysed the last 157 nucleotides at the 3'end of the HCV (-) strand. This sequence is known to be the minimal sequence required for an efficient RNA replication. Using bioinformatics and biophysics, we identified a highly conserved G4-prone sequence located in the stem-loop IIy' of the negative strand. We also showed that the formation of this G-quadruplex inhibits the in vitro RNA synthesis by the RdRp. Furthermore, Phen-DC3, a specific G-quadruplex binder, is able to inhibit HCV viral replication in cells in conditions where no cytotoxicity was measured. Considering that this domain of the negative RNA strand is well conserved among HCV genotypes, G4 ligands could be of interest for new antiviral therapies.

Deciphering KRAS and NRAS mutated clone dynamics in MLL-AF4 paediatric leukaemia by ultra deep sequencing analysis.

PubMed

Trentin, Luca; Bresolin, Silvia; Giarin, Emanuela; Bardini, Michela; Serafin, Valentina; Accordi, Benedetta; Fais, Franco; Tenca, Claudya; De Lorenzo, Paola; Valsecchi, Maria Grazia; Cazzaniga, Giovanni; Kronnie, Geertruy Te; Basso, Giuseppe

2016-10-04

To induce and sustain the leukaemogenic process, MLL-AF4+ leukaemia seems to require very few genetic alterations in addition to the fusion gene itself. Studies of infant and paediatric patients with MLL-AF4+ B cell precursor acute lymphoblastic leukaemia (BCP-ALL) have reported mutations in KRAS and NRAS with incidences ranging from 25 to 50%. Whereas previous studies employed Sanger sequencing, here we used next generation amplicon deep sequencing for in depth evaluation of RAS mutations in 36 paediatric patients at diagnosis of MLL-AF4+ leukaemia. RAS mutations including those in small sub-clones were detected in 63.9% of patients. Furthermore, the mutational analysis of 17 paired samples at diagnosis and relapse revealed complex RAS clone dynamics and showed that the mutated clones present at relapse were almost all originated from clones that were already detectable at diagnosis and survived to the initial therapy. Finally, we showed that mutated patients were indeed characterized by a RAS related signature at both transcriptional and protein levels and that the targeting of the RAS pathway could be of beneficial for treatment of MLL-AF4+ BCP-ALL clones carrying somatic RAS mutations.

Impact of sequencing depth in ChIP-seq experiments

PubMed Central

Jung, Youngsook L.; Luquette, Lovelace J.; Ho, Joshua W.K.; Ferrari, Francesco; Tolstorukov, Michael; Minoda, Aki; Issner, Robbyn; Epstein, Charles B.; Karpen, Gary H.; Kuroda, Mitzi I.; Park, Peter J.

2014-01-01

In a chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) experiment, an important consideration in experimental design is the minimum number of sequenced reads required to obtain statistically significant results. We present an extensive evaluation of the impact of sequencing depth on identification of enriched regions for key histone modifications (H3K4me3, H3K36me3, H3K27me3 and H3K9me2/me3) using deep-sequenced datasets in human and fly. We propose to define sufficient sequencing depth as the number of reads at which detected enrichment regions increase <1% for an additional million reads. Although the required depth depends on the nature of the mark and the state of the cell in each experiment, we observe that sufficient depth is often reached at <20 million reads for fly. For human, there are no clear saturation points for the examined datasets, but our analysis suggests 40–50 million reads as a practical minimum for most marks. We also devise a mathematical model to estimate the sufficient depth and total genomic coverage of a mark. Lastly, we find that the five algorithms tested do not agree well for broad enrichment profiles, especially at lower depths. Our findings suggest that sufficient sequencing depth and an appropriate peak-calling algorithm are essential for ensuring robustness of conclusions derived from ChIP-seq data. PMID:24598259

Whole-Genome Sequencing for Optimized Patient Management

PubMed Central

Bainbridge, Matthew N.; Wiszniewski, Wojciech; Murdock, David R.; Friedman, Jennifer; Gonzaga-Jauregui, Claudia; Newsham, Irene; Reid, Jeffrey G.; Fink, John K.; Morgan, Margaret B.; Gingras, Marie-Claude; Muzny, Donna M.; Hoang, Linh D.; Yousaf, Shahed; Lupski, James R.; Gibbs, Richard A.

2012-01-01

Whole-genome sequencing of patient DNA can facilitate diagnosis of a disease, but its potential for guiding treatment has been under-realized. We interrogated the complete genome sequences of a 14-year-old fraternal twin pair diagnosed with dopa (3,4-dihydroxyphenylalanine)–responsive dystonia (DRD; Mendelian Inheritance in Man #128230). DRD is a genetically heterogeneous and clinically complex movement disorder that is usually treated with l-dopa, a precursor of the neurotransmitter dopamine. Whole-genome sequencing identified compound heterozygous mutations in the SPR gene encoding sepiapterin reductase. Disruption of SPR causes a decrease in tetrahydrobiopterin, a cofactor required for the hydroxylase enzymes that synthesize the neurotransmitters dopamine and serotonin. Supplementation of l-dopa therapy with 5-hydroxytryptophan, a serotonin precursor, resulted in clinical improvements in both twins. PMID:21677200

Isolation and characterization of the promoter sequence of a cassava gene coding for Pt2L4, a glutamic acid-rich protein differentially expressed in storage roots.

PubMed

de Souza, C R; Aragão, F J; Moreira, E C O; Costa, C N M; Nascimento, S B; Carvalho, L J

2009-03-24

Cassava is one of the most important tropical food crops for more than 600 million people worldwide. Transgenic technologies can be useful for increasing its nutritional value and its resistance to viral diseases and insect pests. However, tissue-specific promoters that guarantee correct expression of transgenes would be necessary. We used inverse polymerase chain reaction to isolate a promoter sequence of the Mec1 gene coding for Pt2L4, a glutamic acid-rich protein differentially expressed in cassava storage roots. In silico analysis revealed putative cis-acting regulatory elements within this promoter sequence, including root-specific elements that may be required for its expression in vascular tissues. Transient expression experiments showed that the Mec1 promoter is functional, since this sequence was able to drive GUS expression in bean embryonic axes. Results from our computational analysis can serve as a guide for functional experiments to identify regions with tissue-specific Mec1 promoter activity. The DNA sequence that we identified is a new promoter that could be a candidate for genetic engineering of cassava roots.

R2 & NE Block Group - 2010 Census; Housing and Population Summary

EPA Pesticide Factsheets

The TIGER/Line Files are shapefiles and related database files (.dbf) that are an extract of selected geographic and cartographic information from the U.S. Census Bureau's Master Address File / Topologically Integrated Geographic Encoding and Referencing (MAF/TIGER) Database (MTDB). The MTDB represents a seamless national file with no overlaps or gaps between parts, however, each TIGER/Line File is designed to stand alone as an independent data set, or they can be combined to cover the entire nation. Block Groups (BGs) are defined before tabulation block delineation and numbering, but are clusters of blocks within the same census tract that have the same first digit of their 4-digit census block number from the same decennial census. For example, Census 2000 tabulation blocks 3001, 3002, 3003,.., 3999 within Census 2000 tract 1210.02 are also within BG 3 within that census tract. Census 2000 BGs generally contained between 600 and 3,000 people, with an optimum size of 1,500 people. Most BGs were delineated by local participants in the Census Bureau's Participant Statistical Areas Program (PSAP). The Census Bureau delineated BGs only where the PSAP participant declined to delineate BGs or where the Census Bureau could not identify any local PSAP participant. A BG usually covers a contiguous area. Each census tract contains at least one BG, and BGs are uniquely numbered within census tract. Within the standard census geographic hierarchy, BGs never cross

Major- and Trace-Element Concentrations in Rock Samples Collected in 2006 from the Taylor Mountains 1:250,000-scale Quadrangle, Alaska

USGS Publications Warehouse

Klimasauskas, Edward P.; Miller, Marti L.; Bradley, Dwight C.

2007-01-01

Introduction The Kuskokwim mineral belt of Bundtzen and Miller (1997) forms an important metallogenic region in southwestern Alaska that has yielded more than 3.22 million ounces of gold and 400,000 ounces of silver. Precious-metal and related deposits in this region associated with Late Cretaceous to early Tertiary igneous complexes extend into the Taylor Mountains 1:250,000-scale quadrangle. The U.S. Geological Survey is in the process of conducting a mineral resource assessment of this region. This report presents analytical data collected during the third year of this multiyear study. A total of 138 rock geochemistry samples collected during the 2006 field season were analyzed using the ICP-AES/MS42, ICP-AES10, fire assay, and cold vapor atomic absorption methods described in more detail below. Analytical values are provided in percent (% or pct: 1 gram per 100 grams), parts per million (ppm: 1 gram per 1,000,000 grams), or parts per billion (ppb: 1 gram per 1,000,000,000 grams) as indicated in the column heading of the data table. Data are provided for download in Excel (*.xls), comma delimited (*.csv), dBase 4 (*.dbf) and as a point coverage in ArcInfo interchange (*.e00) formats available at http://pubs.usgs.gov/of/2007/1386/.

R2 & NE Tract - 2010 Census; Housing and Population Summary

EPA Pesticide Factsheets

The TIGER/Line Files are shapefiles and related database files (.dbf) that are an extract of selected geographic and cartographic information from the U.S. Census Bureau's Master Address File / Topologically Integrated Geographic Encoding and Referencing (MAF/TIGER) Database (MTDB). The MTDB represents a seamless national file with no overlaps or gaps between parts, however, each TIGER/Line File is designed to stand alone as an independent data set, or they can be combined to cover the entire nation. Census tracts are small, relatively permanent statistical subdivisions of a county or equivalent entity, and were defined by local participants as part of the 2010 Census Participant Statistical Areas Program. The Census Bureau delineated the census tracts in situations where no local participant existed or where all the potential participants declined to participate. The primary purpose of census tracts is to provide a stable set of geographic units for the presentation of census data and comparison back to previous decennial censuses. Census tracts generally have a population size between 1,200 and 8,000 people, with an optimum size of 4,000 people. When first delineated, census tracts were designed to be homogeneous with respect to population characteristics, economic status, and living conditions. The spatial size of census tracts varies widely depending on the density of settlement. Physical changes in street patterns caused by highway construction, new

Quantification of soil respiration in forest ecosystems across China

NASA Astrophysics Data System (ADS)

Song, Xinzhang; Peng, Changhui; Zhao, Zhengyong; Zhang, Zhiting; Guo, Baohua; Wang, Weifeng; Jiang, Hong; Zhu, Qiuan

2014-09-01

We collected 139 estimates of the annual forest soil CO2 flux and 173 estimates of the Q10 value (the temperature sensitivity) assembled from 90 published studies across Chinese forest ecosystems. We analyzed the annual soil respiration (Rs) rates and the temperature sensitivities of seven forest ecosystems, including evergreen broadleaf forests (EBF), deciduous broadleaf forests (DBF), broadleaf and needleleaf mixed forests (BNMF), evergreen needleleaf forests (ENF), deciduous needleleaf forests (DNF), bamboo forests (BF) and shrubs (SF). The results showed that the mean annual Rs rate was 33.65 t CO2 ha-1 year-1 across Chinese forest ecosystems. Rs rates were significantly different (P < 0.001) among the seven forest types, and were significantly and positively influenced by mean annual temperature (MAT), mean annual precipitation (MAP), and actual evapotranspiration (AET); but negatively affected by latitude and elevation. The mean Q10 value of 1.28 was lower than the world average (1.4-2.0). The Q10 values derived from the soil temperature at a depth of 5 cm varied among forest ecosystems by an average of 2.46 and significantly decreased with the MAT but increased with elevation and latitude. Moreover, our results suggested that an artificial neural network (ANN) model can effectively predict Rs across Chinese forest ecosystems. This study contributes to better understanding of Rs across Chinese forest ecosystems and their possible responses to global warming.

Nanoplatforms for highly sensitive fluorescence detection of cancer-related proteases.

PubMed

Wang, Hongwang; Udukala, Dinusha N; Samarakoon, Thilani N; Basel, Matthew T; Kalita, Mausam; Abayaweera, Gayani; Manawadu, Harshi; Malalasekera, Aruni; Robinson, Colette; Villanueva, David; Maynez, Pamela; Bossmann, Leonie; Riedy, Elizabeth; Barriga, Jenny; Wang, Ni; Li, Ping; Higgins, Daniel A; Zhu, Gaohong; Troyer, Deryl L; Bossmann, Stefan H

2014-02-01

Numerous proteases are known to be necessary for cancer development and progression including matrix metalloproteinases (MMPs), tissue serine proteases, and cathepsins. The goal of this research is to develop an Fe/Fe3O4 nanoparticle-based system for clinical diagnostics, which has the potential to measure the activity of cancer-associated proteases in biospecimens. Nanoparticle-based "light switches" for measuring protease activity consist of fluorescent cyanine dyes and porphyrins that are attached to Fe/Fe3O4 nanoparticles via consensus sequences. These consensus sequences can be cleaved in the presence of the correct protease, thus releasing a fluorescent dye from the Fe/Fe3O4 nanoparticle, resulting in highly sensitive (down to 1 × 10(-16) mol l(-1) for 12 proteases), selective, and fast nanoplatforms (required time: 60 min).

A method to determine fault vectors in 4H-SiC from stacking sequences observed on high resolution transmission electron microscopy images

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

Wu, Fangzhen; Wang, Huanhuan; Raghothamachar, Balaji

A new method has been developed to determine the fault vectors associated with stacking faults in 4H-SiC from their stacking sequences observed on high resolution TEM images. This method, analogous to the Burgers circuit technique for determination of dislocation Burgers vector, involves determination of the vectors required in the projection of the perfect lattice to correct the deviated path constructed in the faulted material. Results for several different stacking faults were compared with fault vectors determined from X-ray topographic contrast analysis and were found to be consistent. This technique is expected to applicable to all structures comprising corner shared tetrahedra.

77 FR 65537 - Requirements for Patent Applications Containing Nucleotide Sequence and/or Amino Acid Sequence...

Federal Register 2010, 2011, 2012, 2013, 2014

2012-10-29

... DEPARTMENT OF COMMERCE Patent and Trademark Office Requirements for Patent Applications Containing Nucleotide Sequence and/or Amino Acid Sequence Disclosures ACTION: Proposed collection; comment request... Patent applications that contain nucleotide and/or amino acid sequence disclosures must include a copy of...

Genomics and introgression: discovery and mapping of thousands of species-diagnostic SNPs using RAD sequencing

USGS Publications Warehouse

Hand, Brian K.; Hether, Tyler D; Kovach, Ryan P.; Muhlfeld, Clint C.; Amish, Stephen J.; Boyer, Matthew C.; O’Rourke, Sean M.; Miller, Michael R.; Lowe, Winsor H.; Hohenlohe, Paul A.; Luikart, Gordon

2015-01-01

Invasive hybridization and introgression pose a serious threat to the persistence of many native species. Understanding the effects of hybridization on native populations (e.g., fitness consequences) requires numerous species-diagnostic loci distributed genome-wide. Here we used RAD sequencing to discover thousands of single-nucleotide polymorphisms (SNPs) that are diagnostic between rainbow trout (RBT, Oncorhynchus mykiss), the world’s most widely introduced fish, and native westslope cutthroat trout (WCT, O. clarkii lewisi) in the northern Rocky Mountains, USA. We advanced previous work that identified 4,914 species-diagnostic loci by using longer sequence reads (100 bp vs. 60 bp) and a larger set of individuals (n = 84). We sequenced RAD libraries for individuals from diverse sampling sources, including native populations of WCT and hatchery broodstocks of WCT and RBT. We also took advantage of a newly released reference genome assembly for RBT to align our RAD loci. In total, we discovered 16,788 putatively diagnostic SNPs, 10,267 of which we mapped to anchored chromosome locations on the RBT genome. A small portion of previously discovered putative diagnostic loci (325 of 4,914) were no longer diagnostic (i.e., fixed between species) based on our wider survey of non-hybridized RBT and WCT individuals. Our study suggests that RAD loci mapped to a draft genome assembly could provide the marker density required to identify genes and chromosomal regions influencing selection in admixed populations of conservation concern and evolutionary interest.

A novel class of plant-specific zinc-dependent DNA-binding protein that binds to A/T-rich DNA sequences

PubMed Central

Nagano, Yukio; Furuhashi, Hirofumi; Inaba, Takehito; Sasaki, Yukiko

2001-01-01

Complementary DNA encoding a DNA-binding protein, designated PLATZ1 (plant AT-rich sequence- and zinc-binding protein 1), was isolated from peas. The amino acid sequence of the protein is similar to those of other uncharacterized proteins predicted from the genome sequences of higher plants. However, no paralogous sequences have been found outside the plant kingdom. Multiple alignments among these paralogous proteins show that several cysteine and histidine residues are invariant, suggesting that these proteins are a novel class of zinc-dependent DNA-binding proteins with two distantly located regions, C-x2-H-x11-C-x2-C-x(4–5)-C-x2-C-x(3–7)-H-x2-H and C-x2-C-x(10–11)-C-x3-C. In an electrophoretic mobility shift assay, the zinc chelator 1,10-o-phenanthroline inhibited DNA binding, and two distant zinc-binding regions were required for DNA binding. A protein blot with 65ZnCl2 showed that both regions are required for zinc-binding activity. The PLATZ1 protein non-specifically binds to A/T-rich sequences, including the upstream region of the pea GTPase pra2 and plastocyanin petE genes. Expression of the PLATZ1 repressed those of the reporter constructs containing the coding sequence of luciferase gene driven by the cauliflower mosaic virus (CaMV) 35S90 promoter fused to the tandem repeat of the A/T-rich sequences. These results indicate that PLATZ1 is a novel class of plant-specific zinc-dependent DNA-binding protein responsible for A/T-rich sequence-mediated transcriptional repression. PMID:11600698

A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function.

PubMed

Catling, A D; Schaeffer, H J; Reuter, C W; Reddy, G R; Weber, M J

1995-10-01

Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs.

A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function.

PubMed Central

Catling, A D; Schaeffer, H J; Reuter, C W; Reddy, G R; Weber, M J

1995-01-01

Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs. PMID:7565670

Molecular cloning of human protein 4.2: a major component of the erythrocyte membrane.

PubMed Central

Sung, L A; Chien, S; Chang, L S; Lambert, K; Bliss, S A; Bouhassira, E E; Nagel, R L; Schwartz, R S; Rybicki, A C

1990-01-01

Protein 4.2 (P4.2) comprises approximately 5% of the protein mass of human erythrocyte (RBC) membranes. Anemia occurs in patients with RBCs deficient in P4.2, suggesting a role for this protein in maintaining RBC stability and integrity. We now report the molecular cloning and characterization of human RBC P4.2 cDNAs. By immunoscreening a human reticulocyte cDNA library and by using the polymerase chain reaction, two cDNA sequences of 2.4 and 2.5 kilobases (kb) were obtained. These cDNAs differ only by a 90-base-pair insert in the longer isoform located three codons downstream from the putative initiation site. The 2.4- and 2.5-kb cDNAs predict proteins of approximately 77 and approximately 80 kDa, respectively, and the authenticity was confirmed by sequence identity with 46 amino acids of three cyanogen bromide-cleaved peptides of P4.2. Northern blot analysis detected a major 2.4-kb RNA species in reticulocytes. Isolation of two P4.2 cDNAs implies existence of specific regulation of P4.2 expression in human RBCs. Human RBC P4.2 has significant homology with human factor XIII subunit a and guinea pig liver transglutaminase. Sequence alignment of P4.2 with these two transglutaminases, however, revealed that P4.2 lacks the critical cysteine residue required for the enzymatic crosslinking of substrates. Images PMID:1689063

Do you remember where sounds, pictures and words came from? The role of the stimulus format in object location memory.

PubMed

Delogu, Franco; Lilla, Christopher C

2017-11-01

Contrasting results in visual and auditory spatial memory stimulate the debate over the role of sensory modality and attention in identity-to-location binding. We investigated the role of sensory modality in the incidental/deliberate encoding of the location of a sequence of items. In 4 separated blocks, 88 participants memorised sequences of environmental sounds, spoken words, pictures and written words, respectively. After memorisation, participants were asked to recognise old from new items in a new sequence of stimuli. They were also asked to indicate from which side of the screen (visual stimuli) or headphone channel (sounds) the old stimuli were presented in encoding. In the first block, participants were not aware of the spatial requirement while, in blocks 2, 3 and 4 they knew that their memory for item location was going to be tested. Results show significantly lower accuracy of object location memory for the auditory stimuli (environmental sounds and spoken words) than for images (pictures and written words). Awareness of spatial requirement did not influence localisation accuracy. We conclude that: (a) object location memory is more effective for visual objects; (b) object location is implicitly associated with item identity during encoding and (c) visual supremacy in spatial memory does not depend on the automaticity of object location binding.

Efficient algorithms for accurate hierarchical clustering of huge datasets: tackling the entire protein space.

PubMed

Loewenstein, Yaniv; Portugaly, Elon; Fromer, Menachem; Linial, Michal

2008-07-01

UPGMA (average linking) is probably the most popular algorithm for hierarchical data clustering, especially in computational biology. However, UPGMA requires the entire dissimilarity matrix in memory. Due to this prohibitive requirement, UPGMA is not scalable to very large datasets. We present a novel class of memory-constrained UPGMA (MC-UPGMA) algorithms. Given any practical memory size constraint, this framework guarantees the correct clustering solution without explicitly requiring all dissimilarities in memory. The algorithms are general and are applicable to any dataset. We present a data-dependent characterization of hardness and clustering efficiency. The presented concepts are applicable to any agglomerative clustering formulation. We apply our algorithm to the entire collection of protein sequences, to automatically build a comprehensive evolutionary-driven hierarchy of proteins from sequence alone. The newly created tree captures protein families better than state-of-the-art large-scale methods such as CluSTr, ProtoNet4 or single-linkage clustering. We demonstrate that leveraging the entire mass embodied in all sequence similarities allows to significantly improve on current protein family clusterings which are unable to directly tackle the sheer mass of this data. Furthermore, we argue that non-metric constraints are an inherent complexity of the sequence space and should not be overlooked. The robustness of UPGMA allows significant improvement, especially for multidomain proteins, and for large or divergent families. A comprehensive tree built from all UniProt sequence similarities, together with navigation and classification tools will be made available as part of the ProtoNet service. A C++ implementation of the algorithm is available on request.

Characterization of genetic elements required for site-specific integration of Lactobacillus delbrueckii subsp. bulgaricus bacteriophage mv4 and construction of an integration-proficient vector for Lactobacillus plantarum.

PubMed Central

Dupont, L; Boizet-Bonhoure, B; Coddeville, M; Auvray, F; Ritzenthaler, P

1995-01-01

Temperate phage mv4 integrates its DNA into the chromosome of Lactobacillus delbrueckii subsp. bulgaricus strains via site-specific recombination. Nucleotide sequencing of a 2.2-kb attP-containing phage fragment revealed the presence of four open reading frames. The larger open reading frame, close to the attP site, encoded a 427-amino-acid polypeptide with similarity in its C-terminal domain to site-specific recombinases of the integrase family. Comparison of the sequences of attP, bacterial attachment site attB, and host-phage junctions attL and attR identified a 17-bp common core sequence, where strand exchange occurs during recombination. Analysis of the attB sequence indicated that the core region overlaps the 3' end of a tRNA(Ser) gene. Phage mv4 DNA integration into the tRNA(Ser) gene preserved an intact tRNA(Ser) gene at the attL site. An integration vector based on the mv4 attP site and int gene was constructed. This vector transforms a heterologous host, L. plantarum, through site-specific integration into the tRNA(Ser) gene of the genome and will be useful for development of an efficient integration system for a number of additional bacterial species in which an identical tRNA gene is present. PMID:7836291

Effects of learning duration on implicit transfer.

PubMed

Tanaka, Kanji; Watanabe, Katsumi

2015-10-01

Implicit learning and transfer in sequence acquisition play important roles in daily life. Several previous studies have found that even when participants are not aware that a transfer sequence has been transformed from the learning sequence, they are able to perform the transfer sequence faster and more accurately; this suggests implicit transfer of visuomotor sequences. Here, we investigated whether implicit transfer could be modulated by the number of trials completed in a learning session. Participants learned a sequence through trial and error, known as the m × n task (Hikosaka et al. in J Neurophysiol 74:1652-1661, 1995). In the learning session, participants were required to successfully perform the same sequence 4, 12, 16, or 20 times. In the transfer session, participants then learned one of two other sequences: one where the button configuration Vertically Mirrored the learning sequence, or a randomly generated sequence. Our results show that even when participants did not notice the alternation rule (i.e., vertical mirroring), their total working time was less and their total number of errors was lower in the transfer session compared with those who performed a Random sequence, irrespective of the number of trials completed in the learning session. This result suggests that implicit transfer likely occurs even over a shorter learning duration.

Developmental and Subcellular Organization of Single-Cell C₄ Photosynthesis in Bienertia sinuspersici Determined by Large-Scale Proteomics and cDNA Assembly from 454 DNA Sequencing.

PubMed

Offermann, Sascha; Friso, Giulia; Doroshenk, Kelly A; Sun, Qi; Sharpe, Richard M; Okita, Thomas W; Wimmer, Diana; Edwards, Gerald E; van Wijk, Klaas J

2015-05-01

Kranz C4 species strictly depend on separation of primary and secondary carbon fixation reactions in different cell types. In contrast, the single-cell C4 (SCC4) species Bienertia sinuspersici utilizes intracellular compartmentation including two physiologically and biochemically different chloroplast types; however, information on identity, localization, and induction of proteins required for this SCC4 system is currently very limited. In this study, we determined the distribution of photosynthesis-related proteins and the induction of the C4 system during development by label-free proteomics of subcellular fractions and leaves of different developmental stages. This was enabled by inferring a protein sequence database from 454 sequencing of Bienertia cDNAs. Large-scale proteome rearrangements were observed as C4 photosynthesis developed during leaf maturation. The proteomes of the two chloroplasts are different with differential accumulation of linear and cyclic electron transport components, primary and secondary carbon fixation reactions, and a triose-phosphate shuttle that is shared between the two chloroplast types. This differential protein distribution pattern suggests the presence of a mRNA or protein-sorting mechanism for nuclear-encoded, chloroplast-targeted proteins in SCC4 species. The combined information was used to provide a comprehensive model for NAD-ME type carbon fixation in SCC4 species.

From Sequences to Insights in Microbial Ecology

PubMed Central

Knight, R.

2010-01-01

s4-3 Rapid declines in the cost of sequencing have made large volumes of DNA sequence data available to individual investigators. Now, data analysis is the rate-limiting step: providing a user with sequences alone typically leads to bewilderment, frustration, and skepticism about the technology. In this talk, I focus on how to extract insights from 16S rRNA data, including key lab steps (barcoding and normalization) and on which tools are available to perform routine but essential processing steps such as denoising, chimera detection, taxonomy assignment, and diversity analyses (including detection of biological clusters and gradients in the samples). Providing users with advice on these points and with a standard pipeline they can exploit (but modify if circumstances require) can greatly accelerate the rate of understanding, publication, and acquisition of funding for further studies.

A versatile pulse programmer for pulsed nuclear magnetic resonance spectroscopy.

NASA Technical Reports Server (NTRS)

Tarr, C. E.; Nickerson, M. A.

1972-01-01

A digital pulse programmer producing the standard pulse sequences required for pulsed nuclear magnetic resonance spectroscopy is described. In addition, a 'saturation burst' sequence, useful in the measurement of long relaxation times in solids, is provided. Both positive and negative 4 V trigger pulses are produced that are fully synchronous with a crystal-controlled time base, and the pulse programmer may be phase-locked with a maximum pulse jitter of 3 ns to the oscillator of a coherent pulse spectrometer. Medium speed TTL integrated circuits are used throughout.

Geochemical Constraints on the Distribution and Function of Thermoproteales Populations in Yellowstone National Park

NASA Astrophysics Data System (ADS)

Jay, Z.; Rusch, D.; Romine, M.; Beam, J.; Inskeep, W.

2014-12-01

Metagenome surveys in Yellowstone National Park (YNP) indicate that members of the order Thermoproteales (phylum Crenarchaeota) are abundant in high-temperature (> 70 °C) geothermal systems. The goals of this study were to compare Thermoproteales sequences from different geothermal environments across YNP, and determine the variation in metabolic potential associated with their distribution. Thermoproteales sequence assemblies (> 0.5 Mbases) were curated from 10 habitats ranging in pH from 3 - 9 (with or without dissolved sulfide). The distribution of specific Thermoproteales is constrained by pH: Vulcanisaeta-like sequences are the most abundant Thermoproteales at pH < 6, Caldivirga-like sequences more important from pH 4 - 6, and Thermoproteus-like sequences abundant from ~ pH 5 - 7, and at pH > 7, Pyrobaculum­-like sequences are nearly the only Thermoproteales present. Thermoproteales populations are generally found in hypoxic systems where reduced forms of S and As often limit concentrations of dissolved oxygen. These environmental conditions are correlated with the presence or absence of system-defined respiratory complexes including different terminal oxidases (e.g., aa3 or bd), numerous DMSO-molybdopterins, and dissimilatory sulfate reductases. Metabolic reconstruction of different genera revealed similar pathways for the degradation of carbohydrates, amino acids, and lipids across sites. Only the Thermoproteus and Pyrobaculum populations contained the three marker genes for the dicarboxylate/4-hyhdroxybutyrate cycle, which is responsible for the fixation of inorganic carbon. Most Thermoproteales populations have the metabolic capacity to synthesize their requirements for vitamins, cofactors, amino acids, and/or nucleotides. Our results indicate that Thermoproteales populations are important members of high-temperature microbial communities across a wide pH range, are responsible for the degradation of organic carbon, and may also serve as a source of metabolites required by other community members. Thermoproteales genera are abundant thermophiles in many hypoxic (and especially sulfidic) systems; however, the presence of introns in the 16S rRNA gene of many Thermoproteales often precludes accurate abundance estimates using universal primers.

Structural evolution of the 4/1 genes and proteins in non-vascular and lower vascular plants.

PubMed

Morozov, Sergey Y; Milyutina, Irina A; Bobrova, Vera K; Ryazantsev, Dmitry Y; Erokhina, Tatiana N; Zavriev, Sergey K; Agranovsky, Alexey A; Solovyev, Andrey G; Troitsky, Alexey V

2015-12-01

The 4/1 protein of unknown function is encoded by a single-copy gene in most higher plants. The 4/1 protein of Nicotiana tabacum (Nt-4/1 protein) has been shown to be alpha-helical and predominantly expressed in conductive tissues. Here, we report the analysis of 4/1 genes and the encoded proteins of lower land plants. Sequences of a number of 4/1 genes from liverworts, lycophytes, ferns and gymnosperms were determined and analyzed together with sequences available in databases. Most of the vascular plants were found to encode Magnoliophyta-like 4/1 proteins exhibiting previously described gene structure and protein properties. Identification of the 4/1-like proteins in hornworts, liverworts and charophyte algae (sister lineage to all land plants) but not in mosses suggests that 4/1 proteins are likely important for plant development but not required for a primary metabolic function of plant cell. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

A space-efficient algorithm for local similarities.

PubMed

Huang, X Q; Hardison, R C; Miller, W

1990-10-01

Existing dynamic-programming algorithms for identifying similar regions of two sequences require time and space proportional to the product of the sequence lengths. Often this space requirement is more limiting than the time requirement. We describe a dynamic-programming local-similarity algorithm that needs only space proportional to the sum of the sequence lengths. The method can also find repeats within a single long sequence. To illustrate the algorithm's potential, we discuss comparison of a 73,360 nucleotide sequence containing the human beta-like globin gene cluster and a corresponding 44,594 nucleotide sequence for rabbit, a problem well beyond the capabilities of other dynamic-programming software.

49 CFR 236.1011 - PTC Implementation Plan content requirements.

Code of Federal Regulations, 2010 CFR

2010-10-01

... the following risk factors by track segment: (i) Segment traffic characteristics such as typical...; (4) How, to the extent practical, the PTC system will be implemented to address areas of greater risk to the public and railroad employees before areas of lesser risk; (5) The sequence and schedule in...

49 CFR 173.62 - Specific packaging requirements for explosives.

Code of Federal Regulations, 2013 CFR

2013-10-01

... first column in numerical sequence by their identification number (ID #), which is listed in column 4 of the § 172.101 table, of this subchapter. The second column of the Explosives Table specifies the...) Explosives must be packaged in accordance with the following table: (1) The first column lists, in...

RDE-4 preferentially binds long dsRNA and its dimerization is necessary for cleavage of dsRNA to siRNA.

PubMed

Parker, Greg S; Eckert, Debra M; Bass, Brenda L

2006-05-01

In organisms ranging from Arabidopsis to humans, Dicer requires dsRNA-binding proteins (dsRBPs) to carry out its roles in RNA interference (RNAi) and micro-RNA (miRNA) processing. In Caenorhabditis elegans, the dsRBP RDE-4 acts with Dicer during the initiation of RNAi, when long dsRNA is cleaved to small interfering RNAs (siRNAs). RDE-4 is not required in subsequent steps, and how RDE-4 distinguishes between long dsRNA and short siRNA is unclear. We report the first detailed analysis of RDE-4 binding, using purified recombinant RDE-4 and various truncated proteins. We find that, similar to other dsRBPs, RDE-4 is not sequence-specific. However, consistent with its in vivo roles, RDE-4 binds with higher affinity to long dsRNA. We also observe that RDE-4 is a homodimer in solution, and that the C-terminal domain of the protein is required for dimerization. Using extracts from wild-type and rde-4 mutant C. elegans, we show that the C-terminal dimerization domain is required for the production of siRNA. Our findings suggest a model for RDE-4 function during the initiation of RNAi.

RDE-4 preferentially binds long dsRNA and its dimerization is necessary for cleavage of dsRNA to siRNA

PubMed Central

Parker, Greg S.; Eckert, Debra M.; Bass, Brenda L.

2006-01-01

In organisms ranging from Arabidopsis to humans, Dicer requires dsRNA-binding proteins (dsRBPs) to carry out its roles in RNA interference (RNAi) and micro-RNA (miRNA) processing. In Caenorhabditis elegans, the dsRBP RDE-4 acts with Dicer during the initiation of RNAi, when long dsRNA is cleaved to small interfering RNAs (siRNAs). RDE-4 is not required in subsequent steps, and how RDE-4 distinguishes between long dsRNA and short siRNA is unclear. We report the first detailed analysis of RDE-4 binding, using purified recombinant RDE-4 and various truncated proteins. We find that, similar to other dsRBPs, RDE-4 is not sequence-specific. However, consistent with its in vivo roles, RDE-4 binds with higher affinity to long dsRNA. We also observe that RDE-4 is a homodimer in solution, and that the C-terminal domain of the protein is required for dimerization. Using extracts from wild-type and rde-4 mutant C. elegans, we show that the C-terminal dimerization domain is required for the production of siRNA. Our findings suggest a model for RDE-4 function during the initiation of RNAi. PMID:16603715

Simultaneous two-voxel localized 1H-observed 13C-edited spectroscopy for in vivo MRS on rat brain at 9.4 T: Application to the investigation of excitotoxic lesions

NASA Astrophysics Data System (ADS)

Doan, Bich-Thuy; Autret, Gwennhael; Mispelter, Joël; Méric, Philippe; Même, William; Montécot-Dubourg, Céline; Corrèze, Jean-Loup; Szeremeta, Frédéric; Gillet, Brigitte; Beloeil, Jean-Claude

2009-05-01

13C spectroscopy combined with the injection of 13C-labeled substrates is a powerful method for the study of brain metabolism in vivo. Since highly localized measurements are required in a heterogeneous organ such as the brain, it is of interest to augment the sensitivity of 13C spectroscopy by proton acquisition. Furthermore, as focal cerebral lesions are often encountered in animal models of disorders in which the two brain hemispheres are compared, we wished to develop a bi-voxel localized sequence for the simultaneous bilateral investigation of rat brain metabolism, with no need for external additional references. Two sequences were developed at 9.4 T: a bi-voxel 1H-( 13C) STEAM-POCE (Proton Observed Carbon Edited) sequence and a bi-voxel 1H-( 13C) PRESS-POCE adiabatically decoupled sequence with Hadamard encoding. Hadamard encoding allows both voxels to be recorded simultaneously, with the same acquisition time as that required for a single voxel. The method was validated in a biological investigation into the neuronal damage and the effect on the Tri Carboxylic Acid cycle in localized excitotoxic lesions. Following an excitotoxic quinolinate-induced localized lesion in the rat cortex and the infusion of U- 13C glucose, two 1H-( 13C) spectra of distinct (4 × 4 × 4 mm 3) voxels, one centred on the injured hemisphere and the other on the contralateral hemisphere, were recorded simultaneously. Two 1H bi-voxel spectra were also recorded and showed a significant decrease in N-acetyl aspartate, and an accumulation of lactate in the ipsilateral hemisphere. The 1H-( 13C) spectra could be recorded dynamically as a function of time, and showed a fall in the glutamate/glutamine ratio and the presence of a stable glutamine pool, with a permanent increase of lactate in the ipsilateral hemisphere. This bi-voxel 1H-( 13C) method can be used to investigate simultaneously both brain hemispheres, and to perform dynamic studies. We report here the neuronal damage and the effect on the Tri Carboxylic Acid cycle in localized excitotoxic lesions.

Ebola virus RNA editing depends on the primary editing site sequence and an upstream secondary structure.

PubMed

Mehedi, Masfique; Hoenen, Thomas; Robertson, Shelly; Ricklefs, Stacy; Dolan, Michael A; Taylor, Travis; Falzarano, Darryl; Ebihara, Hideki; Porcella, Stephen F; Feldmann, Heinz

2013-01-01

Ebolavirus (EBOV), the causative agent of a severe hemorrhagic fever and a biosafety level 4 pathogen, increases its genome coding capacity by producing multiple transcripts encoding for structural and nonstructural glycoproteins from a single gene. This is achieved through RNA editing, during which non-template adenosine residues are incorporated into the EBOV mRNAs at an editing site encoding for 7 adenosine residues. However, the mechanism of EBOV RNA editing is currently not understood. In this study, we report for the first time that minigenomes containing the glycoprotein gene editing site can undergo RNA editing, thereby eliminating the requirement for a biosafety level 4 laboratory to study EBOV RNA editing. Using a newly developed dual-reporter minigenome, we have characterized the mechanism of EBOV RNA editing, and have identified cis-acting sequences that are required for editing, located between 9 nt upstream and 9 nt downstream of the editing site. Moreover, we show that a secondary structure in the upstream cis-acting sequence plays an important role in RNA editing. EBOV RNA editing is glycoprotein gene-specific, as a stretch encoding for 7 adenosine residues located in the viral polymerase gene did not serve as an editing site, most likely due to an absence of the necessary cis-acting sequences. Finally, the EBOV protein VP30 was identified as a trans-acting factor for RNA editing, constituting a novel function for this protein. Overall, our results provide novel insights into the RNA editing mechanism of EBOV, further understanding of which might result in novel intervention strategies against this viral pathogen.

Grundlegende Steuerungsverfahren im heterogenen Logistiknetz mit Kanban

NASA Astrophysics Data System (ADS)

Dickmann, Eva; Dickmann, Philipp; Lödding, Hermann; Möller, Niklas; Rücker, Thomas; Schneider, Herfried M.; Zäh, Michael F.

In vielen Unternehmen werden heterogene (verschiedene) Steuerungen in einem abgestimmten Konzept kombiniert. Je nach Anwendungsfall und Rahmenbedingungen werden Kombinationen allgemein bekannter Steuerungen oder Steuerungsvarianten gemischt eingesetzt, um eine optimale Steuerung für unterschiedliche Fälle zu erreichen. Hierbei stehen neben den bekannten und weit verbreiteten Methoden, wie Material Requirements Planning (MRP) oder Kanban, auch weniger bekannte oder neue Methoden zur Auswahl, wie die Produktionssteuerung mit dezentraler, bestandsorientierter Fertigungsregelung (DBF). Kanban ist ein simples und effizientes Steuerungskonzept, das in der klassischen Form für spezifische einfache Anwendungsfälle umsetzbar ist. Hochentwickelte Steuerungsalgorithmen können helfen, komplexe Abläufe optimal abzubilden. Mit einer grundlegenden Vereinfachung der Abläufe kann allerdings in vielen Fällen ein wesentlich stärkerer und umfassender Verbesserungseffekt erzielt werden. Die wesentliche Fragestellung sollte folglich lauten: Warum ist der Ablauf nicht mit einer einfachen Steuerung wie Kanban abzubilden? Um die Vorteile des Konzepts auch in untypischen Bereichen anwenden zu können, sind jedoch verschiedene Varianten oder Kanban-ähnliche Steuerungsmethoden entstanden. Darüber hinaus sind in der Praxis hybride Steuerungen im Einsatz, welche so kombiniert werden, dass die Zusammensetzung anspruchsvolle Eigenschaftsbilder noch exakt abbildet. In der Praxis basieren die Steuerungsentscheidungen nur zu einem kleinen Teil auf den eigentlichen Steuerungsalgorithmen, wie sie uns das MRP-System zur Verfügung stellt. Moderne Steuerungswelten" schließen alle relevanten Informationsquellen in eine heterogene Entscheidungsmatrix mit ein. Letztlich zählt nicht, ob die Entscheidung auf den Informationen aus dem MRP-System oder auf Softfacts basierend getroffen wurde, sondern nur, ob die Entscheidung erfolgreich war.

Haplotag: Software for Haplotype-Based Genotyping-by-Sequencing Analysis

PubMed Central

Tinker, Nicholas A.; Bekele, Wubishet A.; Hattori, Jiro

2016-01-01

Genotyping-by-sequencing (GBS), and related methods, are based on high-throughput short-read sequencing of genomic complexity reductions followed by discovery of single nucleotide polymorphisms (SNPs) within sequence tags. This provides a powerful and economical approach to whole-genome genotyping, facilitating applications in genomics, diversity analysis, and molecular breeding. However, due to the complexity of analyzing large data sets, applications of GBS may require substantial time, expertise, and computational resources. Haplotag, the novel GBS software described here, is freely available, and operates with minimal user-investment on widely available computer platforms. Haplotag is unique in fulfilling the following set of criteria: (1) operates without a reference genome; (2) can be used in a polyploid species; (3) provides a discovery mode, and a production mode; (4) discovers polymorphisms based on a model of tag-level haplotypes within sequenced tags; (5) reports SNPs as well as haplotype-based genotypes; and (6) provides an intuitive visual “passport” for each inferred locus. Haplotag is optimized for use in a self-pollinating plant species. PMID:26818073

Centromere-Like Regions in the Budding Yeast Genome

PubMed Central

Lefrançois, Philippe; Auerbach, Raymond K.; Yellman, Christopher M.; Roeder, G. Shirleen; Snyder, Michael

2013-01-01

Accurate chromosome segregation requires centromeres (CENs), the DNA sequences where kinetochores form, to attach chromosomes to microtubules. In contrast to most eukaryotes, which have broad centromeres, Saccharomyces cerevisiae possesses sequence-defined point CENs. Chromatin immunoprecipitation followed by sequencing (ChIP–Seq) reveals colocalization of four kinetochore proteins at novel, discrete, non-centromeric regions, especially when levels of the centromeric histone H3 variant, Cse4 (a.k.a. CENP-A or CenH3), are elevated. These regions of overlapping protein binding enhance the segregation of plasmids and chromosomes and have thus been termed Centromere-Like Regions (CLRs). CLRs form in close proximity to S. cerevisiae CENs and share characteristics typical of both point and regional CENs. CLR sequences are conserved among related budding yeasts. Many genomic features characteristic of CLRs are also associated with these conserved homologous sequences from closely related budding yeasts. These studies provide general and important insights into the origin and evolution of centromeres. PMID:23349633

Are Clay Minerals a Climate Constraint? A Review of Prior Data and New Insights on Martian "Weathering Sequences"

NASA Astrophysics Data System (ADS)

Ehlmann, B. L.; Dundar, M.

2016-12-01

Most clay minerals on Mars are Fe/Mg smectites or chlorites, which typically form from mafic protoliths in aqueous chemical systems that are relatively closed and thus require liquid water but not large amounts of water throughput and large-scale chemical leaching. They may thus form either in the subsurface or under select conditions at the surface. However, Al clay minerals, discovered in multiple locations on Mars (Arabia Terra, Northeast Syrtis, Libya Montes Terra Sirenum, Eridania, circum-Hellas, Valles Marineris) may provide evidence of substantial water throughput, if their protolith materials were basaltic. This is because formation of Al clays from a mafic protolith requires removal of Mg and either formation of accompanying Fe oxides or removal of Fe. Thus, the observed sequences of Al clays atop Fe/Mg clays were proposed to represent open system weathering and possibly a late climate optimum around the late Noachian/early Hesperian [1]. Later, they were comprehensively cataloged and reported to represent "weathering sequences" similar to those in terrestrial tropical environments [2]. However, key questions remain; in particular, how much water throughput over what time scale is required? The answer to this question has substantial bearing on the climate of early Mars. Recently, we employed a newly developed, non-parametric Bayesian algorithm [3,4] for semi-automatic identification of rare spectral classes on 139 CRISM images in areas with reported regional-scale occurrences of Al clays. Dozens of detections of the minerals alunite and jarosite were made with the algorithm and then verified by manual analysis. These sulfate hydroxides form only at low pHs, and thus their presence tightly constrains water chemistry. Here, we discuss the evidence for low pH surface waters associated with the weathering sequences and their implications for the cumulative duration of surface weathering. [1] Ehlmann et al., 2011, Nature | [2] Carter et al., 2015, Icarus | [3] Dundar et al., 2016, IEEE WHISPERS proceedings | [4] Ehlmann & Dundar, submitted

Major- and trace-element concentrations in rock samples collected in 2004 from the Taylor Mountains 1:250,000-scale quadrangle, Alaska

USGS Publications Warehouse

Klimasauskas, Edward P.; Miller, Marti L.; Bradley, Dwight C.; Karl, Sue M.; Baichtal, James F.; Blodgett, Robert B.

2006-01-01

The Kuskokwim mineral belt of Bundtzen and Miller (1997) forms an important metallogenic region in southwestern Alaska that has yielded more than 3.22 million ounces of gold and 400,000 ounces of silver. Precious-metal and related deposits in this region associated with Late Cretaceous to early Tertiary igneous complexes extend into the Taylor Mountains 1:250,000-scale quadrangle. The U.S. Geological Survey is conducting geologic mapping and a mineral resource assessment of this area that will provide a better understanding of the geologic framework, regional geochemistry, and may provide targets for mineral exploration and development. During the 2004 field season 137 rock samples were collected for a variety of purposes. The 4 digital files accompanying this report reflect the type of analysis performed and its intended purpose and are available for download as an Excel workbook, comma delimited format (*.csv), dBase 4 files (*.dbf) or as point coverages in ArcInfo interchange format (*.e00). Data values are provided in percent, pct (1gram per 100grams), or parts per million, ppm (1gram per 1,000,000grams) per the column heading in the table. All samples were analyzed for a suite of 42 trace-elements (icp42.*) to provide data for use in geochemical exploration as well as some baseline data. Selected samples were analyzed by additional methods; 104 targeted geochemical exploration samples were analyzed for gold, arsenic, and mercury (auashg.*); 21 of these samples were also analyzed to obtain concentrations of 10 loosely bound metals (icp10.*); 33 rock samples were analyzed for major element oxides to support the regional mapping program (reg.*), of which 28 sedimentary rock samples were also analyzed for total carbon, and carbonate carbon.

Single Spatial-Mode Room-Temperature-Operated 3.0 to 3.4 micrometer Diode Lasers

NASA Technical Reports Server (NTRS)

Frez, Clifford F.; Soibel, Alexander; Belenky, Gregory; Shterengas, Leon; Kipshidze, Gela

2010-01-01

Compact, highly efficient, 3.0 to 3.4 m light emitters are in demand for spectroscopic analysis and identification of chemical substances (including methane and formaldehyde), infrared countermeasures technologies, and development of advanced infrared scene projectors. The need for these light emitters can be currently addressed either by bulky solid-state light emitters with limited power conversion efficiency, or cooled Interband Cascade (IC) semiconductor lasers. Researchers here have developed a breakthrough approach to fabrication of diode mid-IR lasers that have several advantages over IC lasers used for the Mars 2009 mission. This breakthrough is due to a novel design utilizing the strain-engineered quantum-well (QW) active region and quinternary barriers, and due to optimization of device material composition and growth conditions (growth temperatures and rates). However, in their present form, these GaSb-based laser diodes cannot be directly used as a part of sensor systems. The device spectrum is too broad to perform spectroscopic analysis of gas species, and operating currents and voltages are too high. In the current work, the emitters were fabricated as narrow-ridge waveguide index-guided lasers rather than broad stripe-gain guided multimode Fabry-Perot (FP) lasers as was done previously. These narrow-ridge waveguide mid-IR lasers exhibit much lower power consumptions, and can operate in a single spatial mode that is necessary for demonstration of single-mode distributed feedback (DBF) devices for spectroscopic applications. These lasers will enable a new generation of compact, tunable diode laser spectrometers with lower power consumption, reduced complexity, and significantly reduced development costs. These lasers can be used for the detection of HCN, C2H2, methane, and ethane.

Efficient algorithms for accurate hierarchical clustering of huge datasets: tackling the entire protein space

PubMed Central

Loewenstein, Yaniv; Portugaly, Elon; Fromer, Menachem; Linial, Michal

2008-01-01

Motivation: UPGMA (average linking) is probably the most popular algorithm for hierarchical data clustering, especially in computational biology. However, UPGMA requires the entire dissimilarity matrix in memory. Due to this prohibitive requirement, UPGMA is not scalable to very large datasets. Application: We present a novel class of memory-constrained UPGMA (MC-UPGMA) algorithms. Given any practical memory size constraint, this framework guarantees the correct clustering solution without explicitly requiring all dissimilarities in memory. The algorithms are general and are applicable to any dataset. We present a data-dependent characterization of hardness and clustering efficiency. The presented concepts are applicable to any agglomerative clustering formulation. Results: We apply our algorithm to the entire collection of protein sequences, to automatically build a comprehensive evolutionary-driven hierarchy of proteins from sequence alone. The newly created tree captures protein families better than state-of-the-art large-scale methods such as CluSTr, ProtoNet4 or single-linkage clustering. We demonstrate that leveraging the entire mass embodied in all sequence similarities allows to significantly improve on current protein family clusterings which are unable to directly tackle the sheer mass of this data. Furthermore, we argue that non-metric constraints are an inherent complexity of the sequence space and should not be overlooked. The robustness of UPGMA allows significant improvement, especially for multidomain proteins, and for large or divergent families. Availability: A comprehensive tree built from all UniProt sequence similarities, together with navigation and classification tools will be made available as part of the ProtoNet service. A C++ implementation of the algorithm is available on request. Contact: lonshy@cs.huji.ac.il PMID:18586742

40 CFR 86.1230-96 - Test sequence; general requirements.

Code of Federal Regulations, 2010 CFR

2010-07-01

... Petroleum Gas-Fueled and Methanol-Fueled Heavy-Duty Vehicles § 86.1230-96 Test sequence; general requirements. (a)(1) Gasoline- and methanol-fueled vehicles. The test sequence shown in figure M96-1 of this...

40 CFR 86.1230-96 - Test sequence; general requirements.

Code of Federal Regulations, 2011 CFR

2011-07-01

... Petroleum Gas-Fueled and Methanol-Fueled Heavy-Duty Vehicles § 86.1230-96 Test sequence; general requirements. (a)(1) Gasoline- and methanol-fueled vehicles. The test sequence shown in figure M96-1 of this...

Selective recognition of N4-methylcytosine in DNA by engineered transcription-activator-like effectors.

PubMed

Rathi, Preeti; Maurer, Sara; Summerer, Daniel

2018-06-05

The epigenetic DNA nucleobases 5-methylcytosine (5mC) and N 4-methylcytosine (4mC) coexist in bacterial genomes and have important functions in host defence and transcription regulation. To better understand the individual biological roles of both methylated nucleobases, analytical strategies for distinguishing unmodified cytosine (C) from 4mC and 5mC are required. Transcription-activator-like effectors (TALEs) are programmable DNA-binding repeat proteins, which can be re-engineered for the direct detection of epigenetic nucleobases in user-defined DNA sequences. We here report the natural, cytosine-binding TALE repeat to not strongly differentiate between 5mC and 4mC. To engineer repeats with selectivity in the context of C, 5mC and 4mC, we developed a homogeneous fluorescence assay and screened a library of size-reduced TALE repeats for binding to all three nucleobases. This provided insights into the requirements of size-reduced TALE repeats for 4mC binding and revealed a single mutant repeat as a selective binder of 4mC. Employment of a TALE with this repeat in affinity enrichment enabled the isolation of a user-defined DNA sequence containing a single 4mC but not C or 5mC from the background of a bacterial genome. Comparative enrichments with TALEs bearing this or the natural C-binding repeat provides an approach for the complete, programmable decoding of all cytosine nucleobases found in bacterial genomes.This article is part of a discussion meeting issue 'Frontiers in epigenetic chemical biology'. © 2018 The Author(s).

A Simple and Efficient Method for Assembling TALE Protein Based on Plasmid Library

PubMed Central

Xu, Huarong; Xin, Ying; Zhang, Tingting; Ma, Lixia; Wang, Xin; Chen, Zhilong; Zhang, Zhiying

2013-01-01

DNA binding domain of the transcription activator-like effectors (TALEs) from Xanthomonas sp. consists of tandem repeats that can be rearranged according to a simple cipher to target new DNA sequences with high DNA-binding specificity. This technology has been successfully applied in varieties of species for genome engineering. However, assembling long TALE tandem repeats remains a big challenge precluding wide use of this technology. Although several new methodologies for efficiently assembling TALE repeats have been recently reported, all of them require either sophisticated facilities or skilled technicians to carry them out. Here, we described a simple and efficient method for generating customized TALE nucleases (TALENs) and TALE transcription factors (TALE-TFs) based on TALE repeat tetramer library. A tetramer library consisting of 256 tetramers covers all possible combinations of 4 base pairs. A set of unique primers was designed for amplification of these tetramers. PCR products were assembled by one step of digestion/ligation reaction. 12 TALE constructs including 4 TALEN pairs targeted to mouse Gt(ROSA)26Sor gene and mouse Mstn gene sequences as well as 4 TALE-TF constructs targeted to mouse Oct4, c-Myc, Klf4 and Sox2 gene promoter sequences were generated by using our method. The construction routines took 3 days and parallel constructions were available. The rate of positive clones during colony PCR verification was 64% on average. Sequencing results suggested that all TALE constructs were performed with high successful rate. This is a rapid and cost-efficient method using the most common enzymes and facilities with a high success rate. PMID:23840477

A simple and efficient method for assembling TALE protein based on plasmid library.

PubMed

Zhang, Zhiqiang; Li, Duo; Xu, Huarong; Xin, Ying; Zhang, Tingting; Ma, Lixia; Wang, Xin; Chen, Zhilong; Zhang, Zhiying

2013-01-01

DNA binding domain of the transcription activator-like effectors (TALEs) from Xanthomonas sp. consists of tandem repeats that can be rearranged according to a simple cipher to target new DNA sequences with high DNA-binding specificity. This technology has been successfully applied in varieties of species for genome engineering. However, assembling long TALE tandem repeats remains a big challenge precluding wide use of this technology. Although several new methodologies for efficiently assembling TALE repeats have been recently reported, all of them require either sophisticated facilities or skilled technicians to carry them out. Here, we described a simple and efficient method for generating customized TALE nucleases (TALENs) and TALE transcription factors (TALE-TFs) based on TALE repeat tetramer library. A tetramer library consisting of 256 tetramers covers all possible combinations of 4 base pairs. A set of unique primers was designed for amplification of these tetramers. PCR products were assembled by one step of digestion/ligation reaction. 12 TALE constructs including 4 TALEN pairs targeted to mouse Gt(ROSA)26Sor gene and mouse Mstn gene sequences as well as 4 TALE-TF constructs targeted to mouse Oct4, c-Myc, Klf4 and Sox2 gene promoter sequences were generated by using our method. The construction routines took 3 days and parallel constructions were available. The rate of positive clones during colony PCR verification was 64% on average. Sequencing results suggested that all TALE constructs were performed with high successful rate. This is a rapid and cost-efficient method using the most common enzymes and facilities with a high success rate.

Molecular diagnosis of putative Stargardt disease probands by exome sequencing

PubMed Central

2012-01-01

Background The commonest genetic form of juvenile or early adult onset macular degeneration is Stargardt Disease (STGD) caused by recessive mutations in the gene ABCA4. However, high phenotypic and allelic heterogeneity and a small but non-trivial amount of locus heterogeneity currently impede conclusive molecular diagnosis in a significant proportion of cases. Methods We performed whole exome sequencing (WES) of nine putative Stargardt Disease probands and searched for potentially disease-causing genetic variants in previously identified retinal or macular dystrophy genes. Follow-up dideoxy sequencing was performed for confirmation and to screen for mutations in an additional set of affected individuals lacking a definitive molecular diagnosis. Results Whole exome sequencing revealed seven likely disease-causing variants across four genes, providing a confident genetic diagnosis in six previously uncharacterized participants. We identified four previously missed mutations in ABCA4 across three individuals. Likely disease-causing mutations in RDS/PRPH2, ELOVL, and CRB1 were also identified. Conclusions Our findings highlight the enormous potential of whole exome sequencing in Stargardt Disease molecular diagnosis and research. WES adequately assayed all coding sequences and canonical splice sites of ABCA4 in this study. Additionally, WES enables the identification of disease-related alleles in other genes. This work highlights the importance of collecting parental genetic material for WES testing as the current knowledge of human genome variation limits the determination of causality between identified variants and disease. While larger sample sizes are required to establish the precision and accuracy of this type of testing, this study supports WES for inherited early onset macular degeneration disorders as an alternative to standard mutation screening techniques. PMID:22863181

Detailed Test Plan Redundant Sensor Strapdown IMU Evaluation Program

NASA Technical Reports Server (NTRS)

Hartwell, T.; Miyatake, Y.; Wedekind, D. E.

1971-01-01

The test plan for a redundant sensor strapdown inertial measuring unit evaluation program is presented. The subjects discussed are: (1) test philosophy and limitations, (2) test sequence, (3) equipment specifications, (4) general operating procedures, (5) calibration procedures, (6) alignment test phase, and (7) navigation test phase. The data and analysis requirements are analyzed.

The bifurcated stem loop 4 (SL4) is crucial for efficient packaging of mouse mammary tumor virus (MMTV) genomic RNA.

PubMed

Mustafa, Farah; Vivet-Boudou, Valérie; Jabeen, Ayesha; Ali, Lizna M; Kalloush, Rawan M; Marquet, Roland; Rizvi, Tahir A

2018-06-21

Packaging the mouse mammary tumor virus (MMTV) genomic RNA (gRNA) requires the entire 5' untranslated region (UTR) in conjunction with the first 120 nucleotides of the gag gene. This region includes several palindromic (pal) sequence(s) and stable stem loops (SLs). Among these, stem loop 4 (SL4) adopts a bifurcated structure consisting of three stems, two apical loops, and an internal loop. Pal II, located in one of the apical loops, mediates gRNA dimerization, a process intricately linked to packaging. We thus hypothesized that the bifurcated SL4 structure could constitute the major gRNA packaging determinant. To test this hypothesis, the two apical loops and the flanking sequences forming the bifurcated SL4 were individually mutated. These mutations all had deleterious effects on gRNA packaging and propagation. Next, single and compensatory mutants were designed to destabilize then recreate the bifurcated SL4 structure. A structure-function analysis using bioinformatics predictions and RNA chemical probing revealed that mutations that led to the loss of the SL4 bifurcated structure abrogated RNA packaging and propagation, while compensatory mutations that recreated the native SL4 structure restored RNA packaging and propagation to wild type levels. Altogether, our results demonstrate that SL4 constitutes the principal packaging determinant of MMTV gRNA. Our findings further suggest that SL4 acts as a structural switch that can not only differentiate between RNA for translation versus packaging/dimerization, but its location also allows differentiation between spliced and unspliced RNAs during gRNA encapsidation.

The standard operating procedure of the DOE-JGI Microbial Genome Annotation Pipeline (MGAP v.4).

PubMed

Huntemann, Marcel; Ivanova, Natalia N; Mavromatis, Konstantinos; Tripp, H James; Paez-Espino, David; Palaniappan, Krishnaveni; Szeto, Ernest; Pillay, Manoj; Chen, I-Min A; Pati, Amrita; Nielsen, Torben; Markowitz, Victor M; Kyrpides, Nikos C

2015-01-01

The DOE-JGI Microbial Genome Annotation Pipeline performs structural and functional annotation of microbial genomes that are further included into the Integrated Microbial Genome comparative analysis system. MGAP is applied to assembled nucleotide sequence datasets that are provided via the IMG submission site. Dataset submission for annotation first requires project and associated metadata description in GOLD. The MGAP sequence data processing consists of feature prediction including identification of protein-coding genes, non-coding RNAs and regulatory RNA features, as well as CRISPR elements. Structural annotation is followed by assignment of protein product names and functions.

A Study of the Comparative Effectiveness of Zoology Prerequisites at Slippery Rock State College.

ERIC Educational Resources Information Center

Morrison, William Sechler

This study compared the effectiveness of three sequences of prerequisite courses required before taking zoology. Sequence 1 prerequisite courses consisted of general biology and human biology; Sequence 2 consisted of general biology; and Sequence 3 required cell biology. Zoology students in the spring of 1972 were pretest and a posttest. The mean…

Hierarchical Traces for Reduced NSM Memory Requirements

NASA Astrophysics Data System (ADS)

Dahl, Torbjørn S.

This paper presents work on using hierarchical long term memory to reduce the memory requirements of nearest sequence memory (NSM) learning, a previously published, instance-based reinforcement learning algorithm. A hierarchical memory representation reduces the memory requirements by allowing traces to share common sub-sequences. We present moderated mechanisms for estimating discounted future rewards and for dealing with hidden state using hierarchical memory. We also present an experimental analysis of how the sub-sequence length affects the memory compression achieved and show that the reduced memory requirements do not effect the speed of learning. Finally, we analyse and discuss the persistence of the sub-sequences independent of specific trace instances.

The Minimum M3-M4 Loop Length of Neurotransmitter-activated Pentameric Receptors Is Critical for the Structural Integrity of Cytoplasmic Portals*

PubMed Central

Baptista-Hon, Daniel T.; Deeb, Tarek Z.; Lambert, Jeremy J.; Peters, John A.; Hales, Tim G.

2013-01-01

The 5-HT3A receptor homology model, based on the partial structure of the nicotinic acetylcholine receptor from Torpedo marmorata, reveals an asymmetric ion channel with five portals framed by adjacent helical amphipathic (HA) stretches within the 114-residue loop between the M3 and M4 membrane-spanning domains. The positive charge of Arg-436, located within the HA stretch, is a rate-limiting determinant of single channel conductance (γ). Further analysis reveals that positive charge and volume of residue 436 are determinants of 5-HT3A receptor inward rectification, exposing an additional role for portals. A structurally unresolved stretch of 85 residues constitutes the bulk of the M3-M4 loop, leaving a >45-Å gap in the model between M3 and the HA stretch. There are no additional structural data for this loop, which is vestigial in bacterial pentameric ligand-gated ion channels and was largely removed for crystallization of the Caenorhabditis elegans glutamate-activated pentameric ligand-gated ion channels. We created 5-HT3A subunit loop truncation mutants, in which sequences framing the putative portals were retained, to determine the minimum number of residues required to maintain their functional integrity. Truncation to between 90 and 75 amino acids produced 5-HT3A receptors with unaltered rectification. Truncation to 70 residues abolished rectification and increased γ. These findings reveal a critical M3-M4 loop length required for functions attributable to cytoplasmic portals. Examination of all 44 subunits of the human neurotransmitter-activated Cys-loop receptors reveals that, despite considerable variability in their sequences and lengths, all M3-M4 loops exceed 70 residues, suggesting a fundamental requirement for portal integrity. PMID:23740249

Distribution of hepatitis B virus subgenotype F2a in São Paulo, Brazil.

PubMed

Alvarado-Mora, Mónica V; Botelho-Lima, Livia S; Santana, Rubia A; Sitnik, Roberta; Ferreira, Paulo Abrão; do Amaral Mello, Francisco; Mangueira, Cristovão P; Carrilho, Flair J; Rebello Pinho, João R

2013-10-21

HBV genotype F is primarily found in indigenous populations from South America and is classified in four subgenotypes (F1 to F4). Subgenotype F2a is the most common in Brazil among genotype F cases. The aim of this study was to characterize HBV genotype F2a circulating in 16 patients from São Paulo, Brazil. Samples were collected between 2006 and 2012 and sent to Hospital Israelita Albert Einstein. A fragment of 1306 bp partially comprising HBsAg and DNA polymerase coding regions was amplified and sequenced. Viral sequences were genotyped by phylogenetic analysis using reference sequences from GenBank (n=198), including 80 classified as subgenotype F2a. Bayesian Markov chain Monte Carlo simulation implemented in BEAST v.1.5.4 was applied to obtain the best possible estimates using the model of nucleotide substitutions GTR+G+I. It were identified three groups of sequences of subgenotype F2a: 1) 10 sequences from São Paulo state; 2) 3 sequences from Rio de Janeiro and one from São Paulo states; 3) 8 sequences from the West Amazon Basin. These results showing for the first time the distribution of F2a subgenotype in Brazil. The spreading and the dynamic of subgenotype F2a in Brazil requires the study of a higher number of samples from different regions as it is unfold in almost all Brazilian populations studied so far. We cannot infer with certainty the origin of these different groups due to the lack of available sequences. Nevertheless, our data suggest that the common origin of these groups probably occurred a long time ago.

Identification of Alternative Splicing and Fusion Transcripts in Non-Small Cell Lung Cancer by RNA Sequencing.

PubMed

Hong, Yoonki; Kim, Woo Jin; Bang, Chi Young; Lee, Jae Cheol; Oh, Yeon-Mok

2016-04-01

Lung cancer is the most common cause of cancer related death. Alterations in gene sequence, structure, and expression have an important role in the pathogenesis of lung cancer. Fusion genes and alternative splicing of cancer-related genes have the potential to be oncogenic. In the current study, we performed RNA-sequencing (RNA-seq) to investigate potential fusion genes and alternative splicing in non-small cell lung cancer. RNA was isolated from lung tissues obtained from 86 subjects with lung cancer. The RNA samples from lung cancer and normal tissues were processed with RNA-seq using the HiSeq 2000 system. Fusion genes were evaluated using Defuse and ChimeraScan. Candidate fusion transcripts were validated by Sanger sequencing. Alternative splicing was analyzed using multivariate analysis of transcript sequencing and validated using quantitative real time polymerase chain reaction. RNA-seq data identified oncogenic fusion genes EML4-ALK and SLC34A2-ROS1 in three of 86 normal-cancer paired samples. Nine distinct fusion transcripts were selected using DeFuse and ChimeraScan; of which, four fusion transcripts were validated by Sanger sequencing. In 33 squamous cell carcinoma, 29 tumor specific skipped exon events and six mutually exclusive exon events were identified. ITGB4 and PYCR1 were top genes that showed significant tumor specific splice variants. In conclusion, RNA-seq data identified novel potential fusion transcripts and splice variants. Further evaluation of their functional significance in the pathogenesis of lung cancer is required.

Four-dimensional guidance algorithms for aircraft in an air traffic control environment

NASA Technical Reports Server (NTRS)

Pecsvaradi, T.

1975-01-01

Theoretical development and computer implementation of three guidance algorithms are presented. From a small set of input parameters the algorithms generate the ground track, altitude profile, and speed profile required to implement an experimental 4-D guidance system. Given a sequence of waypoints that define a nominal flight path, the first algorithm generates a realistic, flyable ground track consisting of a sequence of straight line segments and circular arcs. Each circular turn is constrained by the minimum turning radius of the aircraft. The ground track and the specified waypoint altitudes are used as inputs to the second algorithm which generates the altitude profile. The altitude profile consists of piecewise constant flight path angle segments, each segment lying within specified upper and lower bounds. The third algorithm generates a feasible speed profile subject to constraints on the rate of change in speed, permissible speed ranges, and effects of wind. Flight path parameters are then combined into a chronological sequence to form the 4-D guidance vectors. These vectors can be used to drive the autopilot/autothrottle of the aircraft so that a 4-D flight path could be tracked completely automatically; or these vectors may be used to drive the flight director and other cockpit displays, thereby enabling the pilot to track a 4-D flight path manually.

Genomic anatomy of the Tyrp1 (brown) deletion complex

PubMed Central

Smyth, Ian M.; Wilming, Laurens; Lee, Angela W.; Taylor, Martin S.; Gautier, Phillipe; Barlow, Karen; Wallis, Justine; Martin, Sancha; Glithero, Rebecca; Phillimore, Ben; Pelan, Sarah; Andrew, Rob; Holt, Karen; Taylor, Ruth; McLaren, Stuart; Burton, John; Bailey, Jonathon; Sims, Sarah; Squares, Jan; Plumb, Bob; Joy, Ann; Gibson, Richard; Gilbert, James; Hart, Elizabeth; Laird, Gavin; Loveland, Jane; Mudge, Jonathan; Steward, Charlie; Swarbreck, David; Harrow, Jennifer; North, Philip; Leaves, Nicholas; Greystrong, John; Coppola, Maria; Manjunath, Shilpa; Campbell, Mark; Smith, Mark; Strachan, Gregory; Tofts, Calli; Boal, Esther; Cobley, Victoria; Hunter, Giselle; Kimberley, Christopher; Thomas, Daniel; Cave-Berry, Lee; Weston, Paul; Botcherby, Marc R. M.; White, Sharon; Edgar, Ruth; Cross, Sally H.; Irvani, Marjan; Hummerich, Holger; Simpson, Eleanor H.; Johnson, Dabney; Hunsicker, Patricia R.; Little, Peter F. R.; Hubbard, Tim; Campbell, R. Duncan; Rogers, Jane; Jackson, Ian J.

2006-01-01

Chromosome deletions in the mouse have proven invaluable in the dissection of gene function. The brown deletion complex comprises >28 independent genome rearrangements, which have been used to identify several functional loci on chromosome 4 required for normal embryonic and postnatal development. We have constructed a 172-bacterial artificial chromosome contig that spans this 22-megabase (Mb) interval and have produced a contiguous, finished, and manually annotated sequence from these clones. The deletion complex is strikingly gene-poor, containing only 52 protein-coding genes (of which only 39 are supported by human homologues) and has several further notable genomic features, including several segments of >1 Mb, apparently devoid of a coding sequence. We have used sequence polymorphisms to finely map the deletion breakpoints and identify strong candidate genes for the known phenotypes that map to this region, including three lethal loci (l4Rn1, l4Rn2, and l4Rn3) and the fitness mutant brown-associated fitness (baf). We have also characterized misexpression of the basonuclin homologue, Bnc2, associated with the inversion-mediated coat color mutant white-based brown (Bw). This study provides a molecular insight into the basis of several characterized mouse mutants, which will allow further dissection of this region by targeted or chemical mutagenesis. PMID:16505357

[Morphological differentiations of the gills of two Gymnocypris przewa-lskii subspecies in different habitats and their functional adaptations].

PubMed

Zhang, Ren-Yi; Li, Guo-Gang; Zhang, Cun-Fang; Tang, Yong-Tao; Zhao, Kai

2013-08-01

Gill morphologies of two subspecies of Gymnocypris przewalskii (Gymnocypris przewalskii przewalskii and Gymnocypris przewalskii ganzihonensis) in different habitats were analyzed under scanning electron microscope. Results indicated that G. p. przewalskii had numerous long and dense-lined gill rakers while G. p. ganzihonensis had few short and scatter-lined gill rakers. There were no significant differences in distance between gill filaments (DBF) and distance gill lamella (DBL) between the two subspecies, but gill filaments of G. p. przewalskii were longer than in G. p. ganzihonensis. The electron microscopic study indicated that the pavement epithelium cells of G. p. przewalskii were well defined as irregular ovals, but were hexagonal in G. p. ganzihonensis. Moreover, G. p. przewalskii had more chloride cells than G. p. ganzihonensis, and mucous cells were only found on the surface of gill filaments of G. p. przewalskii. The morphological differences between the two subspecies of G. przewalskii are adaptations to their corresponding diets and habitats.

Utah FORGE Gravity Data Shapefile

DOE Data Explorer

Joe Moore

2016-03-13

This is a zipped GIS compatible shapefile of gravity data points used in the Milford, Utah FORGE project as of March 21st, 2016. The shapefile is native to ArcGIS, but can be used with many GIS software packages. Additionally, there is a .dbf (dBase) file that contains the dataset which can be read with Microsoft Excel. The Data was downloaded from the PACES (Pan American Center for Earth and Environmental Studies) hosted by University of Texas El Paso (http://research.utep.edu/Default.aspx?alias=research.utep.edu/paces) Explanation:Source: data source code if available LatNAD83: latitude in NAD83 [decimal degrees] LonNAD83: longitude in NAD83 [decimal degrees]zWGS84: elevation in WGS84 (ellipsoidal) [m]OBSless976: observed gravity minus 976000 mGalIZTC: inner zone terrain correction [mGal]OZTC: outer zone terrain correction [mGal]FA: Free Air anomaly value [mGal]CBGA: Complete Bouguer gravity anomaly value [mGal

NS3 protease polymorphisms and genetic barrier to drug resistance of distinct hepatitis C virus genotypes from worldwide treatment-naïve subjects.

PubMed

Vidal, L L; Soares, M A; Santos, A F

2016-11-01

Hepatitis C virus (HCV) NS3 protease inhibitors have been primarily designed against genotype 1, the one with the lowest response to dual therapy. However, less evidence of their efficacy on non-1 genotypes is available, and any such information is mostly concentrated on genotypes 2-4. This study evaluated HCV protease resistance profiles in the major six HCV genotypes and identified genetic barrier (GB) profiles to each available protease inhibitor across HCV strains from different locations worldwide. We obtained 15 099 HCV sequences from treatment-naïve subjects retrieved at the Los Alamos HCV Sequence Database. The wild-type codons of different HCV genotypes were used to analyse the smallest number of nucleotide substitution steps required for changing that codon to the closest one associated with drug resistance. The 36L and 175L RAVs were found as genetic signatures of genotypes 2-5, while the 80K RAV was found in all genotype 5 sequences. Genotypes 4 and 6 showed a higher GB to RAV mutations conferring resistance to telaprevir, while genotypes 2-5 presented baseline resistance to that drug, carrying the 36L mutation. Genotype 4 had a higher GB to simeprevir resistance, requiring three substitutions to acquire the 155K mutation. Subtype 1b showed a higher GB than subtype 1a to resistance for most PIs, with RAVs at codons 36 and 155. Geographic disparities were also found in frequencies of certain RAVs in genotypes 2 and 3. Under a scenario of unprecedented evolution of anti-HCV direct-acting agents, the genetic composition of the circulating HCV sequences should be evaluated worldwide to choose the most appropriate/feasible therapeutic schemes with the highest genetic barriers to resistance. © 2016 John Wiley & Sons Ltd.

Phage T4 SegB protein is a homing endonuclease required for the preferred inheritance of T4 tRNA gene region occurring in co-infection with a related phage.

PubMed

Brok-Volchanskaya, Vera S; Kadyrov, Farid A; Sivogrivov, Dmitry E; Kolosov, Peter M; Sokolov, Andrey S; Shlyapnikov, Michael G; Kryukov, Valentine M; Granovsky, Igor E

2008-04-01

Homing endonucleases initiate nonreciprocal transfer of DNA segments containing their own genes and the flanking sequences by cleaving the recipient DNA. Bacteriophage T4 segB gene, which is located in a cluster of tRNA genes, encodes a protein of unknown function, homologous to homing endonucleases of the GIY-YIG family. We demonstrate that SegB protein is a site-specific endonuclease, which produces mostly 3' 2-nt protruding ends at its DNA cleavage site. Analysis of SegB cleavage sites suggests that SegB recognizes a 27-bp sequence. It contains 11-bp conserved sequence, which corresponds to a conserved motif of tRNA TpsiC stem-loop, whereas the remainder of the recognition site is rather degenerate. T4-related phages T2L, RB1 and RB3 contain tRNA gene regions that are homologous to that of phage T4 but lack segB gene and several tRNA genes. In co-infections of phages T4 and T2L, segB gene is inherited with nearly 100% of efficiency. The preferred inheritance depends absolutely on the segB gene integrity and is accompanied by the loss of the T2L tRNA gene region markers. We suggest that SegB is a homing endonuclease that functions to ensure spreading of its own gene and the surrounding tRNA genes among T4-related phages.

Sexually-Transmitted/Founder HIV-1 Cannot Be Directly Predicted from Plasma or PBMC-Derived Viral Quasispecies in the Transmitting Partner

PubMed Central

Frange, Pierre; Meyer, Laurence; Jung, Matthieu; Goujard, Cecile; Zucman, David; Abel, Sylvie; Hochedez, Patrick; Gousset, Marine; Gascuel, Olivier; Rouzioux, Christine; Chaix, Marie-Laure

2013-01-01

Objective Characterization of HIV-1 sequences in newly infected individuals is important for elucidating the mechanisms of viral sexual transmission. We report the identification of transmitted/founder viruses in eight pairs of HIV-1 sexually-infected patients enrolled at the time of primary infection (“recipients”) and their transmitting partners (“donors”). Methods Using a single genome-amplification approach, we compared quasispecies in donors and recipients on the basis of 316 and 376 C2V5 env sequences amplified from plasma viral RNA and PBMC-associated DNA, respectively. Results Both DNA and RNA sequences indicated very homogeneous viral populations in all recipients, suggesting transmission of a single variant, even in cases of recent sexually transmitted infections (STIs) in donors (n = 2) or recipients (n = 3). In all pairs, the transmitted/founder virus was derived from an infrequent variant population within the blood of the donor. The donor variant sequences most closely related to the recipient sequences were found in plasma samples in 3/8 cases and/or in PBMC samples in 6/8 cases. Although donors were exclusively (n = 4) or predominantly (n = 4) infected by CCR5-tropic (R5) strains, two recipients were infected with highly homogeneous CXCR4/dual-mixed-tropic (X4/DM) viral populations, identified in both DNA and RNA. The proportion of X4/DM quasispecies in donors was higher in cases of X4/DM than R5 HIV transmission (16.7–22.0% versus 0–2.6%), suggesting that X4/DM transmission may be associated with a threshold population of X4/DM circulating quasispecies in donors. Conclusions These suggest that a severe genetic bottleneck occurs during subtype B HIV-1 heterosexual and homosexual transmission. Sexually-transmitted/founder virus cannot be directly predicted by analysis of the donor’s quasispecies in plasma and/or PBMC. Additional studies are required to fully understand the traits that confer the capacity to transmit and establish infection, and determine the role of concomitant STIs in mitigating the genetic bottleneck in mucosal HIV transmission. PMID:23874894

Statistical properties of filtered pseudorandom digital sequences formed from the sum of maximum-length sequences

NASA Technical Reports Server (NTRS)

Wallace, G. R.; Weathers, G. D.; Graf, E. R.

1973-01-01

The statistics of filtered pseudorandom digital sequences called hybrid-sum sequences, formed from the modulo-two sum of several maximum-length sequences, are analyzed. The results indicate that a relation exists between the statistics of the filtered sequence and the characteristic polynomials of the component maximum length sequences. An analysis procedure is developed for identifying a large group of sequences with good statistical properties for applications requiring the generation of analog pseudorandom noise. By use of the analysis approach, the filtering process is approximated by the convolution of the sequence with a sum of unit step functions. A parameter reflecting the overall statistical properties of filtered pseudorandom sequences is derived. This parameter is called the statistical quality factor. A computer algorithm to calculate the statistical quality factor for the filtered sequences is presented, and the results for two examples of sequence combinations are included. The analysis reveals that the statistics of the signals generated with the hybrid-sum generator are potentially superior to the statistics of signals generated with maximum-length generators. Furthermore, fewer calculations are required to evaluate the statistics of a large group of hybrid-sum generators than are required to evaluate the statistics of the same size group of approximately equivalent maximum-length sequences.

Use of eluted peptide sequence data to identify the binding characteristics of peptides to the insulin-dependent diabetes susceptibility allele HLA-DQ8 (DQ 3.2).

PubMed

Godkin, A; Friede, T; Davenport, M; Stevanovic, S; Willis, A; Jewell, D; Hill, A; Rammensee, H G

1997-06-01

HLA-DQ8 (A1*0301, B1*0302) and -DQ2 (A1*0501, B1*0201) are both associated with diseases such as insulin-dependent diabetes mellitus and coeliac disease. We used the technique of pool sequencing to look at the requirements of peptides binding to HLA-DQ8, and combined these data with naturally sequenced ligands and in vitro binding assays to describe a novel motif for HLA-DQ8. The motif, which has the same basic format as many HLA-DR molecules, consists of four or five anchor regions, in the positions from the N-terminus of the binding core of n, n + 3, n + 5/6 and n + 8, i.e. P1, P4, P6/7 and P9. P1 and P9 require negative or polar residues, with mainly aliphatic residues at P4 and P6/7. The features of the HLA-DQ8 motif were then compared to a pool sequence of peptides eluted from HLA-DQ2. A consensus motif for the binding of a common peptide which may be involved in disease pathogenesis is described. Neither of the disease-associated alleles HLA-DQ2 and -DQ8 have Asp at position 57 of the beta-chain. This Asp, if present, may form a salt bridge with an Arg at position 79 of the alpha-chain and so alter the binding specificity of P9. HLA-DQ2 and -DQ8 both appear to prefer negatively charged amino acids at P9. In contrast, HLA-DQ7 (A1*0301, B1*0301), which is not associated with diabetes, has Asp at beta 57, allowing positively charged amino acids at P9. This analysis of the sequence features of DQ-binding peptides suggests molecular characteristics which may be useful to predict epitopes involved in disease pathogenesis.

Novel methodologies for spectral classification of exon and intron sequences

NASA Astrophysics Data System (ADS)

Kwan, Hon Keung; Kwan, Benjamin Y. M.; Kwan, Jennifer Y. Y.

2012-12-01

Digital processing of a nucleotide sequence requires it to be mapped to a numerical sequence in which the choice of nucleotide to numeric mapping affects how well its biological properties can be preserved and reflected from nucleotide domain to numerical domain. Digital spectral analysis of nucleotide sequences unfolds a period-3 power spectral value which is more prominent in an exon sequence as compared to that of an intron sequence. The success of a period-3 based exon and intron classification depends on the choice of a threshold value. The main purposes of this article are to introduce novel codes for 1-sequence numerical representations for spectral analysis and compare them to existing codes to determine appropriate representation, and to introduce novel thresholding methods for more accurate period-3 based exon and intron classification of an unknown sequence. The main findings of this study are summarized as follows: Among sixteen 1-sequence numerical representations, the K-Quaternary Code I offers an attractive performance. A windowed 1-sequence numerical representation (with window length of 9, 15, and 24 bases) offers a possible speed gain over non-windowed 4-sequence Voss representation which increases as sequence length increases. A winner threshold value (chosen from the best among two defined threshold values and one other threshold value) offers a top precision for classifying an unknown sequence of specified fixed lengths. An interpolated winner threshold value applicable to an unknown and arbitrary length sequence can be estimated from the winner threshold values of fixed length sequences with a comparable performance. In general, precision increases as sequence length increases. The study contributes an effective spectral analysis of nucleotide sequences to better reveal embedded properties, and has potential applications in improved genome annotation.

Multiclass Bayes error estimation by a feature space sampling technique

NASA Technical Reports Server (NTRS)

Mobasseri, B. G.; Mcgillem, C. D.

1979-01-01

A general Gaussian M-class N-feature classification problem is defined. An algorithm is developed that requires the class statistics as its only input and computes the minimum probability of error through use of a combined analytical and numerical integration over a sequence simplifying transformations of the feature space. The results are compared with those obtained by conventional techniques applied to a 2-class 4-feature discrimination problem with results previously reported and 4-class 4-feature multispectral scanner Landsat data classified by training and testing of the available data.

Evaluation of the Abbott Real Time HCV genotype II assay for Hepatitis C virus genotyping.

PubMed

Sariguzel, Fatma Mutlu; Berk, Elife; Gokahmetoglu, Selma; Ercal, Baris Derya; Celik, Ilhami

2015-01-01

The determination of HCV genotypes and subtypes is very important for the selection of antiviral therapy and epidemiological studies. The aim of this study was to evaluate the performance of Abbott Real Time HCV Genotype II assay in HCV genotyping of HCV infected patients in Kayseri, Turkey. One hundred patients with chronic hepatitis C admitted to our hospital were evaluated between June 2012 and December 2012, HCV RNA levels were determined by the COBAS® AmpliPrep/COBAS® TaqMan® 48 HCV test. HCV genotyping was investigated by the Abbott Real Time HCV Genotype II assay. With the exception of genotype 1, subtypes of HCV genotypes could not be determined by Abbott assay. Sequencing analysis was used as the reference method. Genotypes 1, 2, 3 and 4 were observed in 70, 4, 2 and 24 of the 100 patients, respectively, by two methods. The concordance between the two systems to determine HCV major genotypes was 100%. Of 70 patients with genotype 1, 66 showed infection with subtype 1b and 4 with subtype 1a by Abbott Real Time HCV Genotype II assay. Using sequence analysis, 61 showed infection with subtype 1b and 9 with subtype 1a. In determining of HCV genotype 1 subtypes, the difference between the two methods was not statistically significant (P>0.05). HCV genotype 4 and 3 samples were found to be subtype 4d and 3a, respectively, by sequence analysis. There were four patients with genotype 2. Sequence analysis revealed that two of these patients had type 2a and the other two had type 2b. The Abbott Real Time HCV Genotype II assay yielded results consistent with sequence analysis. However, further optimization of the Abbott Real Time HCV Genotype II assay for subtype identification of HCV is required.

Construction of Graphic Symbol Sequences by Preschool-Aged Children: Learning, Training, and Maintenance

ERIC Educational Resources Information Center

Poupart, Annick; Trudeau, Natacha; Sutton, Ann

2013-01-01

The use of augmentative and alternative communication systems based on graphic symbols requires children to learn to combine symbols to convey utterances. The current study investigated how children without disabilities aged 4 to 6 years (n = 74) performed on a simple sentence (subject-verb and subject-verb-object) transposition task (i.e., spoken…

Final Report for LDRD Project 02-ERD-069: Discovering the Unknown Mechanism(s) of Virulence in a BW, Class A Select Agent

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

Chain, P; Garcia, E

2003-02-06

The goal of this proposed effort was to assess the difficulty in identifying and characterizing virulence candidate genes in an organism for which very limited data exists. This was accomplished by first addressing the finishing phase of draft-sequenced F. tularensis genomes and conducting comparative analyses to determine the coding potential of each genome; to discover the differences in genome structure and content, and to identify potential genes whose products may be involved in the F. tularensis virulence process. The project was divided into three parts: (1) Genome finishing: This part involves determining the order and orientation of the consensus sequencesmore » of contigs obtained from Phrap assemblies of random draft genomic sequences. This tedious process consists of linking contig ends using information embedded in each sequence file that relates the sequence to the original cloned insert. Since inserts are sequenced from both ends, we can establish a link between these paired-ends in different contigs and thus order and orient contigs. Since these genomes carry numerous copies of insertion sequences, these repeated elements ''confuse'' the Phrap assembly program. It is thus necessary to break these contigs apart at the repeated sequences and individually join the proper flanking regions using paired-end information, or using results of comparisons against a similar genome. Larger repeated elements such as the small subunit ribosomal RNA operon require verification with PCR. Tandem repeats require manual intervention and typically rely on single nucleotide polymorphisms to be resolved. Remaining gaps require PCR reactions and sequencing. Once the genomes have been ''closed'', low quality regions are addressed by resequencing reactions. (2) Genome analysis: The final consensus sequences are processed by combining the results of three gene modelers: Glimmer, Critica and Generation. The final gene models are submitted to a battery of homology searches and domain prediction programs in order to annotate them (e.g. BLAST, Pfam, TIGRfam, COG, KEGG, InterPro, TMhmm, SignalP). The genome structure is also assessed in terms of G+C content, GC bias (GC skew), and locations of repeated regions (e.g. IS elements) and phage-like genes. (3) Comparative genomics: The results of the various genome analyses are compared between the finished (or almost finished) genomes. Here, we have compared the F. tularensis genomes from the extremely lethal strain Schu4 (subsp. tularensis), the vaccine strain LVS (subsp. holartica), and strain UT01-4992 of the less virulent, opportunistic subsp. novicida. Regions present in the highly virulent strain that are absent from the other less virulent strains may provide insight into what factors are required for the high level of virulence.« less

Emergent biomarker derived from next-generation sequencing to identify pain patients requiring uncommonly high opioid doses

PubMed Central

Kringel, D; Ultsch, A; Zimmermann, M; Jansen, J-P; Ilias, W; Freynhagen, R; Griessinger, N; Kopf, A; Stein, C; Doehring, A; Resch, E; Lötsch, J

2017-01-01

Next-generation sequencing (NGS) provides unrestricted access to the genome, but it produces ‘big data’ exceeding in amount and complexity the classical analytical approaches. We introduce a bioinformatics-based classifying biomarker that uses emergent properties in genetics to separate pain patients requiring extremely high opioid doses from controls. Following precisely calculated selection of the 34 most informative markers in the OPRM1, OPRK1, OPRD1 and SIGMAR1 genes, pattern of genotypes belonging to either patient group could be derived using a k-nearest neighbor (kNN) classifier that provided a diagnostic accuracy of 80.6±4%. This outperformed alternative classifiers such as reportedly functional opioid receptor gene variants or complex biomarkers obtained via multiple regression or decision tree analysis. The accumulation of several genetic variants with only minor functional influences may result in a qualitative consequence affecting complex phenotypes, pointing at emergent properties in genetics. PMID:27139154

Emergent biomarker derived from next-generation sequencing to identify pain patients requiring uncommonly high opioid doses.

PubMed

Kringel, D; Ultsch, A; Zimmermann, M; Jansen, J-P; Ilias, W; Freynhagen, R; Griessinger, N; Kopf, A; Stein, C; Doehring, A; Resch, E; Lötsch, J

2017-10-01

Next-generation sequencing (NGS) provides unrestricted access to the genome, but it produces 'big data' exceeding in amount and complexity the classical analytical approaches. We introduce a bioinformatics-based classifying biomarker that uses emergent properties in genetics to separate pain patients requiring extremely high opioid doses from controls. Following precisely calculated selection of the 34 most informative markers in the OPRM1, OPRK1, OPRD1 and SIGMAR1 genes, pattern of genotypes belonging to either patient group could be derived using a k-nearest neighbor (kNN) classifier that provided a diagnostic accuracy of 80.6±4%. This outperformed alternative classifiers such as reportedly functional opioid receptor gene variants or complex biomarkers obtained via multiple regression or decision tree analysis. The accumulation of several genetic variants with only minor functional influences may result in a qualitative consequence affecting complex phenotypes, pointing at emergent properties in genetics.

Nonstructural proteins nsP3 and nsP4 of Ross River and O'Nyong-nyong viruses: sequence and comparison with those of other alphaviruses.

PubMed

Strauss, E G; Levinson, R; Rice, C M; Dalrymple, J; Strauss, J H

1988-05-01

We have sequenced the nsP3 and nsP4 region of two alphaviruses, Ross River virus and O'Nyong-nyong virus, in order to examine these viruses for the presence or absence of an opal termination codon present between nsP3 and nsP4 in many alphaviruses. We found that Ross River virus possesses an in-phase opal termination codon between nsP3 and nsP4, whereas in O'Nyong-nyong virus this termination codon is replaced by an arginine codon. Previous studies have shown that two other alphaviruses, Sindbis virus and Middelburg virus, possess an opal termination codon separating nsP3 and nsP4 [E.G. Strauss, C.M. Rice, and J.H. Strauss (1983), Proc. Natl. Acad. Sci. USA 80, 5271-5275], whereas Semliki Forest virus possesses an arginine codon in lieu of the opal codon [K. Takkinen (1986), Nucleic Acids Res. 14, 5667-5682]. Thus, of the five alphaviruses examined to date, three possess the opal codon and two do not. Production of nsP4 requires readthrough of the opal codon in those alphaviruses that possess this termination codon and the function of the termination codon may be to regulate the amount of nsP4 produced. It is an open question then as to whether alphaviruses with no termination codon use other mechanisms to regulate the activity of this gene. The nsP4s of these five alphaviruses are highly conserved, sharing 71-76% amino acid sequence similarity, and all five contain the Gly-Asp-Asp motif found in many RNA virus replicases. The nsP3s are somewhat less conserved, sharing 52-73% amino acid sequence similarity throughout most of the protein, but each possesses a nonconserved C-terminal domain of 134 to 246 amino acids of unknown function.

Shark complement: an assessment.

PubMed

Smith, S L

1998-12-01

The classical (CCP) and alternative (ACP) pathways of complement activation have been established for the nurse shark (Ginglymostoma cirratum). The isolation of a cDNA clone encoding a mannan-binding protein-associated serine protease (MASP)-1-like protein from the Japanese dogfish (Triakis scyllia) suggests the presence of a lectin pathway. The CCP consists of six functionally distinct components: C1n, C2n, C3n, C4n, C8n and C9n, and is activated by immune complexes in the presence of Ca++ and Mg++ ions. The ACP is antibody independent, requiring Mg++ ions and a heat-labile 90 kDa factor B-like protein for activity. Proteins considered homologues of C1q, C3 and C4 (C2n) of the mammalian complement system have been isolated from nurse shark serum. Shark C1q is composed of at least two chain types each showing 50% identity to human C1q chains A and B. Partial sequence of the globular domain of one of the chains shows it to be C1q-like rather than like mannan-binding protein. N-terminal amino acid sequences of the alpha and beta chain of shark C3 and C4 molecules show significant identity with corresponding human C3 and C4 chains. A sequence representing shark C4 gamma chain, shows little similarity to human C4 gamma chain. The terminal shark components C8n and C9n are functional analogues of mammalian C8 and C9. Anaphylatoxin activity has been demonstrated in activated shark serum, and porcine C5a desArg induces shark leucocyte chemotaxis. The deduced amino acid sequence of a partial C3 cDNA clone from the nurse shark shows 50%, 30% and 24% homology with the corresponding region of mammalian C3, C4 and alpha 2-macroglobulin. Deduced amino acid sequence data from partial Bf/C2 cDNA clones, two from the nurse shark and one from the Japanese dogfish, suggest that at least one species of elasmobranch has two distinct Bf/C2 genes.

Effects on in vitro and in vivo angiogenesis induced by small peptides carrying adhesion sequences.

PubMed

Conconi, Maria Teresa; Ghezzo, Francesca; Dettin, Monica; Urbani, Luca; Grandi, Claudio; Guidolin, Diego; Nico, Beatrice; Di Bello, Carlo; Ribatti, Domenico; Parnigotto, Pier Paolo

2010-07-01

It is well known that tumor growth is strictly dependent on neo-vessel formation inside the tumor mass and that cell adhesion is required to allow EC proliferation and migration inside the tumor. In this work, we have evaluated the in vitro and in vivo effects on angiogenesis of some peptides, originally designed to promote cell adhesion on biomaterials, containing RGD motif mediating cell adhesion via integrin receptors [RGD, GRGDSPK, and (GRGDSP)(4)K] or the heparin-binding sequence of human vitronectin that interacts with HSPGs [HVP(351-359)]. Cell adhesion, proliferation, migration, and capillary-like tube formation in Matrigel were determined on HUVECs, whereas the effects on in vivo angiogenesis were evaluated using the CAM assay. (GRGDSP)(4)K linear sequence inhibited cell adhesion, decreased cell proliferation, migration and morphogenesis in Matrigel, and induced anti-angiogenic responses on CAM at higher degree than that determined after incubation with RGD or GRGDSPK. Moreover, it counteracted both in vitro and in vivo the pro-angiogenic effects induced by the Fibroblast growth factor (FGF-2). On the other hand, HVP was not able to affect cell adhesion and appeared less effective than (GRGDSP)(4)K. Our data indicate that the activity of RGD-containing peptides is related to their adhesive properties, and their effects are modulated by the number of cell adhesion motifs and the aminoacidic residues next to these sequences. The anti-angiogenic properties of (GRGDSP)(4)K seem to depend on its interaction with integrins, whereas the effects of HVP may be partially due to an impairment of HSPGs/FGF-2.

Dual-task interference effects on cross-modal numerical order and sound intensity judgments: the more the louder?

PubMed

Alards-Tomalin, Doug; Walker, Alexander C; Nepon, Hillary; Leboe-McGowan, Launa C

2017-09-01

In the current study, cross-task interactions between number order and sound intensity judgments were assessed using a dual-task paradigm. Participants first categorized numerical sequences composed of Arabic digits as either ordered (ascending, descending) or non-ordered. Following each number sequence, participants then had to judge the intensity level of a target sound. Experiment 1 emphasized processing the two tasks independently (serial processing), while Experiments 2 and 3 emphasized processing the two tasks simultaneously (parallel processing). Cross-task interference occurred only when the task required parallel processing and was specific to ascending numerical sequences, which led to a higher proportion of louder sound intensity judgments. In Experiment 4 we examined whether this unidirectional interaction was the result of participants misattributing enhanced processing fluency experienced on ascending sequences as indicating a louder target sound. The unidirectional finding could not be entirely attributed to misattributed processing fluency, and may also be connected to experientially derived conceptual associations between ascending number sequences and greater magnitude, consistent with conceptual mapping theory.

Pair-barcode high-throughput sequencing for large-scale multiplexed sample analysis

PubMed Central

2012-01-01

Background The multiplexing becomes the major limitation of the next-generation sequencing (NGS) in application to low complexity samples. Physical space segregation allows limited multiplexing, while the existing barcode approach only permits simultaneously analysis of up to several dozen samples. Results Here we introduce pair-barcode sequencing (PBS), an economic and flexible barcoding technique that permits parallel analysis of large-scale multiplexed samples. In two pilot runs using SOLiD sequencer (Applied Biosystems Inc.), 32 independent pair-barcoded miRNA libraries were simultaneously discovered by the combination of 4 unique forward barcodes and 8 unique reverse barcodes. Over 174,000,000 reads were generated and about 64% of them are assigned to both of the barcodes. After mapping all reads to pre-miRNAs in miRBase, different miRNA expression patterns are captured from the two clinical groups. The strong correlation using different barcode pairs and the high consistency of miRNA expression in two independent runs demonstrates that PBS approach is valid. Conclusions By employing PBS approach in NGS, large-scale multiplexed pooled samples could be practically analyzed in parallel so that high-throughput sequencing economically meets the requirements of samples which are low sequencing throughput demand. PMID:22276739

Pair-barcode high-throughput sequencing for large-scale multiplexed sample analysis.

PubMed

Tu, Jing; Ge, Qinyu; Wang, Shengqin; Wang, Lei; Sun, Beili; Yang, Qi; Bai, Yunfei; Lu, Zuhong

2012-01-25

The multiplexing becomes the major limitation of the next-generation sequencing (NGS) in application to low complexity samples. Physical space segregation allows limited multiplexing, while the existing barcode approach only permits simultaneously analysis of up to several dozen samples. Here we introduce pair-barcode sequencing (PBS), an economic and flexible barcoding technique that permits parallel analysis of large-scale multiplexed samples. In two pilot runs using SOLiD sequencer (Applied Biosystems Inc.), 32 independent pair-barcoded miRNA libraries were simultaneously discovered by the combination of 4 unique forward barcodes and 8 unique reverse barcodes. Over 174,000,000 reads were generated and about 64% of them are assigned to both of the barcodes. After mapping all reads to pre-miRNAs in miRBase, different miRNA expression patterns are captured from the two clinical groups. The strong correlation using different barcode pairs and the high consistency of miRNA expression in two independent runs demonstrates that PBS approach is valid. By employing PBS approach in NGS, large-scale multiplexed pooled samples could be practically analyzed in parallel so that high-throughput sequencing economically meets the requirements of samples which are low sequencing throughput demand.

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

The positive regulatory function of the 5'-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences.

PubMed Central

Williams, N P; Mueller, P P; Hinnebusch, A G

1988-01-01

Translational control of GCN4 expression in the yeast Saccharomyces cerevisiae is mediated by multiple AUG codons present in the leader of GCN4 mRNA, each of which initiates a short open reading frame of only two or three codons. Upstream AUG codons 3 and 4 are required to repress GCN4 expression in normal growth conditions; AUG codons 1 and 2 are needed to overcome this repression in amino acid starvation conditions. We show that the regulatory function of AUG codons 1 and 2 can be qualitatively mimicked by the AUG codons of two heterologous upstream open reading frames (URFs) containing the initiation regions of the yeast genes PGK and TRP1. These AUG codons inhibit GCN4 expression when present singly in the mRNA leader; however, they stimulate GCN4 expression in derepressing conditions when inserted upstream from AUG codons 3 and 4. This finding supports the idea that AUG codons 1 and 2 function in the control mechanism as translation initiation sites and further suggests that suppression of the inhibitory effects of AUG codons 3 and 4 is a general consequence of the translation of URF 1 and 2 sequences upstream. Several observations suggest that AUG codons 3 and 4 are efficient initiation sites; however, these sequences do not act as positive regulatory elements when placed upstream from URF 1. This result suggests that efficient translation is only one of the important properties of the 5' proximal URFs in GCN4 mRNA. We propose that a second property is the ability to permit reinitiation following termination of translation and that URF 1 is optimized for this regulatory function. Images PMID:3065626

A population study of the minicircles in Trypanosoma cruzi: predicting guide RNAs in the absence of empirical RNA editing.

PubMed

Thomas, Sean; Martinez, L L Isadora Trejo; Westenberger, Scott J; Sturm, Nancy R

2007-05-24

The structurally complex network of minicircles and maxicircles comprising the mitochondrial DNA of kinetoplastids mirrors the complexity of the RNA editing process that is required for faithful expression of encrypted maxicircle genes. Although a few of the guide RNAs that direct this editing process have been discovered on maxicircles, guide RNAs are mostly found on the minicircles. The nuclear and maxicircle genomes have been sequenced and assembled for Trypanosoma cruzi, the causative agent of Chagas disease, however the complement of 1.4-kb minicircles, carrying four guide RNA genes per molecule in this parasite, has been less thoroughly characterised. Fifty-four CL Brener and 53 Esmeraldo strain minicircle sequence reads were extracted from T. cruzi whole genome shotgun sequencing data. With these sequences and all published T. cruzi minicircle sequences, 108 unique guide RNAs from all known T. cruzi minicircle sequences and two guide RNAs from the CL Brener maxicircle were predicted using a local alignment algorithm and mapped onto predicted or experimentally determined sequences of edited maxicircle open reading frames. For half of the sequences no statistically significant guide RNA could be assigned. Likely positions of these unidentified gRNAs in T. cruzi minicircle sequences are estimated using a simple Hidden Markov Model. With the local alignment predictions as a standard, the HMM had an ~85% chance of correctly identifying at least 20 nucleotides of guide RNA from a given minicircle sequence. Inter-minicircle recombination was documented. Variable regions contain species-specific areas of distinct nucleotide preference. Two maxicircle guide RNA genes were found. The identification of new minicircle sequences and the further characterization of all published minicircles are presented, including the first observation of recombination between minicircles. Extrapolation suggests a level of 4% recombinants in the population, supporting a relatively high recombination rate that may serve to minimize the persistence of gRNA pseudogenes. Characteristic nucleotide preferences observed within variable regions provide potential clues regarding the transcription and maturation of T. cruzi guide RNAs. Based on these preferences, a method of predicting T. cruzi guide RNAs using only primary minicircle sequence data was created.

Sequence Ready Characterization of the Pericentromeric Region of 19p12

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

Evan E. Eichler

2006-08-31

Current mapping and sequencing strategies have been inadequate within the proximal portion of 19p12 due, in part, to the presence of a recently expanded ZNF (zinc-finger) gene family and the presence of large (25-50 kb) inverted beta-satellite repeat structures which bracket this tandemly duplicated gene family. The virtual of absence of classically defined “unique” sequence within the region has hampered efforts to identify and characterize a suitable minimal tiling path of clones which can be used as templates required for finished sequencing of the region. The goal of this proposal is to develop and implement a novel sequence-anchor strategy tomore » generate a contiguous BAC map of the most proximal portion of chromosome 19p12 for the purpose of complete sequence characterization. The target region will be an estimated 4.5 Mb of DNA extending from STS marker D19S450 (the beginning of the ZNF gene cluster) to the centromeric (alpha-satellite) junction of 19p11. The approach will entail 1) pre-selection of 19p12 BAC and cosmid clones (NIH approved library) utilizing both 19p12 -unique and 19p12-SPECIFIC repeat probes (Eichler et al., 1998); 2) the generation of a BAC/cosmid end-sequence map across the region with a density of one marker every 8kb; 3) the development of a second-generation of STS (sequence tagged sites) which will be used to identify and verify clonal overlap at the level of the sequence; 4) incorporation of these sequence-anchored overlapping clones into existing cosmid/BAC restriction maps developed at Livermore National Laboratory; and 5) validation of the organization of this region utilizing high-resolution FISH techniques (extended chromatin analysis) on monochromosomal 19 somatic cell hybrids and parental cell lines of source material. The data generated will be used in the selection of the most parsimonious tiling path of BAC clones to be sequenced as part of the JGI effort on chromosome 19 and should serve as a model for the sequence characterization of other difficult regions of the human genome« less

The standard operating procedure of the DOE-JGI Microbial Genome Annotation Pipeline (MGAP v.4)

DOE PAGES

Huntemann, Marcel; Ivanova, Natalia N.; Mavromatis, Konstantinos; ...

2015-10-26

The DOE-JGI Microbial Genome Annotation Pipeline performs structural and functional annotation of microbial genomes that are further included into the Integrated Microbial Genome comparative analysis system. MGAP is applied to assembled nucleotide sequence datasets that are provided via the IMG submission site. Dataset submission for annotation first requires project and associated metadata description in GOLD. The MGAP sequence data processing consists of feature prediction including identification of protein-coding genes, non-coding RNAs and regulatory RNA features, as well as CRISPR elements. In conclusion, structural annotation is followed by assignment of protein product names and functions.

The standard operating procedure of the DOE-JGI Microbial Genome Annotation Pipeline (MGAP v.4)

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

Huntemann, Marcel; Ivanova, Natalia N.; Mavromatis, Konstantinos

The DOE-JGI Microbial Genome Annotation Pipeline performs structural and functional annotation of microbial genomes that are further included into the Integrated Microbial Genome comparative analysis system. MGAP is applied to assembled nucleotide sequence datasets that are provided via the IMG submission site. Dataset submission for annotation first requires project and associated metadata description in GOLD. The MGAP sequence data processing consists of feature prediction including identification of protein-coding genes, non-coding RNAs and regulatory RNA features, as well as CRISPR elements. In conclusion, structural annotation is followed by assignment of protein product names and functions.

Two sequence-ready contigs spanning the two copies of a 200-kb duplication on human 21q: partial sequence and polymorphisms.

PubMed

Potier, M; Dutriaux, A; Orti, R; Groet, J; Gibelin, N; Karadima, G; Lutfalla, G; Lynn, A; Van Broeckhoven, C; Chakravarti, A; Petersen, M; Nizetic, D; Delabar, J; Rossier, J

1998-08-01

Physical mapping across a duplication can be a tour de force if the region is larger than the size of a bacterial clone. This was the case of the 170- to 275-kb duplication present on the long arm of chromosome 21 in normal human at 21q11.1 (proximal region) and at 21q22.1 (distal region), which we described previously. We have constructed sequence-ready contigs of the two copies of the duplication of which all the clones are genuine representatives of one copy or the other. This required the identification of four duplicon polymorphisms that are copy-specific and nonallelic variations in the sequence of the STSs. Thirteen STSs were mapped inside the duplicated region and 5 outside but close to the boundaries. Among these STSs 10 were end clones from YACs, PACs, or cosmids, and the average interval between two markers in the duplicated region was 16 kb. Eight PACs and cosmids showing minimal overlaps were selected in both copies of the duplication. Comparative sequence analysis along the duplication showed three single-basepair changes between the two copies over 659 bp sequenced (4 STSs), suggesting that the duplication is recent (less than 4 mya). Two CpG islands were located in the duplication, but no genes were identified after a 36-kb cosmid from the proximal copy of the duplication was sequenced. The homology of this chromosome 21 duplicated region with the pericentromeric regions of chromosomes 13, 2, and 18 suggests that the mechanism involved is probably similar to pericentromeric-directed mechanisms described in interchromosomal duplications. Copyright 1998 Academic Press.

Saccharomyces cerevisiae sigma 1278b has novel genes of the N-acetyltransferase gene superfamily required for L-proline analogue resistance.

PubMed

Takagi, H; Shichiri, M; Takemura, M; Mohri, M; Nakamori, S

2000-08-01

We discovered on the chromosome of Saccharomyces cerevisiae Sigma 1278b novel genes involved in L-proline analogue L-azetidine-2-carboxylic acid resistance which are not present in the standard laboratory strains. The 5.4 kb-DNA fragment was cloned from the genomic library of the L-azetidine-2-carboxylic acid-resistant mutant derived from a cross between S. cerevisiae strains S288C and Sigma 1278b. The nucleotide sequence of a 4.5-kb segment exhibited no identity with the sequence in the genome project involving strain S288C. Deletion analysis indicated that one open reading frame encoding a predicted protein of 229 amino acids is indispensable for L-azetidine-2-carboxylic acid resistance. The protein sequence was found to be a member of the N-acetyltransferase superfamily. Genomic Southern analysis and gene disruption showed that two copies of the novel gene with one amino acid change at position 85 required for L-azetidine-2-carboxylic acid resistance were present on chromosomes X and XIV of Sigma 1278b background strains. When this novel MPR1 or MPR2 gene (sigma 1278b gene for L-proline analogue resistance) was introduced into the other S. cerevisiae strains, all of the recombinants were resistant to L-azetidine-2-carboxylic acid, indicating that both MPR1 and MPR2 are expressed and have a global function in S. cerevisiae.

Acceleration of the Smith-Waterman algorithm using single and multiple graphics processors

NASA Astrophysics Data System (ADS)

Khajeh-Saeed, Ali; Poole, Stephen; Blair Perot, J.

2010-06-01

Finding regions of similarity between two very long data streams is a computationally intensive problem referred to as sequence alignment. Alignment algorithms must allow for imperfect sequence matching with different starting locations and some gaps and errors between the two data sequences. Perhaps the most well known application of sequence matching is the testing of DNA or protein sequences against genome databases. The Smith-Waterman algorithm is a method for precisely characterizing how well two sequences can be aligned and for determining the optimal alignment of those two sequences. Like many applications in computational science, the Smith-Waterman algorithm is constrained by the memory access speed and can be accelerated significantly by using graphics processors (GPUs) as the compute engine. In this work we show that effective use of the GPU requires a novel reformulation of the Smith-Waterman algorithm. The performance of this new version of the algorithm is demonstrated using the SSCA#1 (Bioinformatics) benchmark running on one GPU and on up to four GPUs executing in parallel. The results indicate that for large problems a single GPU is up to 45 times faster than a CPU for this application, and the parallel implementation shows linear speed up on up to 4 GPUs.

Combinatorial pulse position modulation for power-efficient free-space laser communications

NASA Technical Reports Server (NTRS)

Budinger, James M.; Vanderaar, M.; Wagner, P.; Bibyk, Steven

1993-01-01

A new modulation technique called combinatorial pulse position modulation (CPPM) is presented as a power-efficient alternative to quaternary pulse position modulation (QPPM) for direct-detection, free-space laser communications. The special case of 16C4PPM is compared to QPPM in terms of data throughput and bit error rate (BER) performance for similar laser power and pulse duty cycle requirements. The increased throughput from CPPM enables the use of forward error corrective (FEC) encoding for a net decrease in the amount of laser power required for a given data throughput compared to uncoded QPPM. A specific, practical case of coded CPPM is shown to reduce the amount of power required to transmit and receive a given data sequence by at least 4.7 dB. Hardware techniques for maximum likelihood detection and symbol timing recovery are presented.

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

Massive programmed translational jumping in mitochondria

PubMed Central

Lang, B. Franz; Jakubkova, Michaela; Hegedusova, Eva; Daoud, Rachid; Forget, Lise; Brejova, Brona; Vinar, Tomas; Kosa, Peter; Fricova, Dominika; Nebohacova, Martina; Griac, Peter; Tomaska, Lubomir; Burger, Gertraud; Nosek, Jozef

2014-01-01

Programmed translational bypassing is a process whereby ribosomes “ignore” a substantial interval of mRNA sequence. Although discovered 25 y ago, the only experimentally confirmed example of this puzzling phenomenon is expression of the bacteriophage T4 gene 60. Bypassing requires translational blockage at a “takeoff codon” immediately upstream of a stop codon followed by a hairpin, which causes peptidyl-tRNA dissociation and reassociation with a matching “landing triplet” 50 nt downstream, where translation resumes. Here, we report 81 translational bypassing elements (byps) in mitochondria of the yeast Magnusiomyces capitatus and demonstrate in three cases, by transcript analysis and proteomics, that byps are retained in mitochondrial mRNAs but not translated. Although mitochondrial byps resemble the bypass sequence in the T4 gene 60, they utilize unused codons instead of stops for translational blockage and have relaxed matching rules for takeoff/landing sites. We detected byp-like sequences also in mtDNAs of several Saccharomycetales, indicating that byps are mobile genetic elements. These byp-like sequences lack bypassing activity and are tolerated when inserted in-frame in variable protein regions. We hypothesize that byp-like elements have the potential to contribute to evolutionary diversification of proteins by adding new domains that allow exploration of new structures and functions. PMID:24711422

Analysis of High Temporal and Spatial Observations of Hurricane Joaquin During TCI-15

NASA Technical Reports Server (NTRS)

Creasey, Robert; Elsberry, Russell L.; Velden, Chris; Cecil, Daniel J.; Bell, Michael; Hendricks, Eric A.

2016-01-01

Objectives: Provide an example of why analysis of high density soundings across Hurricane Joaquin also require highly accurate center positions; Describe technique for calculating 3-D zero-wind center positions from the highly accurate GPS positions of sequences of High-Density Sounding System (HDSS) soundings as they fall from 10 km to the ocean surface; Illustrate the vertical tilt of the vortex above 4-5 km during two center passes through Hurricane Joaquin on 4 October 2015.

Space power system scheduling using an expert system

NASA Technical Reports Server (NTRS)

Bahrami, K. A.; Biefeld, E.; Costello, L.; Klein, J. W.

1986-01-01

A most pressing problem in space exploration is timely spacecraft power system sequence generation, which requires the scheduling of a set of loads given a set of resource constraints. This is particularly important after an anomaly or failure. This paper discusses the power scheduling problem and how the software program, Plan-It, can be used as a consultant for scheduling power system activities. Modeling of power activities, human interface, and two of the many strategies used by Plan-It are discussed. Preliminary results showing the development of a conflict-free sequence from an initial sequence with conflicts is presented. It shows that a 4-day schedule can be generated in a matter of a few minutes, which provides sufficient time in many cases to aid the crew in the replanning of loads and generation use following a failure or anomaly.

Plastoglobule-Targeting Competence of a Putative Transit Peptide Sequence from Rice Phytoene Synthase 2 in Plastids.

PubMed

You, Min Kyoung; Kim, Jin Hwa; Lee, Yeo Jin; Jeong, Ye Sol; Ha, Sun-Hwa

2016-12-22

Plastoglobules (PGs) are thylakoid membrane microdomains within plastids that are known as specialized locations of carotenogenesis. Three rice phytoene synthase proteins (OsPSYs) involved in carotenoid biosynthesis have been identified. Here, the N-terminal 80-amino-acid portion of OsPSY2 (PTp) was demonstrated to be a chloroplast-targeting peptide by displaying cytosolic localization of OsPSY2(ΔPTp):mCherry in rice protoplast, in contrast to chloroplast localization of OsPSY2:mCherry in a punctate pattern. The peptide sequence of a PTp was predicted to harbor two transmembrane domains eligible for a putative PG-targeting signal. To assess and enhance the PG-targeting ability of PTp, the original PTp DNA sequence ( PTp ) was modified to a synthetic DNA sequence ( stPTp ), which had 84.4% similarity to the original sequence. The motivation of this modification was to reduce the GC ratio from 75% to 65% and to disentangle the hairpin loop structures of PTp . These two DNA sequences were fused to the sequence of the synthetic green fluorescent protein (sGFP) and drove GFP expression with different efficiencies. In particular, the RNA and protein levels of stPTp-sGFP were slightly improved to 1.4-fold and 1.3-fold more than those of sGFP, respectively. The green fluorescent signals of their mature proteins were all observed as speckle-like patterns with slightly blurred stromal signals in chloroplasts. These discrete green speckles of PTp - sGFP and stPTp - sGFP corresponded exactly to the red fluorescent signal displayed by OsPSY2:mCherry in both etiolated and greening protoplasts and it is presumed to correspond to distinct PGs. In conclusion, we identified PTp as a transit peptide sequence facilitating preferential translocation of foreign proteins to PGs, and developed an improved PTp sequence, a s tPTp , which is expected to be very useful for applications in plant biotechnologies requiring precise micro-compartmental localization in plastids.

Two RNAs or DNAs May Artificially Fuse Together at a Short Homologous Sequence (SHS) during Reverse Transcription or Polymerase Chain Reactions, and Thus Reporting an SHS-Containing Chimeric RNA Requires Extra Caution

PubMed Central

Xie, Bingkun; Yang, Wei; Ouyang, Yongchang; Chen, Lichan; Jiang, Hesheng; Liao, Yuying; Liao, D. Joshua

2016-01-01

Tens of thousands of chimeric RNAs have been reported. Most of them contain a short homologous sequence (SHS) at the joining site of the two partner genes but are not associated with a fusion gene. We hypothesize that many of these chimeras may be technical artifacts derived from SHS-caused mis-priming in reverse transcription (RT) or polymerase chain reactions (PCR). We cloned six chimeric complementary DNAs (cDNAs) formed by human mitochondrial (mt) 16S rRNA sequences at an SHS, which were similar to several expression sequence tags (ESTs).These chimeras, which could not be detected with cDNA protection assay, were likely formed because some regions of the 16S rRNA are reversely complementary to another region to form an SHS, which allows the downstream sequence to loop back and anneal at the SHS to prime the synthesis of its complementary strand, yielding a palindromic sequence that can form a hairpin-like structure.We identified a 16S rRNA that ended at the 4th nucleotide(nt) of the mt-tRNA-leu was dominant and thus should be the wild type. We also cloned a mouse Bcl2-Nek9 chimeric cDNA that contained a 5-nt unmatchable sequence between the two partners, contained two copies of the reverse primer in the same direction but did not contain the forward primer, making it unclear how this Bcl2-Nek9 was formed and amplified. Moreover, a cDNA was amplified because one primer has 4 nts matched to the template, suggesting that there may be many more artificial cDNAs than we have realized, because the nuclear and mt genomes have many more 4-nt than 5-nt or longer homologues. Altogether, the chimeric cDNAs we cloned are good examples suggesting that many cDNAs may be artifacts due to SHS-caused mis-priming and thus greater caution should be taken when new sequence is obtained from a technique involving DNA polymerization. PMID:27148738

Human adenovirus serotype 12 virion precursors pMu and pVI are cleaved at amino-terminal and carboxy-terminal sites that conform to the adenovirus 2 endoproteinase cleavage consensus sequence.

PubMed

Freimuth, P; Anderson, C W

1993-03-01

The sequence of a 1158-base pair fragment of the human adenovirus serotype 12 (Ad12) genome was determined. This segment encodes the precursors for virion components Mu and VI. Both Ad12 precursors contain two sequences that conform to a consensus sequence motif for cleavage by the endoproteinase of adenovirus 2 (Ad2). Analysis of the amino terminus of VI and of the peptide fragments found in Ad12 virions demonstrated that these sites are cleaved during Ad12 maturation. This observation suggests that the recognition motif for adenovirus endoproteinases is highly conserved among human serotypes. The adenovirus 2 endoproteinase polypeptide requires additional co-factors for activity (C. W. Anderson, Protein Expression Purif., 1993, 4, 8-15). Synthetic Ad12 or Ad2 pVI carboxy-terminal peptides each permitted efficient cleavage of an artificial endoproteinase substrate by recombinant Ad2 endoproteinase polypeptide.

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.

Transcriptional "silencer" element in rat repetitive sequences associated with the rat insulin 1 gene locus.

PubMed Central

Laimins, L; Holmgren-König, M; Khoury, G

1986-01-01

The enhancer elements from either simian virus 40 or murine sarcoma virus activate the expression of a transfected rat insulin 1 (rI1) gene when placed within 2.0 kilobases or less of the rI1 gene cap site. Inclusion of 4.0 kilobases of upstream rI1 sequence, however, results in a substantial reduction in the enhancer-dependent insulin gene expression. These observations suggested that a negative transcriptional regulatory element was present between 2.0 and 4.0 kilobases of the rI1 sequence. To test this notion, we employed a heterologous enhancer-dependent transcription assay in which the simian virus 40 72-base-pair repeat is linked to a human beta-globin gene. Addition of the upstream rI1 element to this system decreased the level of enhancer-dependent beta-globin transcription by a factor of 5 to 15. This rI1 "silencer" element functions in a manner relatively independent of position and orientation and requires a cis-dependent relationship to the transcription unit on which it acts. Thus, the silencer sequence seems to have a number of the characteristics of enhancer elements, and we suggest that it may function by the converse of the enhancer mechanism. The rI1 silencer sequence was identified as a member of a long interspersed rat repetitive family. Thus, a potential role for certain repetitive sequences interspersed throughout the eukaryotic genome may be to regulate gene expression by retaining transcriptional activity within defined domains. Images PMID:3010279

Bihemispheric Transcranial Direct Current Stimulation Enhances Effector-Independent Representations of Motor Synergy and Sequence Learning

PubMed Central

Husain, Masud; Wiestler, Tobias; Diedrichsen, Jörn

2014-01-01

Complex manual tasks—everything from buttoning up a shirt to playing the piano—fundamentally involve two components: (1) generating specific patterns of muscle activity (here, termed “synergies”); and (2) stringing these into purposeful sequences. Although transcranial direct current stimulation (tDCS) of the primary motor cortex (M1) has been found to increase the learning of motor sequences, it is unknown whether it can similarly facilitate motor synergy learning. Here, we determined the effects of tDCS on the learning of motor synergies using a novel hand configuration task that required the production of difficult muscular activation patterns. Bihemispheric tDCS was applied to M1 of healthy, right-handed human participants during 4 d of repetitive left-hand configuration training in a double-blind design. tDCS augmented synergy learning, leading subsequently to faster and more synchronized execution. This effect persisted for at least 4 weeks after training. Qualitatively similar tDCS-associated improvements occurred during training of finger sequences in a separate subject cohort. We additionally determined whether tDCS only improved the acquisition of motor memories for specific synergies/sequences or whether it also facilitated more general parts of the motor representations, which could be transferred to novel movements. Critically, we observed that tDCS effects generalized to untrained hand configurations and untrained finger sequences (i.e., were nonspecific), as well as to the untrained hand (i.e., were effector-independent). Hence, bihemispheric tDCS may be a promising adjunct to neurorehabilitative training regimes, in which broad transfer to everyday tasks is highly desirable. PMID:24431461

Shuttle OFT Level C navigation requirements

NASA Technical Reports Server (NTRS)

1980-01-01

Detailed requirements for the orbital operations computer loads, OPS 2, and OPS 8 are given. These requirements represent the total on-orbit/rendezvous navigation baseline requirements for the following principal functions: on-orbital/rendezvous navigation sequencer; on-orbit/rendezvous UPP sequencer; on-orbit rendezvous navigation; on-orbit prediction; on-orbit user parameter processing; and landing Site update.

Anaerobic sequencing batch reactor in pilot scale for treatment of tofu industry wastewater

NASA Astrophysics Data System (ADS)

Rahayu, Suparni Setyowati; Purwanto, Budiyono

2015-12-01

The small industry of tofu production process releases the waste water without being processed first, and the wastewater is directly discharged into water. In this study, Anaerobic Sequencing Batch Reactor in Pilot Scale for Treatment of Tofu Industry was developed through an anaerobic process to produce biogas as one kind of environmentally friendly renewable energy which can be developed into the countryside. The purpose of this study was to examine the fundamental characteristics of organic matter elimination of industrial wastewater with small tofu effective method and utilize anaerobic active sludge with Anaerobic Sequencing Bath Reactor (ASBR) to get rural biogas as an energy source. The first factor is the amount of the active sludge concentration which functions as the decomposers of organic matter and controlling selectivity allowance to degrade organic matter. The second factor is that HRT is the average period required substrate to react with the bacteria in the Anaerobic Sequencing Bath Reactor (ASBR).The results of processing the waste of tofu production industry using ASBR reactor with active sludge additions as starter generates cumulative volume of 5814.4 mL at HRT 5 days so that in this study it is obtained the conversion 0.16 L of CH4/g COD and produce biogas containing of CH4: 81.23% and CO2: 16.12%. The wastewater treatment of tofu production using ASBR reactor is able to produce renewable energy that has economic value as well as environmentally friendly by nature.

A Universal Next-Generation Sequencing Protocol To Generate Noninfectious Barcoded cDNA Libraries from High-Containment RNA Viruses

PubMed Central

Moser, Lindsey A.; Ramirez-Carvajal, Lisbeth; Puri, Vinita; Pauszek, Steven J.; Matthews, Krystal; Dilley, Kari A.; Mullan, Clancy; McGraw, Jennifer; Khayat, Michael; Beeri, Karen; Yee, Anthony; Dugan, Vivien; Heise, Mark T.; Frieman, Matthew B.; Rodriguez, Luis L.; Bernard, Kristen A.; Wentworth, David E.

2016-01-01

ABSTRACT Several biosafety level 3 and/or 4 (BSL-3/4) pathogens are high-consequence, single-stranded RNA viruses, and their genomes, when introduced into permissive cells, are infectious. Moreover, many of these viruses are select agents (SAs), and their genomes are also considered SAs. For this reason, cDNAs and/or their derivatives must be tested to ensure the absence of infectious virus and/or viral RNA before transfer out of the BSL-3/4 and/or SA laboratory. This tremendously limits the capacity to conduct viral genomic research, particularly the application of next-generation sequencing (NGS). Here, we present a sequence-independent method to rapidly amplify viral genomic RNA while simultaneously abolishing both viral and genomic RNA infectivity across multiple single-stranded positive-sense RNA (ssRNA+) virus families. The process generates barcoded DNA amplicons that range in length from 300 to 1,000 bp, which cannot be used to rescue a virus and are stable to transport at room temperature. Our barcoding approach allows for up to 288 barcoded samples to be pooled into a single library and run across various NGS platforms without potential reconstitution of the viral genome. Our data demonstrate that this approach provides full-length genomic sequence information not only from high-titer virion preparations but it can also recover specific viral sequence from samples with limited starting material in the background of cellular RNA, and it can be used to identify pathogens from unknown samples. In summary, we describe a rapid, universal standard operating procedure that generates high-quality NGS libraries free of infectious virus and infectious viral RNA. IMPORTANCE This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. Our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for NGS library construction. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories. PMID:27822536

Molecular Diagnosis of Long-QT syndrome at 10 Days of Life by Rapid Whole Genome Sequencing

PubMed Central

Priest, James R.; Ceresnak, Scott R.; Dewey, Frederick E.; Malloy-Walton, Lindsey E.; Dunn, Kyla; Grove, Megan E.; Perez, Marco V.; Maeda, Katsuhide; Dubin, Anne M.; Ashley, Euan A.

2014-01-01

Background The advent of clinical next generation sequencing is rapidly changing the landscape of rare disease medicine. Molecular diagnosis of long QT syndrome (LQTS) can impact clinical management, including risk stratification and selection of pharmacotherapy based on the type of ion channel affected, but results from current gene panel testing requires 4 to 16 weeks before return to clinicians. Objective A term female infant presented with 2:1 atrioventricular block and ventricular arrhythmias consistent with perinatal LQTS, requiring aggressive treatment including epicardial pacemaker, and cardioverter-defibrillator implantation and sympathectomy on day of life two. We sought to provide a rapid molecular diagnosis for optimization of treatment strategies. Methods We performed CLIA-certified rapid whole genome sequencing (WGS) with a speed-optimized bioinformatics platform to achieve molecular diagnosis at 10 days of life. Results We detected a known pathogenic variant in KCNH2 that was demonstrated to be paternally inherited by followup genotyping. The unbiased assessment of the entire catalog of human genes provided by whole genome sequencing revealed a maternally inherited variant of unknown significance in a novel gene. Conclusions Rapid clinical WGS provides faster and more comprehensive diagnostic information by 10 days of life than standard gene-panel testing. In selected clinical scenarios such as perinatal LQTS, rapid WGS may be able to provide more timely and clinically actionable information than a standard commercial test. PMID:24973560

RNAi screen for rapid therapeutic target identification in leukemia patients

PubMed Central

Tyner, Jeffrey W.; Deininger, Michael W.; Loriaux, Marc M.; Chang, Bill H.; Gotlib, Jason R.; Willis, Stephanie G.; Erickson, Heidi; Kovacsovics, Tibor; O'Hare, Thomas; Heinrich, Michael C.; Druker, Brian J.

2009-01-01

Targeted therapy has vastly improved outcomes in certain types of cancer. Extension of this paradigm across a broad spectrum of malignancies will require an efficient method to determine the molecular vulnerabilities of cancerous cells. Improvements in sequencing technology will soon enable high-throughput sequencing of entire genomes of cancer patients; however, determining the relevance of identified sequence variants will require complementary functional analyses. Here, we report an RNAi-assisted protein target identification (RAPID) technology that individually assesses targeting of each member of the tyrosine kinase gene family. We demonstrate that RAPID screening of primary leukemia cells from 30 patients identifies targets that are critical to survival of the malignant cells from 10 of these individuals. We identify known, activating mutations in JAK2 and K-RAS, as well as patient-specific sensitivity to down-regulation of FLT1, CSF1R, PDGFR, ROR1, EPHA4/5, JAK1/3, LMTK3, LYN, FYN, PTK2B, and N-RAS. We also describe a previously undescribed, somatic, activating mutation in the thrombopoietin receptor that is sensitive to down-stream pharmacologic inhibition. Hence, the RAPID technique can quickly identify molecular vulnerabilities in malignant cells. Combination of this technique with whole-genome sequencing will represent an ideal tool for oncogenic target identification such that specific therapies can be matched with individual patients. PMID:19433805

Connecting [NiFe]- and [FeFe]-Hydrogenases: Mixed-Valence Nickel-Iron Dithiolates With Rotated Structures

PubMed Central

Schilter, David; Rauchfuss, Thomas B.; Stein, Matthias

2012-01-01

A series of mixed-valence iron-nickel dithiolates is described that exhibits structures similar to those of mixed-valence diiron dithiolates. Interaction of tricarbonyl salt [(dppe)Ni(pdt)Fe(CO)3]BF4 ([1]BF4, dppe = Ph2PCH2CH2PPh2, pdtH2 = HSCH2CH2CH2SH) with P-donor ligands (L) afforded the substituted derivatives [(dppe)Ni(pdt)Fe(CO)2L]BF4 incorporating L = PHCy2 ([1a]BF4), PPh(NEt2)2 ([1b]BF4), P(NMe2)3 ([1c]BF4), P(i-Pr)3 ([1d]BF4) and PCy3 ([1e]BF4). The related precursor [(dcpe)Ni(pdt)Fe(CO)3]BF4 ([2]BF4, dcpe = Cy2PCH2CH2PCy2) gave the more electron-rich family of compounds [(dcpe)Ni(pdt)Fe(CO)2L]BF4 for L = PPh2(2-pyridyl) ([2a]BF4), PPh3 ([2b]BF4) and PCy3 ([2c]BF4). For bulky and strongly basic monophosphorus ligands, the salts feature distorted Fe coordination geometries: crystallographic analyses of [1e]BF4 and [2c]BF4 showed they adopt ‘rotated’ Fe(I) centers, in which PCy3 occupies a basal site and one CO ligand partially bridges the Ni and Fe centers. Like the undistorted mixed-valence derivatives, the new class of complexes are described as Ni(II)Fe(I) (S = ½) systems according to EPR spectroscopy, although with attenuated 31P hyperfine interactions. DFT calculations using the BP86, B3LYP, and PBE0 exchange-correlation functionals agree with the structural and spectroscopic data, suggesting that the spin for [1e]+ is localized in a Fe(I)-centered d(z2) orbital, orthogonal to the Fe-P bond. The PCy3 complexes, rare examples of species featuring ‘rotated’ Fe centers, both structurally and spectroscopically resemble mixed-valence diiron dithiolates. Also reproducing the NiS2Fe core of the [NiFe]-H2ase active site, the hybrid models incorporate key features of the two major classes of H2ase. Furthermore, cyclic voltammetry experiments suggest that the highly basic phosphine ligands enable a second oxidation corresponding to the couple [(dxpe)Ni(pdt)Fe(CO)2L]+/2+. The resulting unsaturated 32e− dications represent the closest approach to modeling the highly electrophilic Ni-SIa state. In the case of L = PPh2(2-pyridyl) chelation of this ligand accompanies the second oxidation. PMID:22838645

Phage T4 SegB protein is a homing endonuclease required for the preferred inheritance of T4 tRNA gene region occurring in co-infection with a related phage

PubMed Central

Brok-Volchanskaya, Vera S.; Kadyrov, Farid A.; Sivogrivov, Dmitry E.; Kolosov, Peter M.; Sokolov, Andrey S.; Shlyapnikov, Michael G.; Kryukov, Valentine M.; Granovsky, Igor E.

2008-01-01

Homing endonucleases initiate nonreciprocal transfer of DNA segments containing their own genes and the flanking sequences by cleaving the recipient DNA. Bacteriophage T4 segB gene, which is located in a cluster of tRNA genes, encodes a protein of unknown function, homologous to homing endonucleases of the GIY-YIG family. We demonstrate that SegB protein is a site-specific endonuclease, which produces mostly 3′ 2-nt protruding ends at its DNA cleavage site. Analysis of SegB cleavage sites suggests that SegB recognizes a 27-bp sequence. It contains 11-bp conserved sequence, which corresponds to a conserved motif of tRNA TψC stem-loop, whereas the remainder of the recognition site is rather degenerate. T4-related phages T2L, RB1 and RB3 contain tRNA gene regions that are homologous to that of phage T4 but lack segB gene and several tRNA genes. In co-infections of phages T4 and T2L, segB gene is inherited with nearly 100% of efficiency. The preferred inheritance depends absolutely on the segB gene integrity and is accompanied by the loss of the T2L tRNA gene region markers. We suggest that SegB is a homing endonuclease that functions to ensure spreading of its own gene and the surrounding tRNA genes among T4-related phages. PMID:18281701

The Second Subunit of DNA Polymerase Delta Is Required for Genomic Stability and Epigenetic Regulation1[OPEN

PubMed Central

Cheng, Jinkui; Lai, Jinsheng; Gong, Zhizhong

2016-01-01

DNA polymerase δ plays crucial roles in DNA repair and replication as well as maintaining genomic stability. However, the function of POLD2, the second small subunit of DNA polymerase δ, has not been characterized yet in Arabidopsis (Arabidopsis thaliana). During a genetic screen for release of transcriptional gene silencing, we identified a mutation in POLD2. Whole-genome bisulfite sequencing indicated that POLD2 is not involved in the regulation of DNA methylation. POLD2 genetically interacts with Ataxia Telangiectasia-mutated and Rad3-related and DNA polymerase α. The pold2-1 mutant exhibits genomic instability with a high frequency of homologous recombination. It also exhibits hypersensitivity to DNA-damaging reagents and short telomere length. Whole-genome chromatin immunoprecipitation sequencing and RNA sequencing analyses suggest that pold2-1 changes H3K27me3 and H3K4me3 modifications, and these changes are correlated with the gene expression levels. Our study suggests that POLD2 is required for maintaining genome integrity and properly establishing the epigenetic markers during DNA replication to modulate gene expression. PMID:27208288

Clinical Validation of Targeted Next Generation Sequencing for Colon and Lung Cancers

PubMed Central

D’Haene, Nicky; Le Mercier, Marie; De Nève, Nancy; Blanchard, Oriane; Delaunoy, Mélanie; El Housni, Hakim; Dessars, Barbara; Heimann, Pierre; Remmelink, Myriam; Demetter, Pieter; Tejpar, Sabine; Salmon, Isabelle

2015-01-01

Objective Recently, Next Generation Sequencing (NGS) has begun to supplant other technologies for gene mutation testing that is now required for targeted therapies. However, transfer of NGS technology to clinical daily practice requires validation. Methods We validated the Ion Torrent AmpliSeq Colon and Lung cancer panel interrogating 1850 hotspots in 22 genes using the Ion Torrent Personal Genome Machine. First, we used commercial reference standards that carry mutations at defined allelic frequency (AF). Then, 51 colorectal adenocarcinomas (CRC) and 39 non small cell lung carcinomas (NSCLC) were retrospectively analyzed. Results Sensitivity and accuracy for detecting variants at an AF >4% was 100% for commercial reference standards. Among the 90 cases, 89 (98.9%) were successfully sequenced. Among the 86 samples for which NGS and the reference test were both informative, 83 showed concordant results between NGS and the reference test; i.e. KRAS and BRAF for CRC and EGFR for NSCLC, with the 3 discordant cases each characterized by an AF <10%. Conclusions Overall, the AmpliSeq colon/lung cancer panel was specific and sensitive for mutation analysis of gene panels and can be incorporated into clinical daily practice. PMID:26366557

The eIF4F and eIFiso4F Complexes of Plants: An Evolutionary Perspective

PubMed Central

Patrick, Ryan M.; Browning, Karen S.

2012-01-01

Translation initiation in eukaryotes requires a number of initiation factors to recruit the assembled ribosome to mRNA. The eIF4F complex plays a key role in initiation and is a common target point for regulation of protein synthesis. Most work on the translation machinery of plants to date has focused on flowering plants, which have both the eIF4F complex (eIF4E and eIF4G) as well as the plant-specific eIFiso4F complex (eIFiso4E and eIFiso4G). The increasing availability of plant genome sequence data has made it possible to trace the evolutionary history of these two complexes in plants, leading to several interesting discoveries. eIFiso4G is conserved throughout plants, while eIFiso4E only appears with the evolution of flowering plants. The eIF4G N-terminus, which has been difficult to annotate, appears to be well conserved throughout the plant lineage and contains two motifs of unknown function. Comparison of eIFiso4G and eIF4G sequence data suggests conserved features unique to eIFiso4G and eIF4G proteins. These findings have answered some questions about the evolutionary history of the two eIF4F complexes of plants, while raising new ones. PMID:22611336

Characterisation of the transcriptome of a wild great tit Parus major population by next generation sequencing

PubMed Central

2011-01-01

Background The recent development of next generation sequencing technologies has made it possible to generate very large amounts of sequence data in species with little or no genome information. Combined with the large phenotypic databases available for wild and non-model species, these data will provide an unprecedented opportunity to "genomicise" ecological model organisms and establish the genetic basis of quantitative traits in natural populations. Results This paper describes the sequencing, de novo assembly and analysis from the transcriptome of eight tissues of ten wild great tits. Approximately 4.6 million sequences and 1.4 billion bases of DNA were generated and assembled into 95,979 contigs, one third of which aligned with known Taeniopygia guttata (zebra finch) and Gallus gallus (chicken) transcripts. The majority (78%) of the remaining contigs aligned within or very close to regions of the zebra finch genome containing known genes, suggesting that they represented precursor mRNA rather than untranscribed genomic DNA. More than 35,000 single nucleotide polymorphisms and 10,000 microsatellite repeats were identified. Eleven percent of contigs were expressed in every tissue, while twenty one percent of contigs were expressed in only one tissue. The function of those contigs with strong evidence for tissue specific expression and contigs expressed in every tissue was inferred from the gene ontology (GO) terms associated with these contigs; heart and pancreas had the highest number of highly tissue specific GO terms (21.4% and 28.5% respectively). Conclusions In summary, the transcriptomic data generated in this study will contribute towards efforts to assemble and annotate the great tit genome, as well as providing the markers required to perform gene mapping studies in wild populations. PMID:21635727

XLID-Causing Mutations and Associated Genes Challenged in Light of Data From Large-Scale Human Exome Sequencing

PubMed Central

Piton, Amélie; Redin, Claire; Mandel, Jean-Louis

2013-01-01

Because of the unbalanced sex ratio (1.3–1.4 to 1) observed in intellectual disability (ID) and the identification of large ID-affected families showing X-linked segregation, much attention has been focused on the genetics of X-linked ID (XLID). Mutations causing monogenic XLID have now been reported in over 100 genes, most of which are commonly included in XLID diagnostic gene panels. Nonetheless, the boundary between true mutations and rare non-disease-causing variants often remains elusive. The sequencing of a large number of control X chromosomes, required for avoiding false-positive results, was not systematically possible in the past. Such information is now available thanks to large-scale sequencing projects such as the National Heart, Lung, and Blood (NHLBI) Exome Sequencing Project, which provides variation information on 10,563 X chromosomes from the general population. We used this NHLBI cohort to systematically reassess the implication of 106 genes proposed to be involved in monogenic forms of XLID. We particularly question the implication in XLID of ten of them (AGTR2, MAGT1, ZNF674, SRPX2, ATP6AP2, ARHGEF6, NXF5, ZCCHC12, ZNF41, and ZNF81), in which truncating variants or previously published mutations are observed at a relatively high frequency within this cohort. We also highlight 15 other genes (CCDC22, CLIC2, CNKSR2, FRMPD4, HCFC1, IGBP1, KIAA2022, KLF8, MAOA, NAA10, NLGN3, RPL10, SHROOM4, ZDHHC15, and ZNF261) for which replication studies are warranted. We propose that similar reassessment of reported mutations (and genes) with the use of data from large-scale human exome sequencing would be relevant for a wide range of other genetic diseases. PMID:23871722

Emergence of Sequence Type 779 Methicillin-Resistant Staphylococcus aureus Harboring a Novel Pseudo Staphylococcal Cassette Chromosome mec (SCCmec)-SCC-SCCCRISPR Composite Element in Irish Hospitals

PubMed Central

Kinnevey, Peter M.; Shore, Anna C.; Brennan, Grainne I.; Sullivan, Derek J.; Ehricht, Ralf; Monecke, Stefan; Slickers, Peter

2013-01-01

Methicillin-resistant Staphylococcus aureus (MRSA) has been a major cause of nosocomial infection in Irish hospitals for 4 decades, and replacement of predominant MRSA clones has occurred several times. An MRSA isolate recovered in 2006 as part of a larger study of sporadic MRSA exhibited a rare spa (t878) and multilocus sequence (ST779) type and was nontypeable by PCR- and DNA microarray-based staphylococcal cassette chromosome mec (SCCmec) element typing. Whole-genome sequencing revealed the presence of a novel 51-kb composite island (CI) element with three distinct domains, each flanked by direct repeat and inverted repeat sequences, including (i) a pseudo SCCmec element (16.3 kb) carrying mecA with a novel mec class region, a fusidic acid resistance gene (fusC), and two copper resistance genes (copB and copC) but lacking ccr genes; (ii) an SCC element (17.5 kb) carrying a novel ccrAB4 allele; and (iii) an SCC element (17.4 kb) carrying a novel ccrC allele and a clustered regularly interspaced short palindromic repeat (CRISPR) region. The novel CI was subsequently identified by PCR in an additional 13 t878/ST779 MRSA isolates, six from bloodstream infections, recovered between 2006 and 2011 in 11 hospitals. Analysis of open reading frames (ORFs) carried by the CI showed amino acid sequence similarity of 44 to 100% to ORFs from S. aureus and coagulase-negative staphylococci (CoNS). These findings provide further evidence of genetic transfer between S. aureus and CoNS and show how this contributes to the emergence of novel SCCmec elements and MRSA strains. Ongoing surveillance of this MRSA strain is warranted and will require updating of currently used SCCmec typing methods. PMID:23147725

A Poisson hierarchical modelling approach to detecting copy number variation in sequence coverage data.

PubMed

Sepúlveda, Nuno; Campino, Susana G; Assefa, Samuel A; Sutherland, Colin J; Pain, Arnab; Clark, Taane G

2013-02-26

The advent of next generation sequencing technology has accelerated efforts to map and catalogue copy number variation (CNV) in genomes of important micro-organisms for public health. A typical analysis of the sequence data involves mapping reads onto a reference genome, calculating the respective coverage, and detecting regions with too-low or too-high coverage (deletions and amplifications, respectively). Current CNV detection methods rely on statistical assumptions (e.g., a Poisson model) that may not hold in general, or require fine-tuning the underlying algorithms to detect known hits. We propose a new CNV detection methodology based on two Poisson hierarchical models, the Poisson-Gamma and Poisson-Lognormal, with the advantage of being sufficiently flexible to describe different data patterns, whilst robust against deviations from the often assumed Poisson model. Using sequence coverage data of 7 Plasmodium falciparum malaria genomes (3D7 reference strain, HB3, DD2, 7G8, GB4, OX005, and OX006), we showed that empirical coverage distributions are intrinsically asymmetric and overdispersed in relation to the Poisson model. We also demonstrated a low baseline false positive rate for the proposed methodology using 3D7 resequencing data and simulation. When applied to the non-reference isolate data, our approach detected known CNV hits, including an amplification of the PfMDR1 locus in DD2 and a large deletion in the CLAG3.2 gene in GB4, and putative novel CNV regions. When compared to the recently available FREEC and cn.MOPS approaches, our findings were more concordant with putative hits from the highest quality array data for the 7G8 and GB4 isolates. In summary, the proposed methodology brings an increase in flexibility, robustness, accuracy and statistical rigour to CNV detection using sequence coverage data.

TH-EF-BRA-06: A Novel Retrospective 3D K-Space Sorting 4D-MRI Technique Using a Radial K-Space Acquisition MRI Sequence

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

Liu, Y; Subashi, E; Yin, F

Purpose: Current retrospective 4D-MRI provides superior tumor-to-tissue contrast and accurate respiratory motion information for radiotherapy motion management. The developed 4D-MRI techniques based on 2D-MRI image sorting require a high frame-rate of the MR sequences. However, several MRI sequences provide excellent image quality but have low frame-rate. This study aims at developing a novel retrospective 3D k-space sorting 4D-MRI technique using radial k-space acquisition MRI sequences to improve 4D-MRI image quality and temporal-resolution for imaging irregular organ/tumor respiratory motion. Methods: The method is based on a RF-spoiled, steady-state, gradient-recalled sequence with minimal echo time. A 3D radial k-space data acquisition trajectorymore » was used for sampling the datasets. Each radial spoke readout data line starts from the 3D center of Field-of-View. Respiratory signal can be extracted from the k-space center data point of each spoke. The spoke data was sorted based on its self-synchronized respiratory signal using phase sorting. Subsequently, 3D reconstruction was conducted to generate the time-resolved 4D-MRI images. As a feasibility study, this technique was implemented on a digital human phantom XCAT. The respiratory motion was controlled by an irregular motion profile. To validate using k-space center data as a respiratory surrogate, we compared it with the XCAT input controlling breathing profile. Tumor motion trajectories measured on reconstructed 4D-MRI were compared to the average input trajectory. The mean absolute amplitude difference (D) was calculated. Results: The signal extracted from k-space center data matches well with the input controlling respiratory profile of XCAT. The relative amplitude error was 8.6% and the relative phase error was 3.5%. XCAT 4D-MRI demonstrated a clear motion pattern with little serrated artifacts. D of tumor trajectories was 0.21mm, 0.23mm and 0.23mm in SI, AP and ML directions, respectively. Conclusion: A novel retrospective 3D k-space sorting 4D-MRI technique has been developed and evaluated on human digital phantom. NIH (1R21CA165384-01A1)« less

Meta4: a web application for sharing and annotating metagenomic gene predictions using web services.

PubMed

Richardson, Emily J; Escalettes, Franck; Fotheringham, Ian; Wallace, Robert J; Watson, Mick

2013-01-01

Whole-genome shotgun metagenomics experiments produce DNA sequence data from entire ecosystems, and provide a huge amount of novel information. Gene discovery projects require up-to-date information about sequence homology and domain structure for millions of predicted proteins to be presented in a simple, easy-to-use system. There is a lack of simple, open, flexible tools that allow the rapid sharing of metagenomics datasets with collaborators in a format they can easily interrogate. We present Meta4, a flexible and extensible web application that can be used to share and annotate metagenomic gene predictions. Proteins and predicted domains are stored in a simple relational database, with a dynamic front-end which displays the results in an internet browser. Web services are used to provide up-to-date information about the proteins from homology searches against public databases. Information about Meta4 can be found on the project website, code is available on Github, a cloud image is available, and an example implementation can be seen at.

Sequence requirement of the ade6-4095 meiotic recombination hotspot in Schizosaccharomyces pombe.

PubMed

Foulis, Steven J; Fowler, Kyle R; Steiner, Walter W

2018-02-01

Homologous recombination occurs at a greatly elevated frequency in meiosis compared to mitosis and is initiated by programmed double-strand DNA breaks (DSBs). DSBs do not occur at uniform frequency throughout the genome in most organisms, but occur preferentially at a limited number of sites referred to as hotspots. The location of hotspots have been determined at nucleotide-level resolution in both the budding and fission yeasts, and while several patterns have emerged regarding preferred locations for DSB hotspots, it remains unclear why particular sites experience DSBs at much higher frequency than other sites with seemingly similar properties. Short sequence motifs, which are often sites for binding of transcription factors, are known to be responsible for a number of hotspots. In this study we identified the minimum sequence required for activity of one of such motif identified in a screen of random sequences capable of producing recombination hotspots. The experimentally determined sequence, GGTCTRGACC, closely matches the previously inferred sequence. Full hotspot activity requires an effective sequence length of 9.5 bp, whereas moderate activity requires an effective sequence length of approximately 8.2 bp and shows significant association with DSB hotspots. In combination with our previous work, this result is consistent with a large number of different sequence motifs capable of producing recombination hotspots, and supports a model in which hotspots can be rapidly regenerated by mutation as they are lost through recombination.

The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing.

PubMed

Binladen, Jonas; Gilbert, M Thomas P; Bollback, Jonathan P; Panitz, Frank; Bendixen, Christian; Nielsen, Rasmus; Willerslev, Eske

2007-02-14

The invention of the Genome Sequence 20 DNA Sequencing System (454 parallel sequencing platform) has enabled the rapid and high-volume production of sequence data. Until now, however, individual emulsion PCR (emPCR) reactions and subsequent sequencing runs have been unable to combine template DNA from multiple individuals, as homologous sequences cannot be subsequently assigned to their original sources. We use conventional PCR with 5'-nucleotide tagged primers to generate homologous DNA amplification products from multiple specimens, followed by sequencing through the high-throughput Genome Sequence 20 DNA Sequencing System (GS20, Roche/454 Life Sciences). Each DNA sequence is subsequently traced back to its individual source through 5'tag-analysis. We demonstrate that this new approach enables the assignment of virtually all the generated DNA sequences to the correct source once sequencing anomalies are accounted for (miss-assignment rate<0.4%). Therefore, the method enables accurate sequencing and assignment of homologous DNA sequences from multiple sources in single high-throughput GS20 run. We observe a bias in the distribution of the differently tagged primers that is dependent on the 5' nucleotide of the tag. In particular, primers 5' labelled with a cytosine are heavily overrepresented among the final sequences, while those 5' labelled with a thymine are strongly underrepresented. A weaker bias also exists with regards to the distribution of the sequences as sorted by the second nucleotide of the dinucleotide tags. As the results are based on a single GS20 run, the general applicability of the approach requires confirmation. However, our experiments demonstrate that 5'primer tagging is a useful method in which the sequencing power of the GS20 can be applied to PCR-based assays of multiple homologous PCR products. The new approach will be of value to a broad range of research areas, such as those of comparative genomics, complete mitochondrial analyses, population genetics, and phylogenetics.

Sequence specificity of the human mRNA N6-adenosine methylase in vitro.

PubMed Central

Harper, J E; Miceli, S M; Roberts, R J; Manley, J L

1990-01-01

N6-adenosine methylation is a frequent modification of mRNAs and their precursors, but little is known about the mechanism of the reaction or the function of the modification. To explore these questions, we developed conditions to examine N6-adenosine methylase activity in HeLa cell nuclear extracts. Transfer of the methyl group from S-[3H methyl]-adenosylmethionine to unlabeled random copolymer RNA substrates of varying ribonucleotide composition revealed a substrate specificity consistent with a previously deduced consensus sequence, Pu[G greater than A]AC[A/C/U]. 32-P labeled RNA substrates of defined sequence were used to examine the minimum sequence requirements for methylation. Each RNA was 20 nucleotides long, and contained either the core consensus sequence GGACU, or some variation of this sequence. RNAs containing GGACU, either in single or multiple copies, were good substrates for methylation, whereas RNAs containing single base substitutions within the GGACU sequence gave dramatically reduced methylation. These results demonstrate that the N6-adenosine methylase has a strict sequence specificity, and that there is no requirement for extended sequences or secondary structures for methylation. Recognition of this sequence does not require an RNA component, as micrococcal nuclease pretreatment of nuclear extracts actually increased methylation efficiency. Images PMID:2216767

Perchlorate reduction by hydrogen autotrophic bacteria and microbial community analysis using high-throughput sequencing.

PubMed

Wan, Dongjin; Liu, Yongde; Niu, Zhenhua; Xiao, Shuhu; Li, Daorong

2016-02-01

Hydrogen autotrophic reduction of perchlorate have advantages of high removal efficiency and harmless to drinking water. But so far the reported information about the microbial community structure was comparatively limited, changes in the biodiversity and the dominant bacteria during acclimation process required detailed study. In this study, perchlorate-reducing hydrogen autotrophic bacteria were acclimated by hydrogen aeration from activated sludge. For the first time, high-throughput sequencing was applied to analyze changes in biodiversity and the dominant bacteria during acclimation process. The Michaelis-Menten model described the perchlorate reduction kinetics well. Model parameters q(max) and K(s) were 2.521-3.245 (mg ClO4(-)/gVSS h) and 5.44-8.23 (mg/l), respectively. Microbial perchlorate reduction occurred across at pH range 5.0-11.0; removal was highest at pH 9.0. The enriched mixed bacteria could use perchlorate, nitrate and sulfate as electron accepter, and the sequence of preference was: NO3(-) > ClO4(-) > SO4(2-). Compared to the feed culture, biodiversity decreased greatly during acclimation process, the microbial community structure gradually stabilized after 9 acclimation cycles. The Thauera genus related to Rhodocyclales was the dominated perchlorate reducing bacteria (PRB) in the mixed culture.

High-quality-draft genome sequence of the fermenting bacterium Anaerobium acetethylicum type strain GluBS11T (DSM 29698)

DOE PAGES

Patil, Yogita; Müller, Nicolai; Schink, Bernhard; ...

2017-02-20

Anaerobium acetethylicum strain GluBS11 T belongs to the family Lachnospiraceae within the order Clostridiales. It is a Gram-positive, non-motile and strictly anaerobic bacterium isolated from biogas slurry that was originally enriched with gluconate as carbon source (Patil, et al., Int J Syst Evol Microbiol 65:3289-3296, 2015). Here we describe the draft genome sequence of strain GluBS11 T and provide a detailed insight into its physiological and metabolic features. The draft genome sequence generated 4,609,043 bp, distributed among 105 scaffolds assembled using the SPAdes genome assembler method. It comprises in total 4,132 genes, of which 4,008 were predicted to be proteinmore » coding genes, 124 RNA genes and 867 pseudogenes. The content was 43.51 mol %. The annotated genome of strain GluBS11 T contains putative genes coding for the pentose phosphate pathway, the Embden-Meyerhoff-Parnas pathway, the Entner-Doudoroff pathway and the tricarboxylic acid cycle. The genome revealed the presence of most of the necessary genes required for the fermentation of glucose and gluconate to acetate, ethanol, and hydrogen gas. However, a candidate gene for production of formate was not identified.« less

High-quality-draft genome sequence of the fermenting bacterium Anaerobium acetethylicum type strain GluBS11T (DSM 29698)

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

Patil, Yogita; Müller, Nicolai; Schink, Bernhard

Anaerobium acetethylicum strain GluBS11 T belongs to the family Lachnospiraceae within the order Clostridiales. It is a Gram-positive, non-motile and strictly anaerobic bacterium isolated from biogas slurry that was originally enriched with gluconate as carbon source (Patil, et al., Int J Syst Evol Microbiol 65:3289-3296, 2015). Here we describe the draft genome sequence of strain GluBS11 T and provide a detailed insight into its physiological and metabolic features. The draft genome sequence generated 4,609,043 bp, distributed among 105 scaffolds assembled using the SPAdes genome assembler method. It comprises in total 4,132 genes, of which 4,008 were predicted to be proteinmore » coding genes, 124 RNA genes and 867 pseudogenes. The content was 43.51 mol %. The annotated genome of strain GluBS11 T contains putative genes coding for the pentose phosphate pathway, the Embden-Meyerhoff-Parnas pathway, the Entner-Doudoroff pathway and the tricarboxylic acid cycle. The genome revealed the presence of most of the necessary genes required for the fermentation of glucose and gluconate to acetate, ethanol, and hydrogen gas. However, a candidate gene for production of formate was not identified.« less

Isolation of a gene (pbsC) required for siderophore biosynthesis in fluorescent Pseudomonas sp. strain M114.

PubMed

Adams, C; Dowling, D N; O'Sullivan, D J; O'Gara, F

1994-06-03

An iron-regulated gene, pbsC, required for siderophore production in fluorescent Pseudomonas sp. strain M114 has been identified. A kanamycin-resistance cassette was inserted at specific restriction sites within a 7 kb genomic fragment of M114 DNA and by marker exchange two siderophore-negative mutants, designated M1 and M2, were isolated. The nucleotide sequence of approximately 4 kb of the region flanking the insertion sites was determined and a large open reading frame (ORF) extending for 2409 bp was identified. This gene was designated pbsC (pseudobactin synthesis C) and its putative protein product termed PbsC. PbsC was found to be homologous to a family of enzymes involved in the biosynthesis of secondary metabolites, including EntF of Escherichia coli. These enzymes are believed to act via ATP-dependent binding of AMP to their substrate. Several areas of high sequence homology between these proteins and PbsC were observed, including a conserved AMP-binding domain. The expression of pbsC is iron-regulated as revealed when a DNA fragment containing the upstream region was cloned in a promoter probe vector and conjugated into the wild-type strain, M114. The nucleotide sequence upstream of the putative translational start site contains a region homologous to previously defined -16 to -25 sequences of iron-regulated genes but did not contain an iron-box consensus sequence. It was noted that inactivation of the pbsC gene also affected other iron-regulated phenotypes of Pseudomonas M114.

A chain-retrieval model for voluntary task switching.

PubMed

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

2012-09-01

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

Inferring Short-Range Linkage Information from Sequencing Chromatograms

PubMed Central

Beggel, Bastian; Neumann-Fraune, Maria; Kaiser, Rolf; Verheyen, Jens; Lengauer, Thomas

2013-01-01

Direct Sanger sequencing of viral genome populations yields multiple ambiguous sequence positions. It is not straightforward to derive linkage information from sequencing chromatograms, which in turn hampers the correct interpretation of the sequence data. We present a method for determining the variants existing in a viral quasispecies in the case of two nearby ambiguous sequence positions by exploiting the effect of sequence context-dependent incorporation of dideoxynucleotides. The computational model was trained on data from sequencing chromatograms of clonal variants and was evaluated on two test sets of in vitro mixtures. The approach achieved high accuracies in identifying the mixture components of 97.4% on a test set in which the positions to be analyzed are only one base apart from each other, and of 84.5% on a test set in which the ambiguous positions are separated by three bases. In silico experiments suggest two major limitations of our approach in terms of accuracy. First, due to a basic limitation of Sanger sequencing, it is not possible to reliably detect minor variants with a relative frequency of no more than 10%. Second, the model cannot distinguish between mixtures of two or four clonal variants, if one of two sets of linear constraints is fulfilled. Furthermore, the approach requires repetitive sequencing of all variants that might be present in the mixture to be analyzed. Nevertheless, the effectiveness of our method on the two in vitro test sets shows that short-range linkage information of two ambiguous sequence positions can be inferred from Sanger sequencing chromatograms without any further assumptions on the mixture composition. Additionally, our model provides new insights into the established and widely used Sanger sequencing technology. The source code of our method is made available at http://bioinf.mpi-inf.mpg.de/publications/beggel/linkageinformation.zip. PMID:24376502

FRESCO: Referential compression of highly similar sequences.

PubMed

Wandelt, Sebastian; Leser, Ulf

2013-01-01

In many applications, sets of similar texts or sequences are of high importance. Prominent examples are revision histories of documents or genomic sequences. Modern high-throughput sequencing technologies are able to generate DNA sequences at an ever-increasing rate. In parallel to the decreasing experimental time and cost necessary to produce DNA sequences, computational requirements for analysis and storage of the sequences are steeply increasing. Compression is a key technology to deal with this challenge. Recently, referential compression schemes, storing only the differences between a to-be-compressed input and a known reference sequence, gained a lot of interest in this field. In this paper, we propose a general open-source framework to compress large amounts of biological sequence data called Framework for REferential Sequence COmpression (FRESCO). Our basic compression algorithm is shown to be one to two orders of magnitudes faster than comparable related work, while achieving similar compression ratios. We also propose several techniques to further increase compression ratios, while still retaining the advantage in speed: 1) selecting a good reference sequence; and 2) rewriting a reference sequence to allow for better compression. In addition,we propose a new way of further boosting the compression ratios by applying referential compression to already referentially compressed files (second-order compression). This technique allows for compression ratios way beyond state of the art, for instance,4,000:1 and higher for human genomes. We evaluate our algorithms on a large data set from three different species (more than 1,000 genomes, more than 3 TB) and on a collection of versions of Wikipedia pages. Our results show that real-time compression of highly similar sequences at high compression ratios is possible on modern hardware.

Plant MicroRNA Prediction by Supervised Machine Learning Using C5.0 Decision Trees.

PubMed

Williams, Philip H; Eyles, Rod; Weiller, Georg

2012-01-01

MicroRNAs (miRNAs) are nonprotein coding RNAs between 20 and 22 nucleotides long that attenuate protein production. Different types of sequence data are being investigated for novel miRNAs, including genomic and transcriptomic sequences. A variety of machine learning methods have successfully predicted miRNA precursors, mature miRNAs, and other nonprotein coding sequences. MirTools, mirDeep2, and miRanalyzer require "read count" to be included with the input sequences, which restricts their use to deep-sequencing data. Our aim was to train a predictor using a cross-section of different species to accurately predict miRNAs outside the training set. We wanted a system that did not require read-count for prediction and could therefore be applied to short sequences extracted from genomic, EST, or RNA-seq sources. A miRNA-predictive decision-tree model has been developed by supervised machine learning. It only requires that the corresponding genome or transcriptome is available within a sequence window that includes the precursor candidate so that the required sequence features can be collected. Some of the most critical features for training the predictor are the miRNA:miRNA(∗) duplex energy and the number of mismatches in the duplex. We present a cross-species plant miRNA predictor with 84.08% sensitivity and 98.53% specificity based on rigorous testing by leave-one-out validation.

De Novo Assembly and Characterization of Four Anthozoan (Phylum Cnidaria) Transcriptomes.

PubMed

Kitchen, Sheila A; Crowder, Camerron M; Poole, Angela Z; Weis, Virginia M; Meyer, Eli

2015-09-17

Many nonmodel species exemplify important biological questions but lack the sequence resources required to study the genes and genomic regions underlying traits of interest. Reef-building corals are famously sensitive to rising seawater temperatures, motivating ongoing research into their stress responses and long-term prospects in a changing climate. A comprehensive understanding of these processes will require extending beyond the sequenced coral genome (Acropora digitifera) to encompass diverse coral species and related anthozoans. Toward that end, we have assembled and annotated reference transcriptomes to develop catalogs of gene sequences for three scleractinian corals (Fungia scutaria, Montastraea cavernosa, Seriatopora hystrix) and a temperate anemone (Anthopleura elegantissima). High-throughput sequencing of cDNA libraries produced ~20-30 million reads per sample, and de novo assembly of these reads produced ~75,000-110,000 transcripts from each sample with size distributions (mean ~1.4 kb, N50 ~2 kb), comparable to the distribution of gene models from the coral genome (mean ~1.7 kb, N50 ~2.2 kb). Each assembly includes matches for more than half the gene models from A. digitifera (54-67%) and many reasonably complete transcripts (~5300-6700) spanning nearly the entire gene (ortholog hit ratios ≥0.75). The catalogs of gene sequences developed in this study made it possible to identify hundreds to thousands of orthologs across diverse scleractinian species and related taxa. We used these sequences for phylogenetic inference, recovering known relationships and demonstrating superior performance over phylogenetic trees constructed using single mitochondrial loci. The resources developed in this study provide gene sequences and genetic markers for several anthozoan species. To enhance the utility of these resources for the research community, we developed searchable databases enabling researchers to rapidly recover sequences for genes of interest. Our analysis of de novo assembly quality highlights metrics that we expect will be useful for evaluating the relative quality of other de novo transcriptome assemblies. The identification of orthologous sequences and phylogenetic reconstruction demonstrates the feasibility of these methods for clarifying the substantial uncertainties in the existing scleractinian phylogeny. Copyright © 2015 Kitchen et al.

Information and redundancy in the burial folding code of globular proteins within a wide range of shapes and sizes.

PubMed

Ferreira, Diogo C; van der Linden, Marx G; de Oliveira, Leandro C; Onuchic, José N; de Araújo, Antônio F Pereira

2016-04-01

Recent ab initio folding simulations for a limited number of small proteins have corroborated a previous suggestion that atomic burial information obtainable from sequence could be sufficient for tertiary structure determination when combined to sequence-independent geometrical constraints. Here, we use simulations parameterized by native burials to investigate the required amount of information in a diverse set of globular proteins comprising different structural classes and a wide size range. Burial information is provided by a potential term pushing each atom towards one among a small number L of equiprobable concentric layers. An upper bound for the required information is provided by the minimal number of layers L(min) still compatible with correct folding behavior. We obtain L(min) between 3 and 5 for seven small to medium proteins with 50 ≤ Nr ≤ 110 residues while for a larger protein with Nr = 141 we find that L ≥ 6 is required to maintain native stability. We additionally estimate the usable redundancy for a given L ≥ L(min) from the burial entropy associated to the largest folding-compatible fraction of "superfluous" atoms, for which the burial term can be turned off or target layers can be chosen randomly. The estimated redundancy for small proteins with L = 4 is close to 0.8. Our results are consistent with the above-average quality of burial predictions used in previous simulations and indicate that the fraction of approachable proteins could increase significantly with even a mild, plausible, improvement on sequence-dependent burial prediction or on sequence-independent constraints that augment the detectable redundancy during simulations. © 2016 Wiley Periodicals, Inc.

Analysis of Multilocus Sequence Typing and Virulence Characterization of Listeria monocytogenes Isolates from Chinese Retail Ready-to-Eat Food

PubMed Central

Wu, Shi; Wu, Qingping; Zhang, Jumei; Chen, Moutong; Guo, Weipeng

2016-01-01

Eighty Listeria monocytogenes isolates were obtained from Chinese retail ready-to-eat (RTE) food and were previously characterized with serotyping and antibiotic susceptibility tests. The aim of this study was to characterize the subtype and virulence potential of these L. monocytogenes isolates by multilocus sequence typing (MLST), virulence-associate genes, epidemic clones (ECs), and sequence analysis of the important virulence factor: internalin A (inlA). The result of MLST revealed that these L. monocytogenes isolates belonged to 14 different sequence types (STs). With the exception of four new STs (ST804, ST805, ST806, and ST807), all other STs observed in this study have been associated with human listeriosis and outbreaks to varying extents. Six virulence-associate genes (inlA, inlB, inlC, inlJ, hly, and llsX) were selected and their presence was investigated using PCR. All strains carried inlA, inlB, inlC, inlJ, and hly, whereas 38.8% (31/80) of strains harbored the listeriolysin S genes (llsX). A multiplex PCR assay was used to evaluate the presence of markers specific to epidemic clones of L. monocytogenes and identified 26.3% (21/80) of ECI in the 4b-4d-4e strains. Further study of inlA sequencing revealed that most strains contained the full-length InlA required for host cell invasion, whereas three mutations lead to premature stop codons (PMSC) within a novel PMSCs at position 326 (GAA → TAA). MLST and inlA sequence analysis results were concordant, and different virulence potentials within isolates were observed. These findings suggest that L. monocytogenes isolates from RTE food in China could be virulent and be capable of causing human illness. Furthermore, the STs and virulence profiles of L. monocytogenes isolates have significant implications for epidemiological and public health studies of this pathogen. PMID:26909076

Analysis of Multilocus Sequence Typing and Virulence Characterization of Listeria monocytogenes Isolates from Chinese Retail Ready-to-Eat Food.

PubMed

Wu, Shi; Wu, Qingping; Zhang, Jumei; Chen, Moutong; Guo, Weipeng

2016-01-01

Eighty Listeria monocytogenes isolates were obtained from Chinese retail ready-to-eat (RTE) food and were previously characterized with serotyping and antibiotic susceptibility tests. The aim of this study was to characterize the subtype and virulence potential of these L. monocytogenes isolates by multilocus sequence typing (MLST), virulence-associate genes, epidemic clones (ECs), and sequence analysis of the important virulence factor: internalin A (inlA). The result of MLST revealed that these L. monocytogenes isolates belonged to 14 different sequence types (STs). With the exception of four new STs (ST804, ST805, ST806, and ST807), all other STs observed in this study have been associated with human listeriosis and outbreaks to varying extents. Six virulence-associate genes (inlA, inlB, inlC, inlJ, hly, and llsX) were selected and their presence was investigated using PCR. All strains carried inlA, inlB, inlC, inlJ, and hly, whereas 38.8% (31/80) of strains harbored the listeriolysin S genes (llsX). A multiplex PCR assay was used to evaluate the presence of markers specific to epidemic clones of L. monocytogenes and identified 26.3% (21/80) of ECI in the 4b-4d-4e strains. Further study of inlA sequencing revealed that most strains contained the full-length InlA required for host cell invasion, whereas three mutations lead to premature stop codons (PMSC) within a novel PMSCs at position 326 (GAA → TAA). MLST and inlA sequence analysis results were concordant, and different virulence potentials within isolates were observed. These findings suggest that L. monocytogenes isolates from RTE food in China could be virulent and be capable of causing human illness. Furthermore, the STs and virulence profiles of L. monocytogenes isolates have significant implications for epidemiological and public health studies of this pathogen.

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.

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.

Evolution of infectious hematopoietic necrosis virus (IHNV), a fish rhabdovirus, in Europe over 20 years: implications for control.

PubMed

Enzmann, Peter-Joachim; Castric, Jeannette; Bovo, Giuseppe; Thiery, Richard; Fichtner, Dieter; Schütze, Heike; Wahli, Thomas

2010-02-24

The fish pathogenic rhabdovirus infectious hematopoietic necrosis virus (IHNV) causes substantial losses in European aquaculture. IHNV was first detected in Europe in 1987 and has since undergone considerable spread. Phylogenetic analyses of the full G-gene sequences of 73 isolates obtained from 4 countries in Europe (France, n = 18; Italy, 9; Switzerland, 4; Germany, 42) enable determination of the evolution of the virus in Europe since the first detection, and identification of characteristic changes within the G-genes of European strains. Further, the database allows us to analyse the pathways of distribution in Europe over time. The results suggest that in most of the recent cases, spread of IHNV was related to trade of infected fish. The data further demonstrate that knowledge of the sequence is required to determine the source of infections in farms.

Next-generation sequencing of the monogenic obesity genes LEP, LEPR, MC4R, PCSK1 and POMC in a Norwegian cohort of patients with morbid obesity and normal weight controls.

PubMed

Nordang, Gry B N; Busk, Øyvind L; Tveten, Kristian; Hanevik, Hans Ivar; Fell, Anne Kristin M; Hjelmesæth, Jøran; Holla, Øystein L; Hertel, Jens K

2017-05-01

Rare sequence variants in at least five genes are known to cause monogenic obesity. In this study we aimed to investigate the prevalence of, and characterize, rare coding and splice site variants in LEP, LEPR, MC4R, PCSK1 and POMC in patients with morbid obesity and normal weight controls. Targeted next-generation sequencing of all exons in LEP, LEPR, MC4R, PCSK1 and POMC was performed in 485 patients with morbid obesity and 327 normal weight population-based controls from Norway. In total 151 variants were detected. Twenty-eight (18.5%) of these were rare, coding or splice variants and five (3.3%) were novel. All individuals, except one control, were heterozygous for the 28 variants, and the distribution of the rare variants showed a significantly higher carrier frequency among cases than controls (9.9% vs. 4.9%, p=0.011). Four variants in MC4R were classified as pathogenic or likely pathogenic. Four cases (0.8%) of monogenic obesity were detected, all due to MC4R variants previously linked to monogenic obesity. Significant differences in carrier frequencies among patients with morbid obesity and normal weight controls suggest an association between heterozygous rare coding variants in these five genes and morbid obesity. However, additional studies in larger cohorts and functional testing of the novel variants identified are required to confirm the findings. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Global Performance Characterization of the Three Burn Trans-Earth Injection Maneuver Sequence over the Lunar Nodal Cycle

NASA Technical Reports Server (NTRS)

Williams, Jacob; Davis, Elizabeth C.; Lee, David E.; Condon, Gerald L.; Dawn, Tim

2009-01-01

The Orion spacecraft will be required to perform a three-burn trans-Earth injection (TEI) maneuver sequence to return to Earth from low lunar orbit. The origin of this approach lies in the Constellation Program requirements for access to any lunar landing site location combined with anytime lunar departure. This paper documents the development of optimized databases used to rapidly model the performance requirements of the TEI three-burn sequence for an extremely large number of mission cases. It also discusses performance results for lunar departures covering a complete 18.6 year lunar nodal cycle as well as general characteristics of the optimized three-burn TEI sequence.

Cruise Missile Engine Program Contract Data Requirements List Sequence Number 95. Qualification Test Report. Volume XXXIX. Vibration and Mission Simulation Testing on Engine 828. XF-107-WR-400 Cruise Missile Engine,

DTIC Science & Technology

1981-06-01

Illinois Page 7 REV. E ACC"T iJ’ , T.-T AUDIT 4 tii~~fs~actor ~ L~~ 4~4’~NDate S "n M. I. M EC RORDRE -MARK 1 95 oh -- Pum Capacity iA 11994 12054 0...2 S.Z 14-. -l_ S. Z.11500 0 60(60-1851 500 515 5.3. At. el 170’f 8061-056 OnY) 1438 1-448 1; 45a ~~:1 462____ 5.4 Acca ? -6S5F 5.4.116400 40 .4595

High density bit transition requirements versus the effects on BCH error correcting code. [bit synchronization

NASA Technical Reports Server (NTRS)

Ingels, F. M.; Schoggen, W. O.

1982-01-01

The design to achieve the required bit transition density for the Space Shuttle high rate multiplexes (HRM) data stream of the Space Laboratory Vehicle is reviewed. It contained a recommended circuit approach, specified the pseudo random (PN) sequence to be used and detailed the properties of the sequence. Calculations showing the probability of failing to meet the required transition density were included. A computer simulation of the data stream and PN cover sequence was provided. All worst case situations were simulated and the bit transition density exceeded that required. The Preliminary Design Review and the critical Design Review are documented. The Cover Sequence Generator (CSG) Encoder/Decoder design was constructed and demonstrated. The demonstrations were successful. All HRM and HRDM units incorporate the CSG encoder or CSG decoder as appropriate.

Performance evaluation of the intra compression in the video coding standards

NASA Astrophysics Data System (ADS)

Abramowski, Andrzej

2015-09-01

The article presents a comparison of the Intra prediction algorithms in the current state-of-the-art video coding standards, including MJPEG 2000, VP8, VP9, H.264/AVC and H.265/HEVC. The effectiveness of techniques employed by each standard is evaluated in terms of compression efficiency and average encoding time. The compression efficiency is measured using BD-PSNR and BD-RATE metrics with H.265/HEVC results as an anchor. Tests are performed on a set of video sequences, composed of sequences gathered by Joint Collaborative Team on Video Coding during the development of the H.265/HEVC standard and 4K sequences provided by Ultra Video Group. According to results, H.265/HEVC provides significant bit-rate savings at the expense of computational complexity, while VP9 may be regarded as a compromise between the efficiency and required encoding time.

GPU-Acceleration of Sequence Homology Searches with Database Subsequence Clustering.

PubMed

Suzuki, Shuji; Kakuta, Masanori; Ishida, Takashi; Akiyama, Yutaka

2016-01-01

Sequence homology searches are used in various fields and require large amounts of computation time, especially for metagenomic analysis, owing to the large number of queries and the database size. To accelerate computing analyses, graphics processing units (GPUs) are widely used as a low-cost, high-performance computing platform. Therefore, we mapped the time-consuming steps involved in GHOSTZ, which is a state-of-the-art homology search algorithm for protein sequences, onto a GPU and implemented it as GHOSTZ-GPU. In addition, we optimized memory access for GPU calculations and for communication between the CPU and GPU. As per results of the evaluation test involving metagenomic data, GHOSTZ-GPU with 12 CPU threads and 1 GPU was approximately 3.0- to 4.1-fold faster than GHOSTZ with 12 CPU threads. Moreover, GHOSTZ-GPU with 12 CPU threads and 3 GPUs was approximately 5.8- to 7.7-fold faster than GHOSTZ with 12 CPU threads.

Identification of a Novel Recycling Sequence in the C-tail of FPR2/ALX Receptor

PubMed Central

Thompson, Dawn; McArthur, Simon; Hislop, James N.; Flower, Roderick J.; Perretti, Mauro

2014-01-01

Formyl-peptide receptor type 2 (FPR2; also called ALX because it is the receptor for lipoxin A4) sustains a variety of biological responses relevant to the development and control of inflammation, yet the cellular regulation of this G-protein-coupled receptor remains unexplored. Here we report that, in response to peptide agonist activation, FPR2/ALX undergoes β-arrestin-mediated endocytosis followed by rapid recycling to the plasma membrane. We identify a transplantable recycling sequence that is both necessary and sufficient for efficient receptor recycling. Furthermore, removal of this C-terminal recycling sequence alters the endocytic fate of FPR2/ALX and evokes pro-apoptotic effects in response to agonist activation. This study demonstrates the importance of endocytic recycling in the anti-apoptotic properties of FPR2/ALX and identifies the molecular determinant required for modulation of this process fundamental for the control of inflammation. PMID:25326384

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

RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer

NASA Astrophysics Data System (ADS)

Wolfe, Andrew L.; Singh, Kamini; Zhong, Yi; Drewe, Philipp; Rajasekhar, Vinagolu K.; Sanghvi, Viraj R.; Mavrakis, Konstantinos J.; Jiang, Man; Roderick, Justine E.; van der Meulen, Joni; Schatz, Jonathan H.; Rodrigo, Christina M.; Zhao, Chunying; Rondou, Pieter; de Stanchina, Elisa; Teruya-Feldstein, Julie; Kelliher, Michelle A.; Speleman, Frank; Porco, John A.; Pelletier, Jerry; Rätsch, Gunnar; Wendel, Hans-Guido

2014-09-01

The translational control of oncoprotein expression is implicated in many cancers. Here we report an eIF4A RNA helicase-dependent mechanism of translational control that contributes to oncogenesis and underlies the anticancer effects of silvestrol and related compounds. For example, eIF4A promotes T-cell acute lymphoblastic leukaemia development in vivo and is required for leukaemia maintenance. Accordingly, inhibition of eIF4A with silvestrol has powerful therapeutic effects against murine and human leukaemic cells in vitro and in vivo. We use transcriptome-scale ribosome footprinting to identify the hallmarks of eIF4A-dependent transcripts. These include 5' untranslated region (UTR) sequences such as the 12-nucleotide guanine quartet (CGG)4 motif that can form RNA G-quadruplex structures. Notably, among the most eIF4A-dependent and silvestrol-sensitive transcripts are a number of oncogenes, superenhancer-associated transcription factors, and epigenetic regulators. Hence, the 5' UTRs of select cancer genes harbour a targetable requirement for the eIF4A RNA helicase.

Mini-midi-mito: adapting the amplification and sequencing strategy of mtDNA to the degradation state of crime scene samples.

PubMed

Berger, Cordula; Parson, Walther

2009-06-01

The degradation state of some biological traces recovered from the crime scene requires the amplification of very short fragments to attain a useful mitochondrial (mt)DNA sequence. We have previously introduced two mini-multiplex assays that amplify 10 overlapping control region (CR) fragments in two separate multiplex PCRs, which brought successful CR consensus sequences from even highly degraded DNA extracts. This procedure requires a total of 20 sequencing reactions per sample, which is laborious and cost intensive. For only moderately degraded samples that we encounter more frequently with typical mtDNA casework material, we developed two new multiplex assays that use a subset of the mini-amplicon primers but embrace larger fragments (midis) and require only 10 sequencing reactions to build a double-stranded CR consensus sequence. We used a preceding mtDNA quantitation step by real-time PCR with two different target fragments (143 and 283 bp) that roughly correspond to the average fragment sizes of the different multiplex approaches to estimate size-dependent mtDNA quantities and to aid the choice of the appropriate PCR multiplexes with respect to quality of the results and required costs.

Next-generation sequencing for genetic testing of familial colorectal cancer syndromes.

PubMed

Simbolo, Michele; Mafficini, Andrea; Agostini, Marco; Pedrazzani, Corrado; Bedin, Chiara; Urso, Emanuele D; Nitti, Donato; Turri, Giona; Scardoni, Maria; Fassan, Matteo; Scarpa, Aldo

2015-01-01

Genetic screening in families with high risk to develop colorectal cancer (CRC) prevents incurable disease and permits personalized therapeutic and follow-up strategies. The advancement of next-generation sequencing (NGS) technologies has revolutionized the throughput of DNA sequencing. A series of 16 probands for either familial adenomatous polyposis (FAP; 8 cases) or hereditary nonpolyposis colorectal cancer (HNPCC; 8 cases) were investigated for intragenic mutations in five CRC familial syndromes-associated genes (APC, MUTYH, MLH1, MSH2, MSH6) applying both a custom multigene Ion AmpliSeq NGS panel and conventional Sanger sequencing. Fourteen pathogenic variants were detected in 13/16 FAP/HNPCC probands (81.3 %); one FAP proband presented two co-existing pathogenic variants, one in APC and one in MUTYH. Thirteen of these 14 pathogenic variants were detected by both NGS and Sanger, while one MSH2 mutation (L280FfsX3) was identified only by Sanger sequencing. This is due to a limitation of the NGS approach in resolving sequences close or within homopolymeric stretches of DNA. To evaluate the performance of our NGS custom panel we assessed its capability to resolve the DNA sequences corresponding to 2225 pathogenic variants reported in the COSMIC database for APC, MUTYH, MLH1, MSH2, MSH6. Our NGS custom panel resolves the sequences where 2108 (94.7 %) of these variants occur. The remaining 117 mutations reside inside or in close proximity to homopolymer stretches; of these 27 (1.2 %) are imprecisely identified by the software but can be resolved by visual inspection of the region, while the remaining 90 variants (4.0 %) are blind spots. In summary, our custom panel would miss 4 % (90/2225) of pathogenic variants that would need a small set of Sanger sequencing reactions to be solved. The multiplex NGS approach has the advantage of analyzing multiple genes in multiple samples simultaneously, requiring only a reduced number of Sanger sequences to resolve homopolymeric DNA regions not adequately assessed by NGS. The implementation of NGS approaches in routine diagnostics of familial CRC is cost-effective and significantly reduces diagnostic turnaround times.

Genetic exchange between endogenous and exogenous LINE-1 repetitive elements in mouse cells.

PubMed Central

Belmaaza, A; Wallenburg, J C; Brouillette, S; Gusew, N; Chartrand, P

1990-01-01

The repetitive LINE (L1) elements of the mouse, which are present at about 10(5) copies per genome and share over 80% of sequence homology, were examined for their ability to undergo genetic exchange with exogenous L1 sequences. The exogenous L1 sequences, carried by a shuttle vector, consisted of an internal fragment from L1Md-A2, a previously described member of the L1 family of the mouse. Using an assay that does not require the reconstitution of a selectable marker we found that this vector, in either circular or linear form, acquired DNA sequences from endogenous L1 elements at a frequency of 10(-3) to 10(-4) per rescued vector. Physical analysis of the acquired L1 sequences revealed that distinct endogenous L1 elements acted as donors and that different subfamilies participated. These results demonstrate that L1 elements are readily capable of genetic exchange. Apart from gene conversion events, the acquisition of L1 sequences outside the region of homology suggested that a second mechanism was also involved in the genetic exchange. A model which accounts for this mechanism is presented and its potential implication on the rearrangement of L1 elements is discussed. Images PMID:1978749

Phylo-VISTA: Interactive visualization of multiple DNA sequence alignments

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

Shah, Nameeta; Couronne, Olivier; Pennacchio, Len A.

The power of multi-sequence comparison for biological discovery is well established. The need for new capabilities to visualize and compare cross-species alignment data is intensified by the growing number of genomic sequence datasets being generated for an ever-increasing number of organisms. To be efficient these visualization algorithms must support the ability to accommodate consistently a wide range of evolutionary distances in a comparison framework based upon phylogenetic relationships. Results: We have developed Phylo-VISTA, an interactive tool for analyzing multiple alignments by visualizing a similarity measure for multiple DNA sequences. The complexity of visual presentation is effectively organized using a frameworkmore » based upon interspecies phylogenetic relationships. The phylogenetic organization supports rapid, user-guided interspecies comparison. To aid in navigation through large sequence datasets, Phylo-VISTA leverages concepts from VISTA that provide a user with the ability to select and view data at varying resolutions. The combination of multiresolution data visualization and analysis, combined with the phylogenetic framework for interspecies comparison, produces a highly flexible and powerful tool for visual data analysis of multiple sequence alignments. Availability: Phylo-VISTA is available at http://www-gsd.lbl. gov/phylovista. It requires an Internet browser with Java Plugin 1.4.2 and it is integrated into the global alignment program LAGAN at http://lagan.stanford.edu« less

Recognition of the Xenopus ribosomal core promoter by the transcription factor xUBF involves multiple HMG box domains and leads to an xUBF interdomain interaction.

PubMed

Leblanc, B; Read, C; Moss, T

1993-02-01

The interaction of the ribosomal transcription factor xUBF with the RNA polymerase I core promoter of Xenopus laevis has been studied both at the DNA and protein levels. It is shown that a single xUBF-DNA complex forms over the 40S initiation site (+1) and involves at least the DNA sequences between -20 and +60 bp. DNA sequences upstream of +10 and downstream of +18 are each sufficient to direct complex formation independently. HMG box 1 of xUBF independently recognizes the sequences -20 to -1 and +1 to +22 and the addition of the N-terminal dimerization domain to HMG box 1 stabilizes its interaction with these sequences approximately 10-fold. HMG boxes 2/3 interact with the DNA downstream of +22 and can independently position xUBF across the initiation site. The C-terminal segment of xUBF, HMG boxes 4, 5 or the acidic domain, directly or indirectly interact with HMG box 1, making the core promoter sequences between -11 and -15 hypersensitive to DNase. This interaction also requires the DNA sequences between +17 and +32, i.e. the HMG box 2/3 binding site. The data suggest extensive folding of the core promoter within the xUBF complex.

Resistance to Change and Preference for Variable versus Fixed Response Sequences

ERIC Educational Resources Information Center

Arantes, Joana; Berg, Mark E.; Le, Dien; Grace, Randolph C.

2012-01-01

In Experiment 1, 4 pigeons were trained on a multiple chain schedule in which the initial link was a variable-interval (VI) 20-s schedule signalled by a red or green center key, and terminal links required four responses made to the left (L) and/or right (R) keys. In the REPEAT component, signalled by red keylights, only LRLR terminal-link…

Identification of the UDP-glucose-4-epimerase required for galactofuranose biosynthesis and galactose metabolism in A. niger.

PubMed

Park, Joohae; Tefsen, Boris; Arentshorst, Mark; Lagendijk, Ellen; van den Hondel, Cees Amjj; van Die, Irma; Ram, Arthur Fj

2014-01-01

Galactofuranose (Gal f )-containing glycoconjugates are important to secure the integrity of the cell wall of filamentous fungi. Mutations that prevent the biosynthesis of Gal f -containing molecules compromise cell wall integrity. In response to cell wall weakening, the cell wall integrity (CWI)-pathway is activated to reinforce the strength of the cell wall. Activation of CWI-pathway in Aspergillus niger is characterized by the specific induction of the agsA gene, which encodes a cell wall α-glucan synthase. In this study, we screened a collection of cell wall mutants with an induced expression of agsA for defects in Gal f biosynthesis using a with anti-Gal f antibody (L10). From this collection of mutants, we previously identified mutants in the UDP-galactopyranose mutase encoding gene ( ugmA ). Here, we have identified six additional UDP-galactopyranose mutase ( ugmA ) mutants and one mutant (named mutant #41) in an additional complementation group that displayed strongly reduced Gal f -levels in the cell wall. By using a whole genome sequencing approach, 21 SNPs in coding regions were identified between mutant #41 and its parental strain which changed the amino acid sequence of the encoded proteins. One of these mutations was in gene An14g03820, which codes for a putative UDP-glucose-4-epimerase (UgeA). The A to G mutation in this gene causes an amino acid change of Asn to Asp at position 191 in the UgeA protein. Targeted deletion of ugeA resulted in an even more severe reduction of Gal f in N-linked glucans, indicating that the UgeA protein in mutant #41 is partially active. The ugeA gene is also required for growth on galactose despite the presence of two UgeA homologs in the A. niger genome. By using a classical mutant screen and whole genome sequencing of a new Gal f -deficient mutant, the UDP-glucose-4-epimerase gene ( ugeA ) has been identified. UgeA is required for the biosynthesis of Gal f as well as for galactose metabolism in Aspergillus niger .

YAMAT-seq: an efficient method for high-throughput sequencing of mature transfer RNAs

PubMed Central

Shigematsu, Megumi; Honda, Shozo; Loher, Phillipe; Telonis, Aristeidis G.; Rigoutsos, Isidore

2017-01-01

Abstract Besides translation, transfer RNAs (tRNAs) play many non-canonical roles in various biological pathways and exhibit highly variable expression profiles. To unravel the emerging complexities of tRNA biology and molecular mechanisms underlying them, an efficient tRNA sequencing method is required. However, the rigid structure of tRNA has been presenting a challenge to the development of such methods. We report the development of Y-shaped Adapter-ligated MAture TRNA sequencing (YAMAT-seq), an efficient and convenient method for high-throughput sequencing of mature tRNAs. YAMAT-seq circumvents the issue of inefficient adapter ligation, a characteristic of conventional RNA sequencing methods for mature tRNAs, by employing the efficient and specific ligation of Y-shaped adapter to mature tRNAs using T4 RNA Ligase 2. Subsequent cDNA amplification and next-generation sequencing successfully yield numerous mature tRNA sequences. YAMAT-seq has high specificity for mature tRNAs and high sensitivity to detect most isoacceptors from minute amount of total RNA. Moreover, YAMAT-seq shows quantitative capability to estimate expression levels of mature tRNAs, and has high reproducibility and broad applicability for various cell lines. YAMAT-seq thus provides high-throughput technique for identifying tRNA profiles and their regulations in various transcriptomes, which could play important regulatory roles in translation and other biological processes. PMID:28108659

Single-primer fluorescent sequencing

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

Ruth, J.L.; Morgan, C.A.; Middendorf, L.R.

Modified linker arm oligonucleotides complementary to standard M13 priming sites were synthesized, labelled with either one, two, or three fluoresceins, and purified by reverse-phase HPLC. When used as primers in standard dideoxy M13 sequencing with /sup 32/P-dNTPs, normal autoradiographic patterns were obtained. To eliminate the radioactivity, direct on-line fluorescence detection was achieved by the use of a scanning 10 mW Argon laser emitting 488 nm light. Fluorescent bands were detected directly in standard 0.2 or 0.35 mm thick polyacrylamide gels at a distance of 24 cm from the loading wells by a photomultiplier tube filtered at 520 nm. Horizontal andmore » temporal location of each band was displayed by computer as a band in real time, providing visual appearance similar to normal 4-lane autoradiograms. Using a single primer labelled with two fluoresceins, sequences of between 500 and 600 bases have been read in a single loading with better than 98% accuracy; up to 400 bases can be read reproducibly with no errors. More than 50 sequences have been determined by this method. This approach requires only 1-2 ug of cloned template, and produces continuous sequence data at about one band per minute.« less

Novel Detection of Coxiella spp., Theileria luwenshuni, and T. ovis Endosymbionts in Deer Keds (Lipoptena fortisetosa).

PubMed

Lee, Seung-Hun; Kim, Kyoo-Tae; Kwon, Oh-Deog; Ock, Younsung; Kim, Taeil; Choi, Donghag; Kwak, Dongmi

2016-01-01

We describe for the first time the detection of Coxiella-like bacteria (CLB), Theileria luwenshuni, and T. ovis endosymbionts in blood-sucking deer keds. Eight deer keds attached to a Korean water deer were identified as Lipoptena fortisetosa (Diptera: Hippoboscidae) by morphological and genetic analyses. Among the endosymbionts assessed, CLB, Theileria luwenshuni, and T. ovis were identified in L. fortisetosa by PCR and nucleotide sequencing. Based on phylogeny, CLB 16S rRNA sequences were classified into clade B, sharing 99.4% identity with CLB from Haemaphysalis longicornis in South Korea. Although the virulence of CLB to vertebrates is still controversial, several studies have reported clinical symptoms in birds due to CLB infections. The 18S rRNA sequences of T. luwenshuni and T. ovis in this study were 98.8-100% identical to those in GenBank, and all of the obtained sequences of T. ovis and T. luwenshuni in this study were 100% identical to each other, respectively. Although further studies are required to positively confirm L. fortisetosa as a biological vector of these pathogens, strong genetic relationships among sequences from this and previous studies suggest potential transmission among mammalian hosts by ticks and keds.

EGG-4 and EGG-5 link events of the oocyte-to-embryo transition with meiotic cell cycle progression in Caenorhabditis elegans

PubMed Central

Parry, Jean M.; Velarde, Nathalie V.; Lefkovith, Ariel J.; Zegarek, Matthew H.; Hang, Julie S.; Ohm, Jonathan; Klancer, Richard; Maruyama, Rika; Druzhinina, Marina K.; Grant, Barth D.; Piano, Fabio; Singson, Andrew

2009-01-01

Summary The molecular underpinnings of the oocyte-to-embryo transition are poorly understood. Here we show that two protein tyrosine phosphatase-like (PTPL) family proteins, EGG-4 and EGG-5, are required for key events of the oocyte-to-embryo transition in Caenorhabditis elegans. The predicted EGG-4 and EGG-5 amino acid sequences are 99% identical and their functions are redundant. In embryos lacking EGG-4 and EGG-5 we observe defects in meiosis, polar body formation, the block to polyspermy, F-actin dynamics, and eggshell deposition. During oogenesis, EGG-4 and EGG-5 assemble at the oocyte cortex with the previously identified regulators or effectors of the oocyte-to-embryo transition EGG-3, CHS-1 and MBK-2 [1, 2]. All of these molecules share a complex interdependence with regards to their dynamics and subcellular localization. Shortly after fertilization, EGG-4 and EGG-5 are required to properly coordinate a redistribution of CHS-1 and EGG-3 away from the cortex during meiotic anaphase I. Therefore EGG-4 and EGG-5 are not only required for critical events of the oocyte-to-embryo transition but also link the dynamics of the regulatory machinery with the advancing cell cycle. PMID:19879147

Analyzing 5'HS3 and 5'HS4 LCR core regions and NF-E2 in Iranian thalassemia intermedia patients with normal or carrier status for beta-globin mutations.

PubMed

Neishabury, Maryam; Azarkeivan, Azita; Oberkanins, Christian; Abedini, Seyedeh Sedigheh; Zamani, Shahbaz; Najmabadi, Hossein

2011-03-15

Our data on 114 Iranian individuals with thalassemia intermedia phenotype revealed homozygous or compound heterozygous beta-globin mutations to be the predominant disease factor in 86.2% of cases. However, 8.2% of these individuals were found to be heterozygous or wild type for beta-globin mutations. In search for determinants outside of the beta-globin gene, which could be responsible for the unexpected thalassemia intermedia phenotype in these subjects, we screened the alpha-globin genes, the 5'HS3 and 5'HS4 regions of the beta-globin LCR, and the NF-E2 transcription factor for sequence variations in selected individuals. The -3.7 deletion was the only alpha-globin mutation detected, and no alterations were found in 5'HS3 and NF-E2. Sequence analysis of the 5'HS4 LCR core region identified three known SNPs in a single patient, who required irregular blood transfusions. The A/G polymorphism in the 5'HS4 palindromic region was also observed to be variable. Family studies were carried out on a female G/G homozygous patient, who received irregular blood transfusions. Her father, who had the same heterozygous IVSII-1 beta-globin mutation but the A/G genotype at the 5'HS4 palindromic site, presented with mild anemia and no requirement for blood transfusions. This suggests an impact of SNPs in the 5'HS4 LCR core region on the thalassemia phenotype and offers an interesting subject for further investigations in the Iranian population. Copyright © 2010 Elsevier Inc. All rights reserved.

Low-Temperature Molecular Layer Deposition Using Monofunctional Aromatic Precursors and Ozone-Based Ring-Opening Reactions.

PubMed

Svärd, Laura; Putkonen, Matti; Kenttä, Eija; Sajavaara, Timo; Krahl, Fabian; Karppinen, Maarit; Van de Kerckhove, Kevin; Detavernier, Christophe; Simell, Pekka

2017-09-26

Molecular layer deposition (MLD) is an increasingly used deposition technique for producing thin coatings consisting of purely organic or hybrid inorganic-organic materials. When organic materials are prepared, low deposition temperatures are often required to avoid decomposition, thus causing problems with low vapor pressure precursors. Monofunctional compounds have higher vapor pressures than traditional bi- or trifunctional MLD precursors, but do not offer the required functional groups for continuing the MLD growth in subsequent deposition cycles. In this study, we have used high vapor pressure monofunctional aromatic precursors in combination with ozone-triggered ring-opening reactions to achieve sustained sequential growth. MLD depositions were carried out by using three different aromatic precursors in an ABC sequence, namely with TMA + phenol + O 3 , TMA + 3-(trifluoromethyl)phenol + O 3 , and TMA + 2-fluoro-4-(trifluoromethyl)benzaldehyde + O 3 . Furthermore, the effect of hydrogen peroxide as a fourth step was evaluated for all studied processes resulting in a four-precursor ABCD sequence. According to the characterization results by ellipsometry, infrared spectroscopy, and X-ray reflectivity, self-limiting MLD processes could be obtained between 75 and 150 °C with each of the three aromatic precursors. In all cases, the GPC (growth per cycle) decreased with increasing temperature. In situ infrared spectroscopy indicated that ring-opening reactions occurred in each ABC sequence. Compositional analysis using time-of-flight elastic recoil detection indicated that fluorine could be incorporated into the film when 3-(trifluoromethyl)phenol and 2-fluoro-4-(trifluoromethyl)benzaldehyde were used as precursors.

Safety and Immune Responses in Children After Concurrent or Sequential 2009 H1N1 and 2009–2010 Seasonal Trivalent Influenza Vaccinations

PubMed Central

Frey, Sharon E.; Bernstein, David I.; Gerber, Michael A.; Keyserling, Harry L.; Munoz, Flor M.; Winokur, Patricia L.; Turley, Christine B.; Rupp, Richard E.; Hill, Heather; Wolff, Mark; Noah, Diana L.; Ross, Allison C.; Cress, Gretchen; Belshe, Robert B.

2012-01-01

Background. Administering 2 separate vaccines for seasonal and pandemic influenza was necessary in 2009. Therefore, we conducted a randomized trial of monovalent 2009 H1N1 influenza vaccine (2009 H1N1 vaccine) and seasonal trivalent inactivated influenza vaccine (TIV; split virion) given sequentially or concurrently in previously vaccinated children. Methods. Children randomized to 4 study groups and stratified by age received 1 dose of seasonal TIV and 2 doses of 2009 H1N1 vaccine in 1 of 4 combinations. Injections were given at 21-day intervals and serum samples for hemagglutination inhibition antibody responses were obtained prior to and 21 days after each vaccination. Reactogenicity and adverse events were monitored. Results. All combinations of vaccines were safe in the 531 children enrolled. Generally, 1 dose of 2009 H1N1 vaccine and 1 dose of TIV, regardless of sequence or concurrency of administration, was immunogenic in children ≥10 years of age; children <10 years of age required 2 doses of 2009 H1N1 vaccine. Conclusions. Vaccines were generally well tolerated. The immune responses to 2009 H1N1 vaccine were adequate regardless of the sequence of vaccination in all age groups but the sequence affected titers to TIV antigens. Two doses of 2009 H1N1 vaccine were required to achieve a protective immune response in children <10 years of age. Clinical Trials Registration. NCT00943202. PMID:22802432

Curriculum Mapping in Higher Education: A Case Study and Proposed Content Scope and Sequence Mapping Tool

ERIC Educational Resources Information Center

Arafeh, Sousan

2016-01-01

Best practice in curriculum development and implementation requires that discipline-based standards or requirements embody both curricular and programme scopes and sequences. Ensuring these are present and aligned in course/programme content, activities and assessments to support student success requires formalised and systematised review and…

Kinetic and Structural Characterization of a Heterohexamer 4-Oxalocrotonate Tautomerase from Chloroflexus aurantiacus J-10-fl: Implications for Functional and Structural Diversity in the Tautomerase Superfamily

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

Burks, Elizabeth A.; Fleming, Christopher D.; Mesecar, Andrew D.

2010-09-30

4-Oxalocrotonate tautomerase (4-OT) isozymes play prominent roles in the bacterial utilization of aromatic hydrocarbons as sole carbon sources. These enzymes catalyze the conversion of 2-hydroxy-2,4-hexadienedioate (or 2-hydroxymuconate) to 2-oxo-3-hexenedioate, where Pro-1 functions as a general base and shuttles a proton from the 2-hydroxyl group of the substrate to the C-5 position of the product. 4-OT, a homohexamer from Pseudomonas putida mt-2, is the most extensively studied 4-OT isozyme and the founding member of the tautomerase superfamily. A search of five thermophilic bacterial genomes identified a coded amino acid sequence in each that had been annotated as a tautomerase-like protein butmore » lacked Pro-1. However, a nearby sequence has Pro-1, but the sequence is not annotated as a tautomerase-like protein. To characterize this group of proteins, two genes from Chloroflexus aurantiacus J-10-fl were cloned, and the corresponding proteins were expressed. Kinetic, biochemical, and X-ray structural analyses show that the two expressed proteins form a functional heterohexamer 4-OT (hh4-OT), composed of three {alpha}{beta} dimers. Like the P. putida enzyme, hh4-OT requires the amino-terminal proline and two arginines for the conversion of 2-hydroxymuconate to the product, implicating an analogous mechanism. In contrast to 4-OT, hh4-OT does not exhibit the low-level activity of another tautomerase superfamily member, the heterohexamer trans-3-chloroacrylic acid dehalogenase (CaaD). Characterization of hh4-OT enables functional assignment of the related enzymes, highlights the diverse ways the {beta}-{alpha}-{beta} building block can be assembled into an active enzyme, and provides further insight into the molecular basis of the low-level CaaD activity in 4-OT.« less

Combustion Stability Characteristics of the Project Morpheus Liquid Oxygen / Liquid Methane Main Engine

NASA Technical Reports Server (NTRS)

Melcher, John C.; Morehead, Robert L.

2014-01-01

The project Morpheus liquid oxygen (LOX) / liquid methane (LCH4) main engine is a Johnson Space Center (JSC) designed 5,000 lbf-thrust, 4:1 throttling, pressure-fed cryogenic engine using an impinging element injector design. The engine met or exceeded all performance requirements without experiencing any in- ight failures, but the engine exhibited acoustic-coupled combustion instabilities during sea-level ground-based testing. First tangential (1T), rst radial (1R), 1T1R, and higher order modes were triggered by conditions during the Morpheus vehicle derived low chamber pressure startup sequence. The instability was never observed to initiate during mainstage, even at low power levels. Ground-interaction acoustics aggravated the instability in vehicle tests. Analysis of more than 200 hot re tests on the Morpheus vehicle and Stennis Space Center (SSC) test stand showed a relationship between ignition stability and injector/chamber pressure. The instability had the distinct characteristic of initiating at high relative injection pressure drop at low chamber pressure during the start sequence. Data analysis suggests that the two-phase density during engine start results in a high injection velocity, possibly triggering the instabilities predicted by the Hewitt stability curves. Engine ignition instability was successfully mitigated via a higher-chamber pressure start sequence (e.g., 50% power level vs 30%) and operational propellant start temperature limits that maintained \\cold LOX" and \\warm methane" at the engine inlet. The main engine successfully demonstrated 4:1 throttling without chugging during mainstage, but chug instabilities were observed during some engine shutdown sequences at low injector pressure drop, especially during vehicle landing.

Structural analysis as an alternative to identify and determine mode of action of antimicrobial peptides: proposition of a kinetic model based on molecular dynamics studies.

PubMed

Robles-Gomez, Edson Edinho; Flores-Villegas, Mirelle Citlali; Gonzalez-Manjarrez, Alicia; Soriano-Garcia, Manuel

2013-05-01

Antimicrobial peptides (AMPs) constitute an important alternative in the search for new treatments against pathogens. We analyzed the sequence variability in cytokine and chemokine proteins to investigate whether these molecules contain a sequence useful in the development of new AMPs. Cluster analysis allowed the identification of tracts, grouped in five categories showing structure and sequence homology. The structure and function relationship among these groups, was analyzed using physicochemical parameters such as length, sequence, charge, hydrophobicity and helicity, which allowed the selection of a candidate that could constitute an AMP. This peptide comprises the C-terminal alpha-helix of chemokines CXCL4/PF-457-70. Far-UV CD spectroscopy showed that this molecule adopts a random conformation in aqueous solution and the addition of 2, 2, 2 trifluoroethanol (TFE) is required to induce a helical secondary structure. The CXCL4/PF-457-70 peptide was found to have antimicrobial activity and very limited hemolytic activity. The mechanism of action was analyzed using model kinetics and molecular dynamics. The kinetic model led to a reasonable assumption about a rate constant and regulatory step on its mechanism of action. Using molecular dynamics simulations, the structural properties the CXCL4/PF-457-70 have been examined in a membrane environment. Our results show that this peptide has a strong preference for binding to the lipid head groups, consequently, increasing the surface density and decreasing the lateral mobility of the lipids alters its functionality.

Systems design study of the Pioneer Venus spacecraft. Volume 1. Technical analyses and tradeoffs, sections 1-4 (part 1 of 4)

NASA Technical Reports Server (NTRS)

1973-01-01

The results are reported of the Pioneer Venus studies from 2 October 1972 through 30 June 1973. Many missions were considered, involving two launch vehicles (Thor/Delta and Atlas/Centaur), and different launch opportunities and spacecraft configurations to meet varying science requirements, all at minimum cost. The sequence of events is described and the specific studies conducted are summarized. The effects of science payload on mission and spacecraft design are discussed along with the mission analyses.

Two divergent endo-beta-1,4-glucanase genes exhibit overlapping expression in ripening fruit and abscising flowers.

PubMed Central

Lashbrook, C C; Gonzalez-Bosch, C; Bennett, A B

1994-01-01

Two structurally divergent endo-beta-1,4-glucanase (EGase) cDNAs were cloned from tomato. Although both cDNAs (Cel1 and Cel2) encode potentially glycosylated, basic proteins of 51 to 53 kD and possess multiple amino acid domains conserved in both plant and microbial EGases, Cel1 and Cel2 exhibit only 50% amino acid identity at the overall sequence level. Amino acid sequence comparisons to other plant EGases indicate that tomato Cel1 is most similar to bean abscission zone EGase (68%), whereas Cel2 exhibits greatest sequence identity to avocado fruit EGase (57%). Sequence comparisons suggest the presence of at least two structurally divergent EGase families in plants. Unlike ripening avocado fruit and bean abscission zones in which a single EGase mRNA predominates, EGase expression in tomato reflects the overlapping accumulation of both Cel1 and Cel2 transcripts in ripening fruit and in plant organs undergoing cell separation. Cel1 mRNA contributes significantly to total EGase mRNA accumulation within plant organs undergoing cell separation (abscission zones and mature anthers), whereas Cel2 mRNA is most abundant in ripening fruit. The overlapping expression of divergent EGase genes within a single species may suggest that multiple activities are required for the cooperative disassembly of cell wall components during fruit ripening, floral abscission, and anther dehiscence. PMID:7994180

The StreamCat Dataset: Accumulated Attributes for NHDPlusV2 (Version 2.1) Catchments Riparian Buffer for the Conterminous United States: 2010 US Census Housing Unit and Population Density

EPA Pesticide Factsheets

This dataset represents the population and housing unit density within individual, local NHDPlusV2 catchments and upstream, contributing watersheds riparian buffers based on 2010 US Census data. Densities are calculated for every block group and watershed averages are calculated for every local NHDPlusV2 catchment(see Data Sources for links to NHDPlusV2 data and Census Data). This data set is derived from The TIGER/Line Files and related database (.dbf) files for the conterminous USA. It was downloaded as Block Group-Level Census 2010 SF1 Data in File Geodatabase Format (ArcGIS version 10.0). The landscape raster (LR) was produced based on the data compiled from the questions asked of all people and about every housing unit. The (block-group population / block group area) and (block-group housing units / block group area) were summarized by local catchment and by watershed to produce local catchment-level and watershed-level metrics as a continuous data type (see Data Structure and Attribute Information for a description).

Spatial control of translation repression and polarized growth by conserved NDR kinase Orb6 and RNA-binding protein Sts5.

PubMed

Nuñez, Illyce; Rodriguez Pino, Marbelys; Wiley, David J; Das, Maitreyi E; Chen, Chuan; Goshima, Tetsuya; Kume, Kazunori; Hirata, Dai; Toda, Takashi; Verde, Fulvia

2016-07-30

RNA-binding proteins contribute to the formation of ribonucleoprotein (RNP) granules by phase transition, but regulatory mechanisms are not fully understood. Conserved fission yeast NDR (Nuclear Dbf2-Related) kinase Orb6 governs cell morphogenesis in part by spatially controlling Cdc42 GTPase. Here we describe a novel, independent function for Orb6 kinase in negatively regulating the recruitment of RNA-binding protein Sts5 into RNPs to promote polarized cell growth. We find that Orb6 kinase inhibits Sts5 recruitment into granules, its association with processing (P) bodies, and degradation of Sts5-bound mRNAs by promoting Sts5 interaction with 14-3-3 protein Rad24. Many Sts5-bound mRNAs encode essential factors for polarized cell growth, and Orb6 kinase spatially and temporally controls the extent of Sts5 granule formation. Disruption of this control system affects cell morphology and alters the pattern of polarized cell growth, revealing a role for Orb6 kinase in the spatial control of translational repression that enables normal cell morphogenesis.

Regulation of Neurospora crassa cell wall remodeling via the cot-1 pathway is mediated by gul-1.

PubMed

Herold, Inbal; Yarden, Oded

2017-02-01

Impairment of the Neurospora crassa Nuclear DBF2-related kinase-encoding gene cot-1 results in pleiotropic effects, including abnormally thick hyphal cell walls and septa. An increase in the transcript abundance of genes encoding chitin and glucan synthases and the chitinase gh18-5, but not the cell wall integrity pathway transcription factor rlm-1, accompany the phenotypic changes observed. Deletion of chs-5 or chs-7 in a cot-1 background results in a reduction of hyperbranching frequency characteristic of the cot-1 parent. gul-1 (a homologue of the yeast SSD1 gene) encodes a translational regulator and has been shown to partially suppress cot-1. We demonstrate that the high expression levels of the cell wall remodeling genes analyzed is curbed, and reaches near wild type levels, when gul-1 is inactivated. This is accompanied by morphological changes that include reduced cell wall thickness and restoration of normal chitin levels. We conclude that gul-1 is a mediator of cell wall remodeling within the cot-1 pathway.

A novel integration system of magnetically immobilized cells and a pair of graphite plate-stainless iron mesh electrodes for the bioremediation of coking wastewater.

PubMed

Jiang, Bei; Tan, Liang; Ning, Shuxiang; Shi, Shengnan

2016-09-01

Magnetically immobilized cells of Comamonas sp. JB coupling with electrode reaction was developed to enhance the treatment efficiency of coking wastewater containing phenol, carbazole (CA), dibenzofuran (DBF), and dibenzothiophene (DBT). The pair of graphite plate-stainless iron mesh electrodes was chosen as the most suitable electrodes. Magnetically immobilized cells coupling with graphite plate-stainless iron mesh electrodes (coupling system) exhibited high degradation activity for all the compounds, which were significantly higher than the sum by single magnetically immobilized cells and electrode reaction at the optimal voltage. Recycling experiments demonstrated that the degradation activity of coupling system increased gradually during eight recycles, indicating that there was a coupling effect between the biodegradation and electrode reaction. Phenol hydroxylase and qPCR assays confirmed that appropriate electrical stimulation could improve phenol hydroxylase activity and promote cells growth. Toxicity assessment suggested the treatment of the coking wastewater by coupling system led to less toxicity than untreated wastewater. Copyright © 2016 Elsevier Ltd. All rights reserved.

A search for new CYP3A4 variants as determinants of tacrolimus dose requirements in renal-transplanted patients.

PubMed

Tavira, Beatriz; Coto, Eliecer; Diaz-Corte, Carmen; Alvarez, Victoria; López-Larrea, Carlos; Ortega, Francisco

2013-08-01

The CYP3A5*3 and CYP3A4*1B alleles have been related with tacrolimus (Tac) dose requirements. The rare CYP3A4*22 variant has also been associated with a significantly lower Tac dose. We genotyped the three single-nucleotide polymorphisms in 206 kidney-transplanted patients who received Tac as the primary immunosuppressor. CYP3A5*1 and CYP3A4*1B allele carriers received a significantly higher Tac dose (P<0.01) compared with wild-type homozygotes. We did not find significant differences between the CYP3A4*22 genotypes, either nominally or according to the CYP3A5 genotype (expressers vs. nonexpressers). Sequencing of CYP3A4 coding exons in a total of 15 patients revealed only one nonreported missense change (p.P227>T) in one patient. We concluded that CYP3A5*3 and CYP3A4*1B were the main determinants of the Tac dose-adjusted blood concentration in our cohort of renal-transplanted patients.

Draft Genome Sequence, and a Sequence-Defined Genetic Linkage Map of the Legume Crop Species Lupinus angustifolius L

PubMed Central

Zheng, Zequn; Zhang, Qisen; Zhou, Gaofeng; Sweetingham, Mark W.; Howieson, John G.; Li, Chengdao

2013-01-01

Lupin (Lupinus angustifolius L.) is the most recently domesticated crop in major agricultural cultivation. Its seeds are high in protein and dietary fibre, but low in oil and starch. Medical and dietetic studies have shown that consuming lupin-enriched food has significant health benefits. We report the draft assembly from a whole genome shotgun sequencing dataset for this legume species with 26.9x coverage of the genome, which is predicted to contain 57,807 genes. Analysis of the annotated genes with metabolic pathways provided a partial understanding of some key features of lupin, such as the amino acid profile of storage proteins in seeds. Furthermore, we applied the NGS-based RAD-sequencing technology to obtain 8,244 sequence-defined markers for anchoring the genomic sequences. A total of 4,214 scaffolds from the genome sequence assembly were aligned into the genetic map. The combination of the draft assembly and a sequence-defined genetic map made it possible to locate and study functional genes of agronomic interest. The identification of co-segregating SNP markers, scaffold sequences and gene annotation facilitated the identification of a candidate R gene associated with resistance to the major lupin disease anthracnose. We demonstrated that the combination of medium-depth genome sequencing and a high-density genetic linkage map by application of NGS technology is a cost-effective approach to generating genome sequence data and a large number of molecular markers to study the genomics, genetics and functional genes of lupin, and to apply them to molecular plant breeding. This strategy does not require prior genome knowledge, which potentiates its application to a wide range of non-model species. PMID:23734219

Draft genome sequence, and a sequence-defined genetic linkage map of the legume crop species Lupinus angustifolius L.

PubMed

Yang, Huaan; Tao, Ye; Zheng, Zequn; Zhang, Qisen; Zhou, Gaofeng; Sweetingham, Mark W; Howieson, John G; Li, Chengdao

2013-01-01

Lupin (Lupinus angustifolius L.) is the most recently domesticated crop in major agricultural cultivation. Its seeds are high in protein and dietary fibre, but low in oil and starch. Medical and dietetic studies have shown that consuming lupin-enriched food has significant health benefits. We report the draft assembly from a whole genome shotgun sequencing dataset for this legume species with 26.9x coverage of the genome, which is predicted to contain 57,807 genes. Analysis of the annotated genes with metabolic pathways provided a partial understanding of some key features of lupin, such as the amino acid profile of storage proteins in seeds. Furthermore, we applied the NGS-based RAD-sequencing technology to obtain 8,244 sequence-defined markers for anchoring the genomic sequences. A total of 4,214 scaffolds from the genome sequence assembly were aligned into the genetic map. The combination of the draft assembly and a sequence-defined genetic map made it possible to locate and study functional genes of agronomic interest. The identification of co-segregating SNP markers, scaffold sequences and gene annotation facilitated the identification of a candidate R gene associated with resistance to the major lupin disease anthracnose. We demonstrated that the combination of medium-depth genome sequencing and a high-density genetic linkage map by application of NGS technology is a cost-effective approach to generating genome sequence data and a large number of molecular markers to study the genomics, genetics and functional genes of lupin, and to apply them to molecular plant breeding. This strategy does not require prior genome knowledge, which potentiates its application to a wide range of non-model species.

Dynamic modeling of normal faults of the 2016 Central Italy earthquake sequence

NASA Astrophysics Data System (ADS)

Aochi, Hideo

2017-04-01

The earthquake sequence of the Central Italy in 2016 are characterized mainly by the Mw6.0 24th August, Mw5.9 26th October and Mw6.4 30th October as well as two Mw5.4 earthquakes (24th August, 26th October) (catalogue INGV). They all show normal faulting mechanisms corresponding to the Apennines's tectonics. They are aligned briefly along NNW-SSE axis, and they may not be on a single continuous fault plane. Therefore, dynamic rupture modeling of sequences should be carried out supposing co-planar normal multiple segments. We apply a Boundary Domain Method (BDM, Goto and Bielak, GJI, 2008) coupling a boundary integral equation method and a domain-based method, namely a finite difference method in this study. The Mw6.0 24th August earthquake is modeled. We use the basic information of hypocenter position, focal mechanism and potential ruptured dimension from the INGV catalogue and Tinti et al., GRL, 2016), and begin with a simple condition (homogeneous boundary condition). From our preliminary simulations, it is shown that a uniformly extended rupture model does not fit the near-field ground motions and localized heterogeneity would be required.

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

Kappler, Ulrike; Davenport, Karen W.; Beatson, Scott

Starkeya novella (Starkey 1934) Kelly et al. 2000 is a member of the family Xanthobacteraceae in the order Rhizobiales, which is thus far poorly characterized at the genome level. Cultures from this spe- cies are most interesting due to their facultatively chemolithoautotrophic lifestyle, which allows them to both consume carbon dioxide and to produce it. This feature makes S. novella an interesting model organism for studying the genomic basis of regulatory networks required for the switch between consumption and production of carbon dioxide, a key component of the global carbon cycle. In addition, S. novella is of interest for itsmore » ability to grow on various inorganic sulfur compounds and several C1- compounds such as methanol. Besides Azorhizobium caulinodans, S. novella is only the second species in the family Xanthobacteraceae with a completely sequenced genome of a type strain. The cur- rent taxonomic classification of this group is in significant conflict with the 16S rRNA data. The genomic data indicate that the physiological capabilities of the organism might have been underestimated. Here, the 4,765,023 bp long chromosome with its 4,511 protein-coding and 52 RNA genes was sequenced as part of the DOE Joint Genome Institute Community Sequencing Program (CSP) 2008.« less

Launch vehicle test and checkout plan. - Volume 2: Saturn 1B launch vehicle Skylab R (rescue) and AS-208 flow plan and listings

NASA Technical Reports Server (NTRS)

1973-01-01

The launch operations test and checkout plan is a planning document that establishes all launch site checkout activity, including the individual tests and sequence of testing required to fulfill the development center and KSC test and checkout requirements. This volume contains the launch vehicle test and checkout plan encompassing S-1B, S-4B, IU stage, and ground support equipment tests. The plan is based upon AS-208 flow utilizing a manned spacecraft, LUT 1, and launch pad 39B facilities.

Eukaryotic Initiation Factor eIFiso4G1 and eIFiso4G2 Are Isoforms Exhibiting Distinct Functional Differences in Supporting Translation in Arabidopsis*

PubMed Central

Gallie, Daniel R.

2016-01-01

The eukaryotic translation initiation factor (eIF) 4G is required during protein synthesis to promote the assembly of several factors involved in the recruitment of a 40S ribosomal subunit to an mRNA. Although many eukaryotes express two eIF4G isoforms that are highly similar, the eIF4G isoforms in plants, referred to as eIF4G and eIFiso4G, are highly divergent in size, sequence, and domain organization but both can interact with eIF4A, eIF4B, eIF4E isoforms, and the poly(A)-binding protein. Nevertheless, eIF4G and eIFiso4G from wheat exhibit preferences in the mRNAs they translate optimally. For example, mRNA containing the 5′-leader (called Ω) of tobacco mosaic virus preferentially uses eIF4G in wheat germ lysate. In this study, the eIF4G isoform specificity of Ω was used to examine functional differences of the eIF4G isoforms in Arabidopsis. As in wheat, Ω-mediated translation was reduced in an eif4g null mutant. Loss of the eIFiso4G1 isoform, which is similar in sequence to wheat eIFiso4G, did not substantially affect Ω-mediated translation. However, loss of the eIFiso4G2 isoform substantially reduced Ω-mediated translation. eIFiso4G2 is substantially divergent from eIFiso4G1 and is present only in the Brassicaceae, suggesting a recent evolution. eIFiso4G2 isoforms exhibit sequence-specific differences in regions representing partner protein and RNA binding sites. Loss of any eIF4G isoform also resulted in a substantial reduction in reporter transcript level. These results suggest that eIFiso4G2 appeared late in plant evolution and exhibits more functional similarity with eIF4G than with eIFiso4G1 during Ω-mediated translation. PMID:26578519

Exploring the sequence-function relationship in transcriptional regulation by the lac O1 operator.

PubMed

Maity, Tuhin S; Jha, Ramesh K; Strauss, Charlie E M; Dunbar, John

2012-07-01

Understanding how binding of a transcription factor to an operator is influenced by the operator sequence is an ongoing quest. It facilitates discovery of alternative binding sites as well as tuning of transcriptional regulation. We investigated the behavior of the Escherichia coli Lac repressor (LacI) protein with a large set of lac O(1) operator variants. The 114 variants examined contained a mean of 2.9 (range 0-4) mutations at positions -4, -2, +2 and +4 in the minimally required 17 bp operator. The relative affinity of LacI for the operators was examined by quantifying expression of a GFP reporter gene and Rosetta structural modeling. The combinations of mutations in the operator sequence created a wide range of regulatory behaviors. We observed variations in the GFP fluorescent signal among the operator variants of more than an order of magnitude under both uninduced and induced conditions. We found that a single nucleotide change may result in changes of up to six- and 12-fold in uninduced and induced GFP signals, respectively. Among the four positions mutated, we found that nucleotide G at position -4 is strongly correlated with strong repression. By Rosetta modeling, we found a significant correlation between the calculated binding energy and the experimentally observed transcriptional repression strength for many operators. However, exceptions were also observed, underscoring the necessity for further improvement in biophysical models of protein-DNA interactions. © 2012 The Authors Journal compilation © 2012 FEBS.

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.

Shifted termination assay (STA) fragment analysis to detect BRAF V600 mutations in papillary thyroid carcinomas

PubMed Central

2013-01-01

Background BRAF mutation is an important diagnostic and prognostic marker in patients with papillary thyroid carcinoma (PTC). To be applicable in clinical laboratories with limited equipment, diverse testing methods are required to detect BRAF mutation. Methods A shifted termination assay (STA) fragment analysis was used to detect common V600 BRAF mutations in 159 PTCs with DNAs extracted from formalin-fixed paraffin-embedded tumor tissue. The results of STA fragment analysis were compared to those of direct sequencing. Serial dilutions of BRAF mutant cell line (SNU-790) were used to calculate limit of detection (LOD). Results BRAF mutations were detected in 119 (74.8%) PTCs by STA fragment analysis. In direct sequencing, BRAF mutations were observed in 118 (74.2%) cases. The results of STA fragment analysis had high correlation with those of direct sequencing (p < 0.00001, κ = 0.98). The LOD of STA fragment analysis and direct sequencing was 6% and 12.5%, respectively. In PTCs with pT3/T4 stages, BRAF mutation was observed in 83.8% of cases. In pT1/T2 carcinomas, BRAF mutation was detected in 65.9% and this difference was statistically significant (p = 0.007). Moreover, BRAF mutation was more frequent in PTCs with extrathyroidal invasion than tumors without extrathyroidal invasion (84.7% versus 62.2%, p = 0.001). To prepare and run the reactions, direct sequencing required 450 minutes while STA fragment analysis needed 290 minutes. Conclusions STA fragment analysis is a simple and sensitive method to detect BRAF V600 mutations in formalin-fixed paraffin-embedded clinical samples. Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/5684057089135749 PMID:23883275

Analysis of Litopenaeus vannamei Transcriptome Using the Next-Generation DNA Sequencing Technique

PubMed Central

Li, Chaozheng; Weng, Shaoping; Chen, Yonggui; Yu, Xiaoqiang; Lü, Ling; Zhang, Haiqing; He, Jianguo; Xu, Xiaopeng

2012-01-01

Background Pacific white shrimp (Litopenaeus vannamei), the major species of farmed shrimps in the world, has been attracting extensive studies, which require more and more genome background knowledge. The now available transcriptome data of L. vannamei are insufficient for research requirements, and have not been adequately assembled and annotated. Methodology/Principal Findings This is the first study that used a next-generation high-throughput DNA sequencing technique, the Solexa/Illumina GA II method, to analyze the transcriptome from whole bodies of L. vannamei larvae. More than 2.4 Gb of raw data were generated, and 109,169 unigenes with a mean length of 396 bp were assembled using the SOAP denovo software. 73,505 unigenes (>200 bp) with good quality sequences were selected and subjected to annotation analysis, among which 37.80% can be matched in NCBI Nr database, 37.3% matched in Swissprot, and 44.1% matched in TrEMBL. Using BLAST and BLAST2Go softwares, 11,153 unigenes were classified into 25 Clusters of Orthologous Groups of proteins (COG) categories, 8171 unigenes were assigned into 51 Gene ontology (GO) functional groups, and 18,154 unigenes were divided into 220 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. To primarily verify part of the results of assembly and annotations, 12 assembled unigenes that are homologous to many embryo development-related genes were chosen and subjected to RT-PCR for electrophoresis and Sanger sequencing analyses, and to real-time PCR for expression profile analyses during embryo development. Conclusions/Significance The L. vannamei transcriptome analyzed using the next-generation sequencing technique enriches the information of L. vannamei genes, which will facilitate our understanding of the genome background of crustaceans, and promote the studies on L. vannamei. PMID:23071809

NF-E2 and GATA binding motifs are required for the formation of DNase I hypersensitive site 4 of the human beta-globin locus control region.

PubMed Central

Stamatoyannopoulos, J A; Goodwin, A; Joyce, T; Lowrey, C H

1995-01-01

The beta-like globin genes require the upstream locus control region (LCR) for proper expression. The active elements of the LCR coincide with strong erythroid-specific DNase I-hypersensitive sites (HSs). We have used 5' HS4 as a model to study the formation of these HSs. Previously, we identified a 101 bp element that is required for the formation of this HS. This element binds six proteins in vitro. We now report a mutational analysis of the HS4 HS-forming element (HSFE). This analysis indicates that binding sites for the hematopoietic transcription factors NF-E2 and GATA-1 are required for the formation of the characteristic chromatin structure of the HS following stable transfection into murine erythroleukemia cells. Similarly arranged NF-E2 and GATA binding sites are present in the other HSs of the human LCR, as well as in the homologous mouse and goat sequences and the chicken beta-globin enhancer. A combination of DNase I and micrococcal nuclease sensitivity assays indicates that the characteristic erythroid-specific hypersensitivity of HS4 to DNase I is the result of tissue-specific alterations in both nucleosome positioning and tertiary DNA structure. Images PMID:7828582

The Neurospora crassa PP2A Regulatory Subunits RGB1 and B56 Are Required for Proper Growth and Development and Interact with the NDR Kinase COT1

PubMed Central

Shomin-Levi, Hila; Yarden, Oded

2017-01-01

COT1 is the founding member of the highly conserved nuclear Dbf2-related (NDR) Ser/Thr kinase family and plays a role in the regulation of polar growth and development in Neurospora crassa and other fungi. Changes in COT1 phosphorylation state have been shown to affect hyphal elongation, branching, and conidiation. The function of NDR protein kinases has been shown to be regulated by type 2A protein phosphatases (PP2As). PP2As are heterotrimers comprised of a catalytic and scaffolding protein along with an interchangeable regulatory subunit involved in determining substrate specificity. Inactivation of the N. crassa PP2A regulatory subunits rgb-1 and b56 conferred severe hyphal growth defects. Partial suppression of defects observed in the rgb-1RIP strain (but not in the Δb56 mutant) was observed in cot-1 phosphomimetic mutants, demonstrating that altering COT1 phosphorylation state can bypass, at least in part, the requirement of a functional RGB1 subunit. The functional fusion proteins RGB1::GFP and B56::GFP predominantly localized to hyphal tips and septa, respectively, indicating that their primary activity is in different cellular locations. COT1 protein forms exhibited a hyperphosphorylated gel migration pattern in an rgb-1RIP mutant background, similar to that observed when the fungus was cultured in the presence of the PP2A inhibitor cantharidin. COT1 was hypophosphorylated in a Δb56 mutant background, suggesting that this regulatory subunit may be involved in determining COT1 phosphorylation state, yet in an indirect manner. Reciprocal co-immunoprecipitation analyses, using tagged COT1, PPH1, RGB1, and B56 subunits established that these proteins physically interact. Taken together, our data determine the presence of a functional and physical link between PP2A and COT1 and show that two of the PP2A regulatory subunits interact with the kinase and determine COT1 phosphorylation state. PMID:28928725

Genome Sequence of Bacillus megaterium Strain YC4-R4, a Plant Growth-Promoting Rhizobacterium Isolated from a High-Salinity Environment.

PubMed

Vílchez, Juan Ignacio; Tang, Qiming; Kaushal, Richa; Wang, Wei; Lv, Suhui; He, Danxia; Chu, Zhaoqing; Zhang, Heng; Liu, Renyi; Zhang, Huiming

2018-06-21

Here, we report the complete genome sequence for Bacillus megaterium strain YC4-R4, a highly salt-tolerant rhizobacterium that promotes growth in plants. The sequencing process was performed by combining pyrosequencing and single-molecule sequencing techniques. The complete genome is estimated to be approximately 5.44 Mb, containing a total of 5,673 predicted protein-coding DNA sequences (CDSs). Copyright © 2018 Vílchez et al.

Simian virus 40 major late promoter: an upstream DNA sequence required for efficient in vitro transcription.

PubMed Central

Brady, J; Radonovich, M; Thoren, M; Das, G; Salzman, N P

1984-01-01

We have previously identified an 11-base DNA sequence, 5'-G-G-T-A-C-C-T-A-A-C-C-3' (simian virus 40 [SV40] map position 294 to 304), which is important in the control of SV40 late RNA expression in vitro and in vivo (Brady et al., Cell 31:625-633, 1982). We report here the identification of another domain of the SV40 late promoter. A series of mutants with deletions extending from SV40 map position 0 to 300 was prepared by nuclease BAL 31 treatment. The cloned templates were then analyzed for efficiency and accuracy of late SV40 RNA expression in the Manley in vitro transcription system. Our studies showed that, in addition to the promoter domain near map position 300, there are essential DNA sequences between nucleotide positions 74 and 95 that are required for efficient expression of late SV40 RNA. Included in this SV40 DNA sequence were two of the six GGGCGG SV40 repeat sequences and an 11-nucleotide segment which showed strong homology with the upstream sequences required for the efficient in vitro and in vivo expression of the histone H2A gene. This upstream promoter sequence supported transcription with the same efficiency even when it was moved 72 nucleotides closer to the major late cap site. In vitro promoter competition analysis demonstrated that the upstream promoter sequence, independent of the 294 to 304 promoter element, is capable of binding polymerase-transcription factors required for SV40 late gene transcription. Finally, we show that DNA sequences which control the specificity of RNA initiation at nucleotide 325 lie downstream of map position 294. Images PMID:6321950

Impact of alemtuzumab on HIV persistence in an HIV-infected individual on antiretroviral therapy with Sezary syndrome.

PubMed

Rasmussen, Thomas A; McMahon, James; Chang, J Judy; Symons, Jori; Roche, Michael; Dantanarayana, Ashanti; Okoye, Afam; Hiener, Bonnie; Palmer, Sarah; Lee, Wen Shi; Kent, Stephen J; Van Der Weyden, Carrie; Prince, H Miles; Cameron, Paul U; Lewin, Sharon R

2017-08-24

To study the effects of alemtuzumab on HIV persistence in an HIV-infected individual on antiretroviral therapy (ART) with Sezary syndrome, a rare malignancy of CD4 T cells. Case report. Blood was collected 30 and 18 months prior to presentation with Sezary syndrome, at the time of presentation and during alemtuzumab. T-cell subsets in malignant (CD7-CD26-TCR-VBeta2+) and nonmalignant cells were quantified by flow cytometry. HIV-DNA in total CD4 T cells, in sorted malignant and nonmalignant CD4 T cells, was quantified by PCR and clonal expansion of HIV-DNA assessed by full-length next-generation sequencing. HIV-hepatitis B virus coinfection was diagnosed and antiretroviral therapy initiated 4 years prior to presentation with Sezary syndrome and primary cutaneous anaplastic large cell lymphoma. The patient received alemtuzumab 10 mg three times per week for 4 weeks but died 6 weeks post alemtuzumab. HIV-DNA was detected in nonmalignant but not in malignant CD4 T cells, consistent with expansion of a noninfected CD4 T-cell clone. Full-length HIV-DNA sequencing demonstrated multiple defective viruses but no identical or expanded sequences. Alemtuzumab extensively depleted T cells, including more than 1 log reduction in total T cells and more than 3 log reduction in CD4 T cells. Finally, alemtuzumab decreased HIV-DNA in CD4 T cells by 57% but HIV-DNA remained detectable at low levels even after depletion of nearly all CD4 T cells. Alemtuzumab extensively depleted multiple T-cell subsets and decreased the frequency of but did not eliminate HIV-infected CD4 T cells. Studying the effects on HIV persistence following immune recovery in HIV-infected individuals who require alemtuzumab for malignancy or in animal studies may provide further insights into novel cure strategies.

Outcomes of Cleft Palate Repair in Patients with Pierre Robin Sequence: A Matched Case-Control Study.

PubMed

Hardwicke, Joseph T; Richards, Helen; Cafferky, Louise; Underwood, Imogen; ter Horst, Britt; Slator, Rona

2016-03-01

Pierre Robin sequence results from a cascade of events that occur during embryologic development and frequently presents with cleft palate. Some studies have shown speech outcomes to be worse in patients with Pierre Robin sequence after cleft palate repair. A cohort of Pierre Robin sequence patients who all required an airway intervention and nasogastric feeding in the neonatal period were identified and speech outcomes assessed at 5 years of age. A cleft- and sex-matched non-Pierre Robin sequence, cleft palate-only comparison group was also identified from the same institution and study period. A total of 24 patients with Pierre Robin sequence that required airway and nutritional support in the neonatal period were matched for age, sex, and cleft type to a group of 24 non-Pierre Robin sequence cleft patients. There was no significant difference in the incidence of oronasal fistula between the groups. Secondary surgery for velopharyngeal incompetence was significantly more (p = 0.017) in the Pierre Robin sequence group, who also had significantly greater nasality (p = 0.031) and cleft speech characteristic (p = 0.023) scores. The authors hypothesize that other factors may exist in Pierre Robin sequence that may lead to poor speech outcomes. The authors would suggest counseling parents of children with Pierre Robin sequence that have required a neonatal airway intervention, that speech development may be poorer than in other children with cleft palate, and that these children will have a significantly higher incidence of secondary speech surgery. Risk, II.

Increased fMRI Sensitivity at Equal Data Burden Using Averaged Shifted Echo Acquisition

PubMed Central

Witt, Suzanne T.; Warntjes, Marcel; Engström, Maria

2016-01-01

There is growing evidence as to the benefits of collecting BOLD fMRI data with increased sampling rates. However, many of the newly developed acquisition techniques developed to collect BOLD data with ultra-short TRs require hardware, software, and non-standard analytic pipelines that may not be accessible to all researchers. We propose to incorporate the method of shifted echo into a standard multi-slice, gradient echo EPI sequence to achieve a higher sampling rate with a TR of <1 s with acceptable spatial resolution. We further propose to incorporate temporal averaging of consecutively acquired EPI volumes to both ameliorate the reduced temporal signal-to-noise inherent in ultra-fast EPI sequences and reduce the data burden. BOLD data were collected from 11 healthy subjects performing a simple, event-related visual-motor task with four different EPI sequences: (1) reference EPI sequence with TR = 1440 ms, (2) shifted echo EPI sequence with TR = 700 ms, (3) shifted echo EPI sequence with every two consecutively acquired EPI volumes averaged and effective TR = 1400 ms, and (4) shifted echo EPI sequence with every four consecutively acquired EPI volumes averaged and effective TR = 2800 ms. Both the temporally averaged sequences exhibited increased temporal signal-to-noise over the shifted echo EPI sequence. The shifted echo sequence with every two EPI volumes averaged also had significantly increased BOLD signal change compared with the other three sequences, while the shifted echo sequence with every four EPI volumes averaged had significantly decreased BOLD signal change compared with the other three sequences. The results indicated that incorporating the method of shifted echo into a standard multi-slice EPI sequence is a viable method for achieving increased sampling rate for collecting event-related BOLD data. Further, consecutively averaging every two consecutively acquired EPI volumes significantly increased the measured BOLD signal change and the subsequently calculated activation map statistics. PMID:27932947

Proceedings of the Conference on Moments and Signal Proceedings Held in Monterey, California on 30-31 March 1992

DTIC Science & Technology

1992-09-21

describt systeiii- atic methodologies for selecting nonlitinr transformiations for blind equal- ization algorithins ,and thus new types of culnulants...nonlinearity is inside the adaptive filter, i.e., the nonlinear filter or neural network. ’We describe methodologies for selecting nonlinear...which do riot require any known training sequence during the startup period. 4 The paper describes systematic methodologies for selecting the

DNA Recognition by a σ 54 Transcriptional Activator from Aquifex aeolicus

DOE PAGES

Vidangos, Natasha K.; Heideker, Johanna; Lyubimov, Artem; ...

2014-08-23

Transcription initiation by bacterial σ 54-polymerase requires the action of a transcriptional activator protein. Activators bind sequence-specifically upstream of the transcription initiation site via a DNA-binding domain. The structurally characterized DNA-binding domains from activators all belong to the Factor for Inversion Stimulation (Fis) family of helix-turn-helix DNA-binding proteins. We report here structures of the free and DNA-bound forms of the DNA-binding domain of NtrC4 (4DBD) from Aquifex aeolicus, a member of the NtrC family of σ 54 activators. Two NtrC4 binding sites were identified upstream (-145 and -85 base pairs) from the start of the lpxC gene, which is responsiblemore » for the first committed step in Lipid A biosynthesis. This is the first experimental evidence for σ 54 regulation in lpxC expression. 4DBD was crystallized both without DNA and in complex with the -145 binding site. The structures, together with biochemical data, indicate that NtrC4 binds to DNA in a manner that is similar to that of its close homologue, Fis. Ultimately, the greater sequence specificity for the binding of 4DBD relative to Fis seems to arise from a larger number of base specific contacts contributing to affinity than for Fis.« less

Intravenous phage display identifies peptide sequences that target the burn-injured intestine.

PubMed

Costantini, Todd W; Eliceiri, Brian P; Putnam, James G; Bansal, Vishal; Baird, Andrew; Coimbra, Raul

2012-11-01

The injured intestine is responsible for significant morbidity and mortality after severe trauma and burn; however, targeting the intestine with therapeutics aimed at decreasing injury has proven difficult. We hypothesized that we could use intravenous phage display technology to identify peptide sequences that target the injured intestinal mucosa in a murine model, and then confirm the cross-reactivity of this peptide sequence with ex vivo human gut. Four hours following 30% TBSA burn we performed an in vivo, intravenous systemic administration of phage library containing 10(12) phage in balb/c mice to biopan for gut-targeting peptides. In vivo assessment of the candidate peptide sequences identified after 4 rounds of internalization was performed by injecting 1×10(12) copies of each selected phage clone into sham or burned animals. Internalization into the gut was assessed using quantitative polymerase chain reaction. We then incubated this gut-targeting peptide sequence with human intestine and visualized fluorescence using confocal microscopy. We identified 3 gut-targeting peptide sequences which caused collapse of the phage library (4-1: SGHQLLLNKMP, 4-5: ILANDLTAPGPR, 4-11: SFKPSGLPAQSL). Sequence 4-5 was internalized into the intestinal mucosa of burned animals 9.3-fold higher than sham animals injected with the same sequence (2.9×10(5)vs. 3.1×10(4) particles per mg tissue). Sequences 4-1 and 4-11 were both internalized into the gut, but did not demonstrate specificity for the injured mucosa. Phage sequence 4-11 demonstrated cross-reactivity with human intestine. In the future, this gut-targeting peptide sequence could serve as a platform for the delivery of biotherapeutics. Copyright © 2012 Elsevier Inc. All rights reserved.

Construction of a multiplex mutation hot spot PCR panel: the first step towards colorectal cancer genotyping on the GS Junior platform.

PubMed

Péterfia, Bálint; Kalmár, Alexandra; Patai, Árpád V; Csabai, István; Bodor, András; Micsik, Tamás; Wichmann, Barnabás; Egedi, Krisztina; Hollósi, Péter; Kovalszky, Ilona; Tulassay, Zsolt; Molnár, Béla

2017-01-01

Background: To support cancer therapy, development of low cost library preparation techniques for targeted next generation sequencing (NGS) is needed. In this study we designed and tested a PCR-based library preparation panel with limited target area for sequencing the top 12 somatic mutation hot spots in colorectal cancer on the GS Junior instrument. Materials and Methods: A multiplex PCR panel was designed to amplify regions of mutation hot spots in 12 selected genes ( APC, BRAF, CTNNB1, EGFR, FBXW7, KRAS, NRAS, MSH6, PIK3CA, SMAD2, SMAD4, TP53 ). Amplicons were sequenced on a GS Junior instrument using ligated and barcoded adaptors. Eight samples were sequenced in a single run. Colonic DNA samples (8 normal mucosa; 33 adenomas; 17 adenocarcinomas) as well as HT-29 and Caco-2 cell lines with known mutation profiles were analyzed. Variants found by the panel on APC, BRAF, KRAS and NRAS genes were validated by conventional sequencing. Results: In total, 34 kinds of mutations were detected including two novel mutations ( FBXW7 c.1740:C>G and SMAD4 c.413C>G) that have not been recorded in mutation databases, and one potential germline mutation ( APC ). The most frequently mutated genes were APC, TP53 and KRAS with 30%, 15% and 21% frequencies in adenomas and 29%, 53% and 29% frequencies in carcinomas, respectively. In cell lines, all the expected mutations were detected except for one located in a homopolymer region. According to re-sequencing results sensitivity and specificity was 100% and 92% respectively. Conclusions: Our NGS-based screening panel denotes a promising step towards low cost colorectal cancer genotyping on the GS Junior instrument. Despite the relatively low coverage, we discovered two novel mutations and obtained mutation frequencies comparable to literature data. Additionally, as an advantage, this panel requires less template DNA than sequence capture colon cancer panels currently available for the GS Junior instrument.

Population-based rare variant detection via pooled exome or custom hybridization capture with or without individual indexing.

PubMed

Ramos, Enrique; Levinson, Benjamin T; Chasnoff, Sara; Hughes, Andrew; Young, Andrew L; Thornton, Katherine; Li, Allie; Vallania, Francesco L M; Province, Michael; Druley, Todd E

2012-12-06

Rare genetic variation in the human population is a major source of pathophysiological variability and has been implicated in a host of complex phenotypes and diseases. Finding disease-related genes harboring disparate functional rare variants requires sequencing of many individuals across many genomic regions and comparing against unaffected cohorts. However, despite persistent declines in sequencing costs, population-based rare variant detection across large genomic target regions remains cost prohibitive for most investigators. In addition, DNA samples are often precious and hybridization methods typically require large amounts of input DNA. Pooled sample DNA sequencing is a cost and time-efficient strategy for surveying populations of individuals for rare variants. We set out to 1) create a scalable, multiplexing method for custom capture with or without individual DNA indexing that was amenable to low amounts of input DNA and 2) expand the functionality of the SPLINTER algorithm for calling substitutions, insertions and deletions across either candidate genes or the entire exome by integrating the variant calling algorithm with the dynamic programming aligner, Novoalign. We report methodology for pooled hybridization capture with pre-enrichment, indexed multiplexing of up to 48 individuals or non-indexed pooled sequencing of up to 92 individuals with as little as 70 ng of DNA per person. Modified solid phase reversible immobilization bead purification strategies enable no sample transfers from sonication in 96-well plates through adapter ligation, resulting in 50% less library preparation reagent consumption. Custom Y-shaped adapters containing novel 7 base pair index sequences with a Hamming distance of ≥2 were directly ligated onto fragmented source DNA eliminating the need for PCR to incorporate indexes, and was followed by a custom blocking strategy using a single oligonucleotide regardless of index sequence. These results were obtained aligning raw reads against the entire genome using Novoalign followed by variant calling of non-indexed pools using SPLINTER or SAMtools for indexed samples. With these pipelines, we find sensitivity and specificity of 99.4% and 99.7% for pooled exome sequencing. Sensitivity, and to a lesser degree specificity, proved to be a function of coverage. For rare variants (≤2% minor allele frequency), we achieved sensitivity and specificity of ≥94.9% and ≥99.99% for custom capture of 2.5 Mb in multiplexed libraries of 22-48 individuals with only ≥5-fold coverage/chromosome, but these parameters improved to ≥98.7 and 100% with 20-fold coverage/chromosome. This highly scalable methodology enables accurate rare variant detection, with or without individual DNA sample indexing, while reducing the amount of required source DNA and total costs through less hybridization reagent consumption, multi-sample sonication in a standard PCR plate, multiplexed pre-enrichment pooling with a single hybridization and lesser sequencing coverage required to obtain high sensitivity.

Quantum sequencing: opportunities and challenges

NASA Astrophysics Data System (ADS)

di Ventra, Massimiliano

Personalized or precision medicine refers to the ability of tailoring drugs to the specific genome and transcriptome of each individual. It is however not yet feasible due the high costs and slow speed of present DNA sequencing methods. I will discuss a sequencing protocol that requires the measurement of the distributions of transverse tunneling currents during the translocation of single-stranded DNA into nanochannels. I will show that such a quantum sequencing approach can reach unprecedented speeds, without requiring any chemical preparation, amplification or labeling. I will discuss recent experiments that support these theoretical predictions, the advantages of this approach over other sequencing methods, and stress the challenges that need to be overcome to render it commercially viable.

Targeted sequencing of plant genomes

Treesearch

Mark D. Huynh

2014-01-01

Next-generation sequencing (NGS) has revolutionized the field of genetics by providing a means for fast and relatively affordable sequencing. With the advancement of NGS, wholegenome sequencing (WGS) has become more commonplace. However, sequencing an entire genome is still not cost effective or even beneficial in all cases. In studies that do not require a whole-...

A Simulation of DNA Sequencing Utilizing 3M Post-It[R] Notes

ERIC Educational Resources Information Center

Christensen, Doug

2009-01-01

An inexpensive and equipment free approach to teaching the technical aspects of DNA sequencing. The activity described requires an instructor with a familiarity of DNA sequencing technology but provides a straight forward method of teaching the technical aspects of sequencing in the absence of expensive sequencing equipment. The final sequence…

The eukaryotic signal sequence, YGRL, targets the chlamydial inclusion

PubMed Central

Kabeiseman, Emily J.; Cichos, Kyle H.; Moore, Elizabeth R.

2014-01-01

Understanding how host proteins are targeted to pathogen-specified organelles, like the chlamydial inclusion, is fundamentally important to understanding the biogenesis of these unique subcellular compartments and how they maintain autonomy within the cell. Syntaxin 6, which localizes to the chlamydial inclusion, contains an YGRL signal sequence. The YGRL functions to return syntaxin 6 to the trans-Golgi from the plasma membrane, and deletion of the YGRL signal sequence from syntaxin 6 also prevents the protein from localizing to the chlamydial inclusion. YGRL is one of three YXXL (YGRL, YQRL, and YKGL) signal sequences which target proteins to the trans-Golgi. We designed various constructs of eukaryotic proteins to test the specificity and propensity of YXXL sequences to target the inclusion. The YGRL signal sequence redirects proteins (e.g., Tgn38, furin, syntaxin 4) that normally do not localize to the chlamydial inclusion. Further, the requirement of the YGRL signal sequence for syntaxin 6 localization to inclusions formed by different species of Chlamydia is conserved. These data indicate that there is an inherent property of the chlamydial inclusion, which allows it to recognize the YGRL signal sequence. To examine whether this “inherent property” was protein or lipid in nature, we asked if deletion of the YGRL signal sequence from syntaxin 6 altered the ability of the protein to interact with proteins or lipids. Deletion or alteration of the YGRL from syntaxin 6 does not appreciably impact syntaxin 6-protein interactions, but does decrease syntaxin 6-lipid interactions. Intriguingly, data also demonstrate that YKGL or YQRL can successfully substitute for YGRL in localization of syntaxin 6 to the chlamydial inclusion. Importantly and for the first time, we are establishing that a eukaryotic signal sequence targets the chlamydial inclusion. PMID:25309881

A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers.

PubMed

Quail, Michael A; Smith, Miriam; Coupland, Paul; Otto, Thomas D; Harris, Simon R; Connor, Thomas R; Bertoni, Anna; Swerdlow, Harold P; Gu, Yong

2012-07-24

Next generation sequencing (NGS) technology has revolutionized genomic and genetic research. The pace of change in this area is rapid with three major new sequencing platforms having been released in 2011: Ion Torrent's PGM, Pacific Biosciences' RS and the Illumina MiSeq. Here we compare the results obtained with those platforms to the performance of the Illumina HiSeq, the current market leader. In order to compare these platforms, and get sufficient coverage depth to allow meaningful analysis, we have sequenced a set of 4 microbial genomes with mean GC content ranging from 19.3 to 67.7%. Together, these represent a comprehensive range of genome content. Here we report our analysis of that sequence data in terms of coverage distribution, bias, GC distribution, variant detection and accuracy. Sequence generated by Ion Torrent, MiSeq and Pacific Biosciences technologies displays near perfect coverage behaviour on GC-rich, neutral and moderately AT-rich genomes, but a profound bias was observed upon sequencing the extremely AT-rich genome of Plasmodium falciparum on the PGM, resulting in no coverage for approximately 30% of the genome. We analysed the ability to call variants from each platform and found that we could call slightly more variants from Ion Torrent data compared to MiSeq data, but at the expense of a higher false positive rate. Variant calling from Pacific Biosciences data was possible but higher coverage depth was required. Context specific errors were observed in both PGM and MiSeq data, but not in that from the Pacific Biosciences platform. All three fast turnaround sequencers evaluated here were able to generate usable sequence. However there are key differences between the quality of that data and the applications it will support.

Parasail: SIMD C library for global, semi-global, and local pairwise sequence alignments.

PubMed

Daily, Jeff

2016-02-10

Sequence alignment algorithms are a key component of many bioinformatics applications. Though various fast Smith-Waterman local sequence alignment implementations have been developed for x86 CPUs, most are embedded into larger database search tools. In addition, fast implementations of Needleman-Wunsch global sequence alignment and its semi-global variants are not as widespread. This article presents the first software library for local, global, and semi-global pairwise intra-sequence alignments and improves the performance of previous intra-sequence implementations. A faster intra-sequence local pairwise alignment implementation is described and benchmarked, including new global and semi-global variants. Using a 375 residue query sequence a speed of 136 billion cell updates per second (GCUPS) was achieved on a dual Intel Xeon E5-2670 24-core processor system, the highest reported for an implementation based on Farrar's 'striped' approach. Rognes's SWIPE optimal database search application is still generally the fastest available at 1.2 to at best 2.4 times faster than Parasail for sequences shorter than 500 amino acids. However, Parasail was faster for longer sequences. For global alignments, Parasail's prefix scan implementation is generally the fastest, faster even than Farrar's 'striped' approach, however the opal library is faster for single-threaded applications. The software library is designed for 64 bit Linux, OS X, or Windows on processors with SSE2, SSE41, or AVX2. Source code is available from https://github.com/jeffdaily/parasail under the Battelle BSD-style license. Applications that require optimal alignment scores could benefit from the improved performance. For the first time, SIMD global, semi-global, and local alignments are available in a stand-alone C library.

Adaptive efficient compression of genomes

PubMed Central

2012-01-01

Modern high-throughput sequencing technologies are able to generate DNA sequences at an ever increasing rate. In parallel to the decreasing experimental time and cost necessary to produce DNA sequences, computational requirements for analysis and storage of the sequences are steeply increasing. Compression is a key technology to deal with this challenge. Recently, referential compression schemes, storing only the differences between a to-be-compressed input and a known reference sequence, gained a lot of interest in this field. However, memory requirements of the current algorithms are high and run times often are slow. In this paper, we propose an adaptive, parallel and highly efficient referential sequence compression method which allows fine-tuning of the trade-off between required memory and compression speed. When using 12 MB of memory, our method is for human genomes on-par with the best previous algorithms in terms of compression ratio (400:1) and compression speed. In contrast, it compresses a complete human genome in just 11 seconds when provided with 9 GB of main memory, which is almost three times faster than the best competitor while using less main memory. PMID:23146997

Cloning and sequencing of the allophycocyanin genes from Spirulina maxima (Cyanophyta)

NASA Astrophysics Data System (ADS)

Qin, Song; Hiroyuki, Kojima; Yoshikazu, Kawata; Shin-Ichi, Yano; Zeng, Cheng-Kui

1998-03-01

The genes coding for the α-and β-subunit of allophycocyanin ( apcA and apcB) from the cyanophyte Spirulina maxima were cloned and sequenced. The results revealed 44.4% of nucleotide sequence similarity and 30.4% of similarity of deduced amino acid sequence between them. The amino acid sequence identities between S. maxima and S. platensis are 99.4% for α subunit and 100% for β subunit.

Bovine viral diarrhea virus NS3 serine proteinase: polyprotein cleavage sites, cofactor requirements, and molecular model of an enzyme essential for pestivirus replication.

PubMed Central

Xu, J; Mendez, E; Caron, P R; Lin, C; Murcko, M A; Collett, M S; Rice, C M

1997-01-01

Members of the Flaviviridae encode a serine proteinase termed NS3 that is responsible for processing at several sites in the viral polyproteins. In this report, we show that the NS3 proteinase of the pestivirus bovine viral diarrhea virus (BVDV) (NADL strain) is required for processing at nonstructural (NS) protein sites 3/4A, 4A/4B, 4B/5A, and 5A/5B but not for cleavage at the junction between NS2 and NS3. Cleavage sites of the proteinase were determined by amino-terminal sequence analysis of the NS4A, NS4B, NS5A, and NS5B proteins. A conserved leucine residue is found at the P1 position of all four cleavage sites, followed by either serine (3/4A, 4B/5A, and 5A/5B sites) or alanine (4A/4B site) at the P1' position. Consistent with this cleavage site preference, a structural model of the pestivirus NS3 proteinase predicts a highly hydrophobic P1 specificity pocket. trans-Processing experiments implicate the 64-residue NS4A protein as an NS3 proteinase cofactor required for cleavage at the 4B/5A and 5A/5B sites. Finally, using a full-length functional BVDV cDNA clone, we demonstrate that a catalytically active NS3 serine proteinase is essential for pestivirus replication. PMID:9188600

All gene-sized DNA molecules in four species of hypotrichs have the same terminal sequence and an unusual 3' terminus.

PubMed Central

Klobutcher, L A; Swanton, M T; Donini, P; Prescott, D M

1981-01-01

In hypotrichous ciliates, all of the macronuclear DNA is in the form of low molecular weight molecules with an average size of approximately 2200 base pairs. Total macronuclear DNA from four hypotrichs has been shown to have inverted terminal repeats by direct sequence analysis. In Oxytricha nova, Oxytricha sp., and Stylonychia pustulata, this terminal sequence may be written as 5'-C4A4C4A4C4 ... 3'-G4T4G4T4G4T4G4T4G4 ... In Euplotes aediculatus, the sequences is similar but differs in the lengths of the duplex region (28 base pairs) and of the putative 3' extension (14 base pairs). Also in Euplotes, a second common sequence of 5 base pairs (A-A-C-T-T-T-T-G-A-A) occurs internal to the terminal repeat and a 17-base-pair heterogeneous region: 5'-C4A4C4A4C4A4C4(X)17T-T-G-A-A ... 3'-G2T4G4T4G4T4G4T4G4T4G4(X)17A-A-C-T-T ... The length of the terminal repeat sequence for O. nova was confirmed in cloned macronuclear DNA molecules. Images PMID:6265931

Reclassification of Actinobacillus muris as Muribacter muris gen. nov., comb. nov.

PubMed

Nicklas, Werner; Bisgaard, Magne; Aalbæk, Bent; Kuhnert, Peter; Christensen, Henrik

2015-10-01

To reinvestigate the taxonomy of [Actinobacillus] muris, 474 strains, mainly from mice and rats, were characterized by phenotype and 130 strains selected for genotypic characterization by 16S rRNA and partial rpoB gene sequencing. The type strain was further investigated by whole-genome sequencing. Phylogenetic analysis of the DNA sequences showed one monophyletic group with intragroup similarities of 96.7 and 97.2 % for the 16S rRNA and rpoB genes, respectively. The highest 16S rRNA gene sequence similarity to a taxon with a validly published name outside the group was 95.9 %, to the type strain of [Pasteurella] pneumotropica. The closest related taxon based on rpoB sequence comparison was 'Haemophilus influenzae-murium', with 88.4 % similarity. A new genus and a new combination, Muribacter muris gen. nov., comb. nov., are proposed based on a distinct phylogenetic position based on 16S rRNA and rpoB gene sequence comparisons, with major divergence from the existing genera of the family Pasteurellaceae. The new genus has the characteristics of [A.] muris with the emendation that acid formation from ( - )-d-mannitol and hydrolysis of aesculin are variable, while the α-glucosidase test is positive. There is no requirement for exogenously supplied NAD (V factor) for the majority of strains investigated; however, one strain was found to require NAD. The major fatty acids of the type strain of Muribacter muris were C14 : 0, C14 : 0 3-OH/iso-C16 : 1 I, C16 : 1ω7c and C16 : 0, which is in line with most genera of the Pasteurellaceae. The type strain of Muribacter muris is CCUG 16938T ( = NCTC 12432T = ATCC 49577T).

BGL4 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

2011-12-06

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl4, and the corresponding BGL4 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL4, recombinant BGL4 proteins and methods for producing the same.

BGL4 .beta.-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel; Goedegebuur, Frits; Ward, Michael; Yao, Jian

2006-05-16

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl4, and the corresponding BGL4 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL4, recombinant BGL4 proteins and methods for producing the same.

BGL4 beta-glucosidase and nucleic acids encoding the same

DOEpatents

Dunn-Coleman, Nigel [Los Gatos, CA; Goedegebuur, Frits [Vlaardingen, NL; Ward, Michael [San Francisco, CA; Yao, Jian [Sunnyvale, CA

2008-01-22

The present invention provides a novel .beta.-glucosidase nucleic acid sequence, designated bgl4, and the corresponding BGL4 amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding BGL4, recombinant BGL4 proteins and methods for producing the same.

Draft Genome Sequence of Sphingobium sp. Strain HDIPO4, an Avid Degrader of Hexachlorocyclohexane

PubMed Central

Mukherjee, Udita; Kumar, Roshan; Mahato, Nitish Kumar; Khurana, J. P.

2013-01-01

Sphingobium sp. strain HDIPO4 was isolated from a hexachlorocyclohexane (HCH) dumpsite and degraded HCH isomers rapidly. The draft genome sequence of HDIPO4 (~4.7 Mbp) contains 143 contigs and 4,646 coding sequences with a G+C content of 65%. PMID:24051321

The Outer Planetary Mission Design Project

NASA Astrophysics Data System (ADS)

Benfield, Michael; Turner, M. W.

2010-10-01

With the recent focus from the planetary science community on the outer planets of the solar system, The University of Alabama in Huntsville Integrated Product Team program is embarking on a new challenge to develop an outer planetary mission for the academic year 2010-2011. Currently four bodies are of interest for this mission: Titan, Europa, Triton, and Enceledus, with one body being chosen by the instructors by the beginning of the fall semester. This project will use the 2010 Discovery Announcement of Opportunity as its Request for Proposal (RFP). All of the teams competing in this project will use the AO to respond with a proposal to the instructors for their proposed mission and spacecraft concept. The project employs the two-semester design sequence of the IPT program to provide a framework for the development of this mission. This sequence is divided into four phases. Phase 1 - Requirements Development - focuses on the development of both the scientific and engineering requirements of the mission. During this phase the teams work very closely with the PI organization, represented by the College of Charleston. Phase 2 - Team Formation and Architecture Development - concentrates on the assessment of the overall mission architecture from the launch vehicle to the ground operations of the proposed spacecraft. Phase 3 - System Definition - provides for spacecraft subsystem trade studies and further refinement of the specific spacecraft to meet the scientific requirements and objectives developed in Phase 1. Phase 4 - Design - is the phase where the engineers provide the spacecraft design that is required for the mission of interest. At the conclusion of Phases 2 and 4, an external review board evaluates the proposed designs and chooses one winner of the competition.

Substrate recognition by ribonucleoprotein ribonuclease MRP

PubMed Central

Esakova, Olga; Perederina, Anna; Quan, Chao; Berezin, Igor; Krasilnikov, Andrey S.

2011-01-01

The ribonucleoprotein complex ribonuclease (RNase) MRP is a site-specific endoribonuclease essential for the survival of the eukaryotic cell. RNase MRP closely resembles RNase P (a universal endoribonuclease responsible for the maturation of the 5′ ends of tRNA) but recognizes distinct substrates including pre-rRNA and mRNA. Here we report the results of an in vitro selection of Saccharomyces cerevisiae RNase MRP substrates starting from a pool of random sequences. The results indicate that RNase MRP cleaves single-stranded RNA and is sensitive to sequences in the immediate vicinity of the cleavage site requiring a cytosine at the position +4 relative to the cleavage site. Structural implications of the differences in substrate recognition by RNases P and MRP are discussed. PMID:21173200

Substrate recognition by ribonucleoprotein ribonuclease MRP.

PubMed

Esakova, Olga; Perederina, Anna; Quan, Chao; Berezin, Igor; Krasilnikov, Andrey S

2011-02-01

The ribonucleoprotein complex ribonuclease (RNase) MRP is a site-specific endoribonuclease essential for the survival of the eukaryotic cell. RNase MRP closely resembles RNase P (a universal endoribonuclease responsible for the maturation of the 5' ends of tRNA) but recognizes distinct substrates including pre-rRNA and mRNA. Here we report the results of an in vitro selection of Saccharomyces cerevisiae RNase MRP substrates starting from a pool of random sequences. The results indicate that RNase MRP cleaves single-stranded RNA and is sensitive to sequences in the immediate vicinity of the cleavage site requiring a cytosine at the position +4 relative to the cleavage site. Structural implications of the differences in substrate recognition by RNases P and MRP are discussed.

Transcriptome analysis of seed dormancy after rinsing and chilling in ornamental peaches (Prunus persica (L.) Batsch).

PubMed

Kanjana, Worarad; Suzuki, Tomohiro; Ishii, Kazuo; Kozaki, Toshinori; Iigo, Masayuki; Yamane, Kenji

2016-08-08

Ornamental peaches cv. 'Yaguchi' (Prunus persica (L.) Batsch) can be propagated via seeds. The establishment of efficient seed treatments for early germination and seedling growth is required to shorten nursery and breeding periods. It is important, therefore, to identify potential candidate genes responsible for the effects of rinsing and chilling on seed germination. We hypothesized that longer rinsing combined with chilling of seeds can alter the genes expression in related to dormancy and then raise the germination rate in the peach. To date, most molecular studies in peaches have involved structural genomics, and few transcriptome studies of seed germination have been conducted. In this study, we investigated the function of key seed dormancy-related genes using next-generation sequencing to profile the transcriptomes involved in seed dormancy in peaches. De novo assembly and analysis of the transcriptome identified differentially expressed and unique genes present in this fruit. De novo RNA-sequencing of peach was performed using the Illumina Miseq 2000 system. Paired-end sequence from mRNAs generated high quality sequence reads (9,049,964, 10,026,362 and 10,101,918 reads) from 'Yaguchi' peach seeds before rinsed (BR) and after rinsed for 2 or 7 days with a chilling period of 4 weeks (termed 2D4W and 7D4W), respectively. The germination rate of 7D4W was significantly higher than that of 2D4W. In total, we obtained 51,366 unique sequences. Differential expression analysis identified 7752, 8469 and 506 differentially expressed genes from BR vs 2D4W, BR vs 7D4W and 2D4W vs 7D4W libraries respectively, filtered based on p-value and an adjusted false discovery rate of less than 0.05. This study identified genes associated with the rinsing and chilling process that included those associated with phytohormones, the stress response and transcription factors. 7D4W treatment downregulated genes involved in ABA synthesis, catabolism and signaling pathways, which eventually suppressed abscisic acid activity and consequently promoted germination and seedling growth. Stress response genes were also downregulated by the 7D4W treatment, suggesting that this treatment released seeds from endodormancy. Transcription factors were upregulated by the BR and 2D4W treatment, suggesting that they play important roles in maintaining seed dormancy. This work indicated that longer rinsing combined with chilling affects gene expression and germination rate, and identified potential candidate genes responsible for dormancy progression in seeds of 'Yaguchi' peach. The results could be used to develop breeding programs and will aid future functional genomic research in peaches and other fruit trees.

Cag3 Is a Novel Essential Component of the Helicobacter pylori Cag Type IV Secretion System Outer Membrane Subcomplex ▿ †

PubMed Central

Pinto-Santini, Delia M.; Salama, Nina R.

2009-01-01

Helicobacter pylori strains harboring the cag pathogenicity island (PAI) have been associated with more severe gastric disease in infected humans. The cag PAI encodes a type IV secretion (T4S) system required for CagA translocation into host cells as well as induction of proinflammatory cytokines, such as interleukin-8 (IL-8). cag PAI genes sharing sequence similarity with T4S components from other bacteria are essential for Cag T4S function. Other cag PAI-encoded genes are also essential for Cag T4S, but lack of sequence-based or structural similarity with genes in existing databases has precluded a functional assignment for the encoded proteins. We have studied the role of one such protein, Cag3 (HP0522), in Cag T4S and determined Cag3 subcellular localization and protein interactions. Cag3 is membrane associated and copurifies with predicted inner and outer membrane Cag T4S components that are essential for Cag T4S as well as putative accessory factors. Coimmunoprecipitation and cross-linking experiments revealed specific interactions with HpVirB7 and CagM, suggesting Cag3 is a new component of the Cag T4S outer membrane subcomplex. Finally, lack of Cag3 lowers HpVirB7 steady-state levels, further indicating Cag3 makes a subcomplex with this protein. PMID:19801411

Automatic Debugging Support for UML Designs

NASA Technical Reports Server (NTRS)

Schumann, Johann; Swanson, Keith (Technical Monitor)

2001-01-01

Design of large software systems requires rigorous application of software engineering methods covering all phases of the software process. Debugging during the early design phases is extremely important, because late bug-fixes are expensive. In this paper, we describe an approach which facilitates debugging of UML requirements and designs. The Unified Modeling Language (UML) is a set of notations for object-orient design of a software system. We have developed an algorithm which translates requirement specifications in the form of annotated sequence diagrams into structured statecharts. This algorithm detects conflicts between sequence diagrams and inconsistencies in the domain knowledge. After synthesizing statecharts from sequence diagrams, these statecharts usually are subject to manual modification and refinement. By using the "backward" direction of our synthesis algorithm. we are able to map modifications made to the statechart back into the requirements (sequence diagrams) and check for conflicts there. Fed back to the user conflicts detected by our algorithm are the basis for deductive-based debugging of requirements and domain theory in very early development stages. Our approach allows to generate explanations oil why there is a conflict and which parts of the specifications are affected.

Pneumocandin biosynthesis: involvement of a trans-selective proline hydroxylase.

PubMed

Houwaart, Stefanie; Youssar, Loubna; Hüttel, Wolfgang

2014-11-03

Echinocandins are cyclic nonribosomal hexapeptides based mostly on nonproteinogenic amino acids and displaying strong antifungal activity. Despite previous studies on their biosynthesis by fungi, the origin of three amino acids, trans-4- and trans-3-hydroxyproline, as well as trans-3-hydroxy-4-methylproline, is still unknown. Here we describe the identification, overexpression, and characterization of GloF, the first eukaryotic α-ketoglutarate/Fe(II) -dependent proline hydroxylase from the pneumocandin biosynthesis cluster of the fungus Glarea lozoyensis ATCC 74030. In in vitro transformations with L-proline, GloF generates trans-4- and trans-3-hydroxyproline simultaneously in a ratio of 8:1; the latter reaction was previously unknown for proline hydroxylase catalysis. trans-4-Methyl-L-proline is converted into the corresponding trans-3-hydroxyproline. All three hydroxyprolines required for the biosynthesis of the echinocandins pneumocandins A0 and B0 in G. lozoyensis are thus provided by GloF. Sequence analyses revealed that GloF is not related to bacterial proline hydroxylases, and none of the putative proteins with high sequence similarity in the databases has been characterized so far. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Fast single-pass alignment and variant calling using sequencing data

USDA-ARS?s Scientific Manuscript database

Sequencing research requires efficient computation. Few programs use already known information about DNA variants when aligning sequence data to the reference map. New program findmap.f90 reads the previous variant list before aligning sequence, calling variant alleles, and summing the allele counts...

Lactobacillus heilongjiangensis sp. nov., isolated from Chinese pickle.

PubMed

Gu, Chun Tao; Li, Chun Yan; Yang, Li Jie; Huo, Gui Cheng

2013-11-01

A Gram-stain-positive bacterial strain, S4-3(T), was isolated from traditional pickle in Heilongjiang Province, China. The bacterium was characterized by a polyphasic approach, including 16S rRNA gene sequence analysis, pheS gene sequence analysis, rpoA gene sequence analysis, dnaK gene sequence analysis, fatty acid methyl ester (FAME) analysis, determination of DNA G+C content, DNA-DNA hybridization and an analysis of phenotypic features. Strain S4-3(T) showed 97.9-98.7 % 16S rRNA gene sequence similarities, 84.4-94.1 % pheS gene sequence similarities and 94.4-96.9 % rpoA gene sequence similarities to the type strains of Lactobacillus nantensis, Lactobacillus mindensis, Lactobacillus crustorum, Lactobacillus futsaii, Lactobacillus farciminis and Lactobacillus kimchiensis. dnaK gene sequence similarities between S4-3(T) and Lactobacillus nantensis LMG 23510(T), Lactobacillus mindensis LMG 21932(T), Lactobacillus crustorum LMG 23699(T), Lactobacillus futsaii JCM 17355(T) and Lactobacillus farciminis LMG 9200(T) were 95.4, 91.5, 90.4, 91.7 and 93.1 %, respectively. Based upon the data obtained in the present study, a novel species, Lactobacillus heilongjiangensis sp. nov., is proposed and the type strain is S4-3(T) ( = LMG 26166(T) = NCIMB 14701(T)).

The Nematode Eukaryotic Translation Initiation Factor 4E/G Complex Works with a trans-Spliced Leader Stem-Loop To Enable Efficient Translation of Trimethylguanosine-Capped RNAs ▿ †

PubMed Central

Wallace, Adam; Filbin, Megan E.; Veo, Bethany; McFarland, Craig; Stepinski, Janusz; Jankowska-Anyszka, Marzena; Darzynkiewicz, Edward; Davis, Richard E.

2010-01-01

Eukaryotic mRNA translation begins with recruitment of the 40S ribosome complex to the mRNA 5′ end through the eIF4F initiation complex binding to the 5′ m7G-mRNA cap. Spliced leader (SL) RNA trans splicing adds a trimethylguanosine (TMG) cap and a sequence, the SL, to the 5′ end of mRNAs. Efficient translation of TMG-capped mRNAs in nematodes requires the SL sequence. Here we define a core set of nucleotides and a stem-loop within the 22-nucleotide nematode SL that stimulate translation of mRNAs with a TMG cap. The structure and core nucleotides are conserved in other nematode SLs and correspond to regions of SL1 required for early Caenorhabditis elegans development. These SL elements do not facilitate translation of m7G-capped RNAs in nematodes or TMG-capped mRNAs in mammalian or plant translation systems. Similar stem-loop structures in phylogenetically diverse SLs are predicted. We show that the nematode eukaryotic translation initiation factor 4E/G (eIF4E/G) complex enables efficient translation of the TMG-SL RNAs in diverse in vitro translation systems. TMG-capped mRNA translation is determined by eIF4E/G interaction with the cap and the SL RNA, although the SL does not increase the affinity of eIF4E/G for capped RNA. These results suggest that the mRNA 5′ untranslated region (UTR) can play a positive and novel role in translation initiation through interaction with the eIF4E/G complex in nematodes and raise the issue of whether eIF4E/G-RNA interactions play a role in the translation of other eukaryotic mRNAs. PMID:20154140

The nematode eukaryotic translation initiation factor 4E/G complex works with a trans-spliced leader stem-loop to enable efficient translation of trimethylguanosine-capped RNAs.

PubMed

Wallace, Adam; Filbin, Megan E; Veo, Bethany; McFarland, Craig; Stepinski, Janusz; Jankowska-Anyszka, Marzena; Darzynkiewicz, Edward; Davis, Richard E

2010-04-01

Eukaryotic mRNA translation begins with recruitment of the 40S ribosome complex to the mRNA 5' end through the eIF4F initiation complex binding to the 5' m(7)G-mRNA cap. Spliced leader (SL) RNA trans splicing adds a trimethylguanosine (TMG) cap and a sequence, the SL, to the 5' end of mRNAs. Efficient translation of TMG-capped mRNAs in nematodes requires the SL sequence. Here we define a core set of nucleotides and a stem-loop within the 22-nucleotide nematode SL that stimulate translation of mRNAs with a TMG cap. The structure and core nucleotides are conserved in other nematode SLs and correspond to regions of SL1 required for early Caenorhabditis elegans development. These SL elements do not facilitate translation of m(7)G-capped RNAs in nematodes or TMG-capped mRNAs in mammalian or plant translation systems. Similar stem-loop structures in phylogenetically diverse SLs are predicted. We show that the nematode eukaryotic translation initiation factor 4E/G (eIF4E/G) complex enables efficient translation of the TMG-SL RNAs in diverse in vitro translation systems. TMG-capped mRNA translation is determined by eIF4E/G interaction with the cap and the SL RNA, although the SL does not increase the affinity of eIF4E/G for capped RNA. These results suggest that the mRNA 5' untranslated region (UTR) can play a positive and novel role in translation initiation through interaction with the eIF4E/G complex in nematodes and raise the issue of whether eIF4E/G-RNA interactions play a role in the translation of other eukaryotic mRNAs.

Fundamental Bounds for Sequence Reconstruction from Nanopore Sequencers.

PubMed

Magner, Abram; Duda, Jarosław; Szpankowski, Wojciech; Grama, Ananth

2016-06-01

Nanopore sequencers are emerging as promising new platforms for high-throughput sequencing. As with other technologies, sequencer errors pose a major challenge for their effective use. In this paper, we present a novel information theoretic analysis of the impact of insertion-deletion (indel) errors in nanopore sequencers. In particular, we consider the following problems: (i) for given indel error characteristics and rate, what is the probability of accurate reconstruction as a function of sequence length; (ii) using replicated extrusion (the process of passing a DNA strand through the nanopore), what is the number of replicas needed to accurately reconstruct the true sequence with high probability? Our results provide a number of important insights: (i) the probability of accurate reconstruction of a sequence from a single sample in the presence of indel errors tends quickly (i.e., exponentially) to zero as the length of the sequence increases; and (ii) replicated extrusion is an effective technique for accurate reconstruction. We show that for typical distributions of indel errors, the required number of replicas is a slow function (polylogarithmic) of sequence length - implying that through replicated extrusion, we can sequence large reads using nanopore sequencers. Moreover, we show that in certain cases, the required number of replicas can be related to information-theoretic parameters of the indel error distributions.

YAMAT-seq: an efficient method for high-throughput sequencing of mature transfer RNAs.

PubMed

Shigematsu, Megumi; Honda, Shozo; Loher, Phillipe; Telonis, Aristeidis G; Rigoutsos, Isidore; Kirino, Yohei

2017-05-19

Besides translation, transfer RNAs (tRNAs) play many non-canonical roles in various biological pathways and exhibit highly variable expression profiles. To unravel the emerging complexities of tRNA biology and molecular mechanisms underlying them, an efficient tRNA sequencing method is required. However, the rigid structure of tRNA has been presenting a challenge to the development of such methods. We report the development of Y-shaped Adapter-ligated MAture TRNA sequencing (YAMAT-seq), an efficient and convenient method for high-throughput sequencing of mature tRNAs. YAMAT-seq circumvents the issue of inefficient adapter ligation, a characteristic of conventional RNA sequencing methods for mature tRNAs, by employing the efficient and specific ligation of Y-shaped adapter to mature tRNAs using T4 RNA Ligase 2. Subsequent cDNA amplification and next-generation sequencing successfully yield numerous mature tRNA sequences. YAMAT-seq has high specificity for mature tRNAs and high sensitivity to detect most isoacceptors from minute amount of total RNA. Moreover, YAMAT-seq shows quantitative capability to estimate expression levels of mature tRNAs, and has high reproducibility and broad applicability for various cell lines. YAMAT-seq thus provides high-throughput technique for identifying tRNA profiles and their regulations in various transcriptomes, which could play important regulatory roles in translation and other biological processes. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Reality of Single Circulating Tumor Cell Sequencing for Molecular Diagnostics in Pancreatic Cancer.

PubMed

Court, Colin M; Ankeny, Jacob S; Sho, Shonan; Hou, Shuang; Li, Qingyu; Hsieh, Carolyn; Song, Min; Liao, Xinfang; Rochefort, Matthew M; Wainberg, Zev A; Graeber, Thomas G; Tseng, Hsian-Rong; Tomlinson, James S

2016-09-01

To understand the potential and limitations of circulating tumor cell (CTC) sequencing for molecular diagnostics, we investigated the feasibility of identifying the ubiquitous KRAS mutation in single CTCs from pancreatic cancer (PC) patients. We used the NanoVelcro/laser capture microdissection CTC platform, combined with whole genome amplification and KRAS Sanger sequencing. We assessed both KRAS codon-12 coverage and the degree that allele dropout during whole genome amplification affected the detection of KRAS mutations from single CTCs. We isolated 385 single cells, 163 from PC cell lines and 222 from the blood of 12 PC patients, and obtained KRAS sequence coverage in 218 of 385 single cells (56.6%). For PC cell lines with known KRAS mutations, single mutations were detected in 67% of homozygous cells but only 37.4% of heterozygous single cells, demonstrating that both coverage and allele dropout are important causes of mutation detection failure from single cells. We could detect KRAS mutations in CTCs from 11 of 12 patients (92%) and 33 of 119 single CTCs sequenced, resulting in a KRAS mutation detection rate of 27.7%. Importantly, KRAS mutations were never found in the 103 white blood cells sequenced. Sequencing of groups of cells containing between 1 and 100 cells determined that at least 10 CTCs are likely required to reliably assess KRAS mutation status from CTCs. Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

Apollo Mission Techniques Lunar Orbit Activities - Part 1a

NASA Technical Reports Server (NTRS)

Interbartolo, Michael A.

2009-01-01

This slide presentation reviews the planned sequence of events and the rationale for all lunar missions, and the flight experiences and lessons learned for the lunar orbit activities from a trajectory perspective. Shown are trajectories which include the moon's position at the various stages in the complete trip from launch, to the return and reentry. Included in the presentation are objectives and the sequence of events,for the Apollo 8, and Apollo 10. This is followed by a discussion of Apollo 11, including: the primary mission objective, the sequence of events, and the flight experience. The next mission discussed was Apollo 12. It reviews the objectives, the ground tracking, procedure changes, and the sequence of events. The aborted Apollo 13 mission is reviewed, including the objectives, and the sequence of events. Brief summaries of the flight experiences for Apollo 14-16 are reviewed. The flight sequence of events of Apollo 17 are discussed. In summary each mission consistently performing precision landings required that Apollo lunar orbit activities devote considerable attention to: (1) Improving fidelity of lunar gravity models, (2) Maximizing availability of ground tracking, (3) Minimizing perturbations on the trajectory, (4) Maximizing LM propellant reserves for hover time. Also the use of radial separation maneuvers (1) allows passive re-rendezvous after each rev, but ... (2) sensitive to small dispersions in initial sep direction

Evolution of Sphingomonad Gene Clusters Related to Pesticide Catabolism Revealed by Genome Sequence and Mobilomics of Sphingobium herbicidovorans MH

PubMed Central

Nielsen, Tue Kjærgaard; Rasmussen, Morten; Demanèche, Sandrine; Cecillon, Sébastien; Vogel, Timothy M.

2017-01-01

Abstract Bacterial degraders of chlorophenoxy herbicides have been isolated from various ecosystems, including pristine environments. Among these degraders, the sphingomonads constitute a prominent group that displays versatile xenobiotic-degradation capabilities. Four separate sequencing strategies were required to provide the complete sequence of the complex and plastic genome of the canonical chlorophenoxy herbicide-degrading Sphingobium herbicidovorans MH. The genome has an intricate organization of the chlorophenoxy-herbicide catabolic genes sdpA, rdpA, and cadABCD that encode the (R)- and (S)-enantiomer-specific 2,4-dichlorophenoxypropionate dioxygenases and four subunits of a Rieske non-heme iron oxygenase involved in 2-methyl-chlorophenoxyacetic acid degradation, respectively. Several major genomic rearrangements are proposed to help understand the evolution and mobility of these important genes and their genetic context. Single-strain mobilomic sequence analysis uncovered plasmids and insertion sequence-associated circular intermediates in this environmentally important bacterium and enabled the description of evolutionary models for pesticide degradation in strain MH and related organisms. The mobilome presented a complex mosaic of mobile genetic elements including four plasmids and several circular intermediate DNA molecules of insertion-sequence elements and transposons that are central to the evolution of xenobiotics degradation. Furthermore, two individual chromosomally integrated prophages were shown to excise and form free circular DNA molecules. This approach holds great potential for improving the understanding of genome plasticity, evolution, and microbial ecology. PMID:28961970

XLID-causing mutations and associated genes challenged in light of data from large-scale human exome sequencing.

PubMed

Piton, Amélie; Redin, Claire; Mandel, Jean-Louis

2013-08-08

Because of the unbalanced sex ratio (1.3-1.4 to 1) observed in intellectual disability (ID) and the identification of large ID-affected families showing X-linked segregation, much attention has been focused on the genetics of X-linked ID (XLID). Mutations causing monogenic XLID have now been reported in over 100 genes, most of which are commonly included in XLID diagnostic gene panels. Nonetheless, the boundary between true mutations and rare non-disease-causing variants often remains elusive. The sequencing of a large number of control X chromosomes, required for avoiding false-positive results, was not systematically possible in the past. Such information is now available thanks to large-scale sequencing projects such as the National Heart, Lung, and Blood (NHLBI) Exome Sequencing Project, which provides variation information on 10,563 X chromosomes from the general population. We used this NHLBI cohort to systematically reassess the implication of 106 genes proposed to be involved in monogenic forms of XLID. We particularly question the implication in XLID of ten of them (AGTR2, MAGT1, ZNF674, SRPX2, ATP6AP2, ARHGEF6, NXF5, ZCCHC12, ZNF41, and ZNF81), in which truncating variants or previously published mutations are observed at a relatively high frequency within this cohort. We also highlight 15 other genes (CCDC22, CLIC2, CNKSR2, FRMPD4, HCFC1, IGBP1, KIAA2022, KLF8, MAOA, NAA10, NLGN3, RPL10, SHROOM4, ZDHHC15, and ZNF261) for which replication studies are warranted. We propose that similar reassessment of reported mutations (and genes) with the use of data from large-scale human exome sequencing would be relevant for a wide range of other genetic diseases. Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

A Poisson hierarchical modelling approach to detecting copy number variation in sequence coverage data

PubMed Central

2013-01-01

Background The advent of next generation sequencing technology has accelerated efforts to map and catalogue copy number variation (CNV) in genomes of important micro-organisms for public health. A typical analysis of the sequence data involves mapping reads onto a reference genome, calculating the respective coverage, and detecting regions with too-low or too-high coverage (deletions and amplifications, respectively). Current CNV detection methods rely on statistical assumptions (e.g., a Poisson model) that may not hold in general, or require fine-tuning the underlying algorithms to detect known hits. We propose a new CNV detection methodology based on two Poisson hierarchical models, the Poisson-Gamma and Poisson-Lognormal, with the advantage of being sufficiently flexible to describe different data patterns, whilst robust against deviations from the often assumed Poisson model. Results Using sequence coverage data of 7 Plasmodium falciparum malaria genomes (3D7 reference strain, HB3, DD2, 7G8, GB4, OX005, and OX006), we showed that empirical coverage distributions are intrinsically asymmetric and overdispersed in relation to the Poisson model. We also demonstrated a low baseline false positive rate for the proposed methodology using 3D7 resequencing data and simulation. When applied to the non-reference isolate data, our approach detected known CNV hits, including an amplification of the PfMDR1 locus in DD2 and a large deletion in the CLAG3.2 gene in GB4, and putative novel CNV regions. When compared to the recently available FREEC and cn.MOPS approaches, our findings were more concordant with putative hits from the highest quality array data for the 7G8 and GB4 isolates. Conclusions In summary, the proposed methodology brings an increase in flexibility, robustness, accuracy and statistical rigour to CNV detection using sequence coverage data. PMID:23442253

Selected exonic sequencing of the AGXT gene provides a genetic diagnosis in 50% of patients with primary hyperoxaluria type 1.

PubMed

Williams, Emma; Rumsby, Gill

2007-07-01

Definitive diagnosis of primary hyperoxaluria type 1 (PH1) requires analysis of alanine:glyoxylate aminotransferase (AGT) activity in the liver. We have previously shown that targeted screening for the 3 most common mutations in the AGXT gene (c.33_34insC, c.508G>A, and c.731T>C) can provide a molecular diagnosis in 34.5% of PH1 patients, eliminating the need for a liver biopsy. Having reviewed the distribution of all AGXT mutations, we have evaluated a diagnostic strategy that uses selected exon sequencing for the molecular diagnosis of PH1. We sequenced exons 1, 4, and 7 for 300 biopsy-confirmed PH1 patients and expressed the identified missense mutations in vitro. Our identification of at least 1 mutation in 224 patients (75%) and 2 mutations in 149 patients increased the diagnostic sensitivity to 50%. We detected 29 kinds of sequence changes, 15 of which were novel. Four of these mutations were in exon 1 (c.2_3delinsAT, c.30_32delCC, c.122G>A, c.126delG), 7 were in exon 4 (c.447_454delGCTGCTGT, c.449T>C, c.473C>T, c.481G>A, c.481G>T, c.497T>C, c.424-2A>G), and 4 were in exon 7 (c.725insT, c.737G>A, c.757T>C, c.776 + 1G>A). The missense changes were associated with severely decreased AGT catalytic activity and negative immunoreactivity when expressed in vitro. Missense mutation c.26C>A, previously described as a pathological mutation, had activity similar to that of the wild-type enzyme. Selective exon sequencing can allow a definitive diagnosis in 50% of PH1 patients. The test offers a rapid turnaround time (15 days) with minimal risk to the patient. Demonstration of the expression of missense changes is essential to demonstrate pathogenicity.

From sequencer to supercomputer: an automatic pipeline for managing and processing next generation sequencing data.

PubMed

Camerlengo, Terry; Ozer, Hatice Gulcin; Onti-Srinivasan, Raghuram; Yan, Pearlly; Huang, Tim; Parvin, Jeffrey; Huang, Kun

2012-01-01

Next Generation Sequencing is highly resource intensive. NGS Tasks related to data processing, management and analysis require high-end computing servers or even clusters. Additionally, processing NGS experiments requires suitable storage space and significant manual interaction. At The Ohio State University's Biomedical Informatics Shared Resource, we designed and implemented a scalable architecture to address the challenges associated with the resource intensive nature of NGS secondary analysis built around Illumina Genome Analyzer II sequencers and Illumina's Gerald data processing pipeline. The software infrastructure includes a distributed computing platform consisting of a LIMS called QUEST (http://bisr.osumc.edu), an Automation Server, a computer cluster for processing NGS pipelines, and a network attached storage device expandable up to 40TB. The system has been architected to scale to multiple sequencers without requiring additional computing or labor resources. This platform provides demonstrates how to manage and automate NGS experiments in an institutional or core facility setting.

Integration of Temporal and Ordinal Information During Serial Interception Sequence Learning

PubMed Central

Gobel, Eric W.; Sanchez, Daniel J.; Reber, Paul J.

2011-01-01

The expression of expert motor skills typically involves learning to perform a precisely timed sequence of movements (e.g., language production, music performance, athletic skills). Research examining incidental sequence learning has previously relied on a perceptually-cued task that gives participants exposure to repeating motor sequences but does not require timing of responses for accuracy. Using a novel perceptual-motor sequence learning task, learning a precisely timed cued sequence of motor actions is shown to occur without explicit instruction. Participants learned a repeating sequence through practice and showed sequence-specific knowledge via a performance decrement when switched to an unfamiliar sequence. In a second experiment, the integration of representation of action order and timing sequence knowledge was examined. When either action order or timing sequence information was selectively disrupted, performance was reduced to levels similar to completely novel sequences. Unlike prior sequence-learning research that has found timing information to be secondary to learning action sequences, when the task demands require accurate action and timing information, an integrated representation of these types of information is acquired. These results provide the first evidence for incidental learning of fully integrated action and timing sequence information in the absence of an independent representation of action order, and suggest that this integrative mechanism may play a material role in the acquisition of complex motor skills. PMID:21417511

Stresses in Implant-Supported Fixed Complete Dentures with Different Screw-Tightening Sequences and Torque Application Modes.

PubMed

Barcellos, Leonardo H; Palmeiro, Marina Lobato; Naconecy, Marcos M; Geremia, Tomás; Cervieri, André; Shinkai, Rosemary S

2018-05-17

To compare the effects of different screw-tightening sequences and torque applications on stresses in implant-supported fixed complete dentures supported by five abutments. Strain gauges fixed to the abutments were used to test the sequences 2-4-3-1-5; 1-2-3-4-5; 3-2-4-1-5; and 2-5-4-1-3 with direct 10-Ncm torque or progressive torque (5 + 10 Ncm). Data were analyzed using analysis of variance and standardized effect size. No effects of tightening sequence or torque application were found except for the sequence 3-2-4-1-5 and some small to moderate effect sizes. Screw-tightening sequences and torque application modes have only a marginal effect on residual stresses.

Improved detection of CXCR4-using HIV by V3 genotyping: application of population-based and "deep" sequencing to plasma RNA and proviral DNA.

PubMed

Swenson, Luke C; Moores, Andrew; Low, Andrew J; Thielen, Alexander; Dong, Winnie; Woods, Conan; Jensen, Mark A; Wynhoven, Brian; Chan, Dennison; Glascock, Christopher; Harrigan, P Richard

2010-08-01

Tropism testing should rule out CXCR4-using HIV before treatment with CCR5 antagonists. Currently, the recombinant phenotypic Trofile assay (Monogram) is most widely utilized; however, genotypic tests may represent alternative methods. Independent triplicate amplifications of the HIV gp120 V3 region were made from either plasma HIV RNA or proviral DNA. These underwent standard, population-based sequencing with an ABI3730 (RNA n = 63; DNA n = 40), or "deep" sequencing with a Roche/454 Genome Sequencer-FLX (RNA n = 12; DNA n = 12). Position-specific scoring matrices (PSSMX4/R5) (-6.96 cutoff) and geno2pheno[coreceptor] (5% false-positive rate) inferred tropism from V3 sequence. These methods were then independently validated with a separate, blinded dataset (n = 278) of screening samples from the maraviroc MOTIVATE trials. Standard sequencing of HIV RNA with PSSM yielded 69% sensitivity and 91% specificity, relative to Trofile. The validation dataset gave 75% sensitivity and 83% specificity. Proviral DNA plus PSSM gave 77% sensitivity and 71% specificity. "Deep" sequencing of HIV RNA detected >2% inferred-CXCR4-using virus in 8/8 samples called non-R5 by Trofile, and <2% in 4/4 samples called R5. Triplicate analyses of V3 standard sequence data detect greater proportions of CXCR4-using samples than previously achieved. Sequencing proviral DNA and "deep" V3 sequencing may also be useful tools for assessing tropism.

SW#db: GPU-Accelerated Exact Sequence Similarity Database Search.

PubMed

Korpar, Matija; Šošić, Martin; Blažeka, Dino; Šikić, Mile

2015-01-01

In recent years we have witnessed a growth in sequencing yield, the number of samples sequenced, and as a result-the growth of publicly maintained sequence databases. The increase of data present all around has put high requirements on protein similarity search algorithms with two ever-opposite goals: how to keep the running times acceptable while maintaining a high-enough level of sensitivity. The most time consuming step of similarity search are the local alignments between query and database sequences. This step is usually performed using exact local alignment algorithms such as Smith-Waterman. Due to its quadratic time complexity, alignments of a query to the whole database are usually too slow. Therefore, the majority of the protein similarity search methods prior to doing the exact local alignment apply heuristics to reduce the number of possible candidate sequences in the database. However, there is still a need for the alignment of a query sequence to a reduced database. In this paper we present the SW#db tool and a library for fast exact similarity search. Although its running times, as a standalone tool, are comparable to the running times of BLAST, it is primarily intended to be used for exact local alignment phase in which the database of sequences has already been reduced. It uses both GPU and CPU parallelization and was 4-5 times faster than SSEARCH, 6-25 times faster than CUDASW++ and more than 20 times faster than SSW at the time of writing, using multiple queries on Swiss-prot and Uniref90 databases.

Identifying and reducing error in cluster-expansion approximations of protein energies.

PubMed

Hahn, Seungsoo; Ashenberg, Orr; Grigoryan, Gevorg; Keating, Amy E

2010-12-01

Protein design involves searching a vast space for sequences that are compatible with a defined structure. This can pose significant computational challenges. Cluster expansion is a technique that can accelerate the evaluation of protein energies by generating a simple functional relationship between sequence and energy. The method consists of several steps. First, for a given protein structure, a training set of sequences with known energies is generated. Next, this training set is used to expand energy as a function of clusters consisting of single residues, residue pairs, and higher order terms, if required. The accuracy of the sequence-based expansion is monitored and improved using cross-validation testing and iterative inclusion of additional clusters. As a trade-off for evaluation speed, the cluster-expansion approximation causes prediction errors, which can be reduced by including more training sequences, including higher order terms in the expansion, and/or reducing the sequence space described by the cluster expansion. This article analyzes the sources of error and introduces a method whereby accuracy can be improved by judiciously reducing the described sequence space. The method is applied to describe the sequence-stability relationship for several protein structures: coiled-coil dimers and trimers, a PDZ domain, and T4 lysozyme as examples with computationally derived energies, and SH3 domains in amphiphysin-1 and endophilin-1 as examples where the expanded pseudo-energies are obtained from experiments. Our open-source software package Cluster Expansion Version 1.0 allows users to expand their own energy function of interest and thereby apply cluster expansion to custom problems in protein design. © 2010 Wiley Periodicals, Inc.

Bacterial community comparisons by taxonomy-supervised analysis independent of sequence alignment and clustering

PubMed Central

Sul, Woo Jun; Cole, James R.; Jesus, Ederson da C.; Wang, Qiong; Farris, Ryan J.; Fish, Jordan A.; Tiedje, James M.

2011-01-01

High-throughput sequencing of 16S rRNA genes has increased our understanding of microbial community structure, but now even higher-throughput methods to the Illumina scale allow the creation of much larger datasets with more samples and orders-of-magnitude more sequences that swamp current analytic methods. We developed a method capable of handling these larger datasets on the basis of assignment of sequences into an existing taxonomy using a supervised learning approach (taxonomy-supervised analysis). We compared this method with a commonly used clustering approach based on sequence similarity (taxonomy-unsupervised analysis). We sampled 211 different bacterial communities from various habitats and obtained ∼1.3 million 16S rRNA sequences spanning the V4 hypervariable region by pyrosequencing. Both methodologies gave similar ecological conclusions in that β-diversity measures calculated by using these two types of matrices were significantly correlated to each other, as were the ordination configurations and hierarchical clustering dendrograms. In addition, our taxonomy-supervised analyses were also highly correlated with phylogenetic methods, such as UniFrac. The taxonomy-supervised analysis has the advantages that it is not limited by the exhaustive computation required for the alignment and clustering necessary for the taxonomy-unsupervised analysis, is more tolerant of sequencing errors, and allows comparisons when sequences are from different regions of the 16S rRNA gene. With the tremendous expansion in 16S rRNA data acquisition underway, the taxonomy-supervised approach offers the potential to provide more rapid and extensive community comparisons across habitats and samples. PMID:21873204

Shewanella gelidii sp. nov., isolated from the red algae Gelidium amansii, and emended description of Shewanella waksmanii.

PubMed

Wang, Yan; Chen, Hongli; Liu, Zhenhua; Ming, Hong; Zhou, Chenyan; Zhu, Xinshu; Zhang, Peng; Jing, Changqin; Feng, Huigen

2016-08-01

A novel Gram-stain-negative, straight or slightly curved rod-shaped, non-spore-forming, facultatively anaerobic bacterium with a single polar flagellum, designated RZB5-4T, was isolated from a sample of the red algae Gelidium amansii collected from the coastal region of Rizhao, PR China (119.625° E 35.517° N). The organism grew optimally between 24 and 28 °C, at pH 7.0 and in the presence of 2-3 % (w/v) NaCl. The strain required seawater or artificial seawater for growth, and NaCl alone did not support growth. Strain RZB5-4T contained C16 : 1ω7c and/or C16 : 1ω6c, C16 : 0 and iso-C15 : 0 as the dominant fatty acids. The respiratory quinones detected in strain RZB5-4T were ubiquinone 7, ubiquinone 8, menaquinone 7 and methylmenaquinone 7. The polar lipids of strain RZB5-4T comprised phosphatidylethanolamine, phosphatidylglycerol, phosphatidylmonomethylethanolamine, one unidentified glycolipid, one unidentified phospholipid and one unknown lipid. The DNA G+C content of strain RZB5-4T was 47 mol %. Phylogenetic analysis based on 16S rRNA and gyrase B (gyrB) gene sequences showed that strain RZB5-4T belonged to the genus Shewanella, clustering with Shewanella waksmanii ATCC BAA-643T. Strain RZB5-4T exhibited the highest 16S rRNA gene sequence similarity value (96.6 %) and the highest gyrB gene sequence similarity value (80.7 %), respectively, to S. waksmanii ATCC BAA-643T. On the basis of polyphasic analyses, strain RZB5-4T represents a novel species of the genus Shewanella, for which the name Shewanella gelidii sp. nov. is proposed. The type strain is RZB5-4T (=JCM 30804T=KCTC 42663T=MCCC 1K00697T).

Microbial characterization of nitrification in a shallow, nitrogen-contaminated aquifer, Cape Cod, Massachusetts and detection of a novel cluster associated with nitrifying Betaproteobacteria

USGS Publications Warehouse

Miller, D.N.; Smith, R.L.

2009-01-01

Groundwater nitrification is a poorly characterized process affecting the speciation and transport of nitrogen. Cores from two sites in a plume of contamination were examined using culture-based and molecular techniques targeting nitrification processes. The first site, located beneath a sewage effluent infiltration bed, received treated effluent containing O2 (> 300????M) and NH4+ (51-800????M). The second site was 2.5??km down-gradient near the leading edge of the ammonium zone within the contaminant plume and featured vertical gradients of O2, NH4+, and NO3- (0-300, 0-500, and 100-200????M with depth, respectively). Ammonia- and nitrite-oxidizers enumerated by the culture-based MPN method were low in abundance at both sites (1.8 to 350??g- 1 and 33 to 35,000??g- 1, respectively). Potential nitrifying activity measured in core material in the laboratory was also very low, requiring several weeks for products to accumulate. Molecular analysis of aquifer DNA (nested PCR followed by cloning and 16S rDNA sequencing) detected primarily sequences associated with the Nitrosospira genus throughout the cores at the down-gradient site and a smaller proportion from the Nitrosomonas genus in the deeper anoxic, NH4+ zone at the down-gradient site. Only a single Nitrosospira sequence was detected beneath the infiltration bed. Furthermore, the majority of Nitrosospira-associated sequences represent an unrecognized cluster. We conclude that an uncharacterized group associated with Nitrosospira dominate at the geochemically stable, down-gradient site, but found little evidence for Betaproteobacteria nitrifiers beneath the infiltration beds where geochemical conditions were more variable.

Microbial characterization of nitrification in a shallow, nitrogen-contaminated aquifer, Cape Cod, Massachusetts and detection of a novel cluster associated with nitrifying Betaproteobacteria

NASA Astrophysics Data System (ADS)

Miller, Daniel N.; Smith, Richard L.

2009-01-01

Groundwater nitrification is a poorly characterized process affecting the speciation and transport of nitrogen. Cores from two sites in a plume of contamination were examined using culture-based and molecular techniques targeting nitrification processes. The first site, located beneath a sewage effluent infiltration bed, received treated effluent containing O 2 (> 300 µM) and NH 4+ (51-800 µM). The second site was 2.5 km down-gradient near the leading edge of the ammonium zone within the contaminant plume and featured vertical gradients of O 2, NH 4+, and NO 3- (0-300, 0-500, and 100-200 µM with depth, respectively). Ammonia- and nitrite-oxidizers enumerated by the culture-based MPN method were low in abundance at both sites (1.8 to 350 g - 1 and 33 to 35,000 g - 1 , respectively). Potential nitrifying activity measured in core material in the laboratory was also very low, requiring several weeks for products to accumulate. Molecular analysis of aquifer DNA (nested PCR followed by cloning and 16S rDNA sequencing) detected primarily sequences associated with the Nitrosospira genus throughout the cores at the down-gradient site and a smaller proportion from the Nitrosomonas genus in the deeper anoxic, NH 4+ zone at the down-gradient site. Only a single Nitrosospira sequence was detected beneath the infiltration bed. Furthermore, the majority of Nitrosospira-associated sequences represent an unrecognized cluster. We conclude that an uncharacterized group associated with Nitrosospira dominate at the geochemically stable, down-gradient site, but found little evidence for Betaproteobacteria nitrifiers beneath the infiltration beds where geochemical conditions were more variable.

Combinatory RNA-Sequencing Analyses Reveal a Dual Mode of Gene Regulation by ADAR1 in Gastric Cancer.

PubMed

Cho, Charles J; Jung, Jaeeun; Jiang, Lushang; Lee, Eun Ji; Kim, Dae-Soo; Kim, Byung Sik; Kim, Hee Sung; Jung, Hwoon-Yong; Song, Ho-June; Hwang, Sung Wook; Park, Yangsoon; Jung, Min Kyo; Pack, Chan Gi; Myung, Seung-Jae; Chang, Suhwan

2018-04-25

Adenosine deaminase acting on RNA 1 (ADAR1) is known to mediate deamination of adenosine-to-inosine through binding to double-stranded RNA, the phenomenon known as RNA editing. Currently, the function of ADAR1 in gastric cancer is unclear. This study was aimed at investigating RNA editing-dependent and editing-independent functions of ADAR1 in gastric cancer, especially focusing on its influence on editing of 3' untranslated regions (UTRs) and subsequent changes in expression of messenger RNAs (mRNAs) as well as microRNAs (miRNAs). RNA-sequencing and small RNA-sequencing were performed on AGS and MKN-45 cells with a stable ADAR1 knockdown. Changed frequencies of editing and mRNA and miRNA expression were then identified by bioinformatic analyses. Targets of RNA editing were further validated in patients' samples. In the Alu region of both gastric cell lines, editing was most commonly of the A-to-I type in 3'-UTR or intron. mRNA and protein levels of PHACTR4 increased in ADAR1 knockdown cells, because of the loss of seed sequences in 3'-UTR of PHACTR4 mRNA that are required for miRNA-196a-3p binding. Immunohistochemical analyses of tumor and paired normal samples from 16 gastric cancer patients showed that ADAR1 expression was higher in tumors than in normal tissues and inversely correlated with PHACTR4 staining. On the other hand, decreased miRNA-148a-3p expression in ADAR1 knockdown cells led to increased mRNA and protein expression of NFYA, demonstrating ADAR1's editing-independent function. ADAR1 regulates post-transcriptional gene expression in gastric cancer through both RNA editing-dependent and editing-independent mechanisms.

Integrating feeding behavior, ecological data, and DNA barcoding to identify developmental differences in invertebrate foraging strategies in wild white-faced capuchins (Cebus capucinus).

PubMed

Mallott, Elizabeth K; Garber, Paul A; Malhi, Ripan S

2017-02-01

Invertebrate foraging strategies in nonhuman primates often require complex extractive foraging or prey detection techniques. As these skills take time to master, juveniles may have reduced foraging efficiency or concentrate their foraging efforts on easier to acquire prey than adults. We use DNA barcoding, behavioral observations, and ecological data to assess age-based differences in invertebrate prey foraging strategies in a group of white-faced capuchins (Cebus capucinus) in northeastern Costa Rica. Invertebrate availability was monitored using canopy traps and sweep netting. Fecal samples were collected from adult female, adult male, and juvenile white-faced capuchins (n = 225). COI mtDNA sequences were compared with known sequences in GenBank and the Barcode of Life Database. Frequencies of Lepidoptera and Hymenoptera consumption were higher in juveniles than in adults. A significantly smaller proportion of juvenile fecal samples contained Gryllidae and Cercopidae sequences, compared with adults (0% and 4.2% vs. 4.6% and 12.5%), and a significantly larger proportion contained Tenthredinidae, Culicidae, and Crambidae (5.6%, 9.7%, and 5.6% vs. 1.3%, 0.7%, and 1.3%). Juveniles spent significantly more time feeding and foraging than adults, and focused their foraging efforts on prey that require different skills to capture or extract. Arthropod availability was not correlated with foraging efficiency, and the rate of consumption of specific orders of invertebrates was not correlated with the availability of those same taxa. Our data support the hypothesis that juveniles are concentrating their foraging efforts on different prey than adults, potentially focusing their foraging efforts on more easily acquired types of prey. © 2016 Wiley Periodicals, Inc.

The PARIGA server for real time filtering and analysis of reciprocal BLAST results.

PubMed

Orsini, Massimiliano; Carcangiu, Simone; Cuccuru, Gianmauro; Uva, Paolo; Tramontano, Anna

2013-01-01

BLAST-based similarity searches are commonly used in several applications involving both nucleotide and protein sequences. These applications span from simple tasks such as mapping sequences over a database to more complex procedures as clustering or annotation processes. When the amount of analysed data increases, manual inspection of BLAST results become a tedious procedure. Tools for parsing or filtering BLAST results for different purposes are then required. We describe here PARIGA (http://resources.bioinformatica.crs4.it/pariga/), a server that enables users to perform all-against-all BLAST searches on two sets of sequences selected by the user. Moreover, since it stores the two BLAST output in a python-serialized-objects database, results can be filtered according to several parameters in real-time fashion, without re-running the process and avoiding additional programming efforts. Results can be interrogated by the user using logical operations, for example to retrieve cases where two queries match same targets, or when sequences from the two datasets are reciprocal best hits, or when a query matches a target in multiple regions. The Pariga web server is designed to be a helpful tool for managing the results of sequence similarity searches. The design and implementation of the server renders all operations very fast and easy to use.

Secondary structure prediction and structure-specific sequence analysis of single-stranded DNA.

PubMed

Dong, F; Allawi, H T; Anderson, T; Neri, B P; Lyamichev, V I

2001-08-01

DNA sequence analysis by oligonucleotide binding is often affected by interference with the secondary structure of the target DNA. Here we describe an approach that improves DNA secondary structure prediction by combining enzymatic probing of DNA by structure-specific 5'-nucleases with an energy minimization algorithm that utilizes the 5'-nuclease cleavage sites as constraints. The method can identify structural differences between two DNA molecules caused by minor sequence variations such as a single nucleotide mutation. It also demonstrates the existence of long-range interactions between DNA regions separated by >300 nt and the formation of multiple alternative structures by a 244 nt DNA molecule. The differences in the secondary structure of DNA molecules revealed by 5'-nuclease probing were used to design structure-specific probes for mutation discrimination that target the regions of structural, rather than sequence, differences. We also demonstrate the performance of structure-specific 'bridge' probes complementary to non-contiguous regions of the target molecule. The structure-specific probes do not require the high stringency binding conditions necessary for methods based on mismatch formation and permit mutation detection at temperatures from 4 to 37 degrees C. Structure-specific sequence analysis is applied for mutation detection in the Mycobacterium tuberculosis katG gene and for genotyping of the hepatitis C virus.

The full genome sequences of 8 equine herpesvirus type 4 isolates from horses in Japan.

PubMed

Izume, Satoko; Kirisawa, Rikio; Ohya, Kenji; Ohnuma, Aiko; Kimura, Takashi; Omatsu, Tsutomu; Katayama, Yukie; Mizutani, Tetsuya; Fukushi, Hideto

2017-01-24

Equine herpesvirus type 4 (EHV-4) is one of the most important pathogens in horses. To clarify the key genes of the EHV-4 genome that cause abortion in female horses, we determined the whole genome sequences of a laboratory strain and 7 Japanese EHV-4 isolates that were isolated from 2 aborted fetuses and nasal swabs of 5 horses with respiratory disease. The full genome sequences and predicted amino acid sequences of each gene of these isolates were compared with of the reference EHV-4 strain NS80567 and Australian isolates that were reported in 2015. The EHV-4 isolates clustered in 2 groups which did not reflect their pathogenicity. A comparison of the predicted amino acid sequences of the genes did not reveal any genes that were associated with EHV-4-induced abortion.

Driving on the surface of Mars with the rover sequencing and visualization program

NASA Technical Reports Server (NTRS)

Wright, J.; Hartman, F.; Cooper, B.; Maxwell, S.; Yen, J.; Morrison, J.

2005-01-01

Operating a rover on Mars is not possible using teleoperations due to the distance involved and the bandwith limitations. To operate these rovers requires sophisticated tools to make operators knowledgeable of the terrain, hazards, features of interest, and rover state and limitations, and to support building command sequences and rehearsing expected operations. This paper discusses how the Rover Sequencing and Visualization program and a small set of associated tools support this requirement.

Proficiency testing for bacterial whole genome sequencing: an end-user survey of current capabilities, requirements and priorities.

PubMed

Moran-Gilad, Jacob; Sintchenko, Vitali; Pedersen, Susanne Karlsmose; Wolfgang, William J; Pettengill, James; Strain, Errol; Hendriksen, Rene S

2015-04-03

The advent of next-generation sequencing (NGS) has revolutionised public health microbiology. Given the potential impact of NGS, it is paramount to ensure standardisation of 'wet' laboratory and bioinformatic protocols and promote comparability of methods employed by different laboratories and their outputs. Therefore, one of the ambitious goals of the Global Microbial Identifier (GMI) initiative (http://www.globalmicrobialidentifier.org/) has been to establish a mechanism for inter-laboratory NGS proficiency testing (PT). This report presents findings from the survey recently conducted by Working Group 4 among GMI members in order to ascertain NGS end-use requirements and attitudes towards NGS PT. The survey identified the high professional diversity of laboratories engaged in NGS-based public health projects and the wide range of capabilities within institutions, at a notable range of costs. The priority pathogens reported by respondents reflected the key drivers for NGS use (high burden disease and 'high profile' pathogens). The performance of and participation in PT was perceived as important by most respondents. The wide range of sequencing and bioinformatics practices reported by end-users highlights the importance of standardisation and harmonisation of NGS in public health and underpins the use of PT as a means to assuring quality. The findings of this survey will guide the design of the GMI PT program in relation to the spectrum of pathogens included, testing frequency and volume as well as technical requirements. The PT program for external quality assurance will evolve and inform the introduction of NGS into clinical and public health microbiology practice in the post-genomic era.

A survey and evaluations of histogram-based statistics in alignment-free sequence comparison.

PubMed

Luczak, Brian B; James, Benjamin T; Girgis, Hani Z

2017-12-06

Since the dawn of the bioinformatics field, sequence alignment scores have been the main method for comparing sequences. However, alignment algorithms are quadratic, requiring long execution time. As alternatives, scientists have developed tens of alignment-free statistics for measuring the similarity between two sequences. We surveyed tens of alignment-free k-mer statistics. Additionally, we evaluated 33 statistics and multiplicative combinations between the statistics and/or their squares. These statistics are calculated on two k-mer histograms representing two sequences. Our evaluations using global alignment scores revealed that the majority of the statistics are sensitive and capable of finding similar sequences to a query sequence. Therefore, any of these statistics can filter out dissimilar sequences quickly. Further, we observed that multiplicative combinations of the statistics are highly correlated with the identity score. Furthermore, combinations involving sequence length difference or Earth Mover's distance, which takes the length difference into account, are always among the highest correlated paired statistics with identity scores. Similarly, paired statistics including length difference or Earth Mover's distance are among the best performers in finding the K-closest sequences. Interestingly, similar performance can be obtained using histograms of shorter words, resulting in reducing the memory requirement and increasing the speed remarkably. Moreover, we found that simple single statistics are sufficient for processing next-generation sequencing reads and for applications relying on local alignment. Finally, we measured the time requirement of each statistic. The survey and the evaluations will help scientists with identifying efficient alternatives to the costly alignment algorithm, saving thousands of computational hours. The source code of the benchmarking tool is available as Supplementary Materials. © The Author 2017. Published by Oxford University Press.

Liquid rocket engine centrifugal flow turbopumps. [design criteria

NASA Technical Reports Server (NTRS)

1973-01-01

Design criteria and recommended practices are discussed for the following configurations selected from the design sequence of a liquid rocket engine centrifugal flow turbopump: (1) pump performance including speed, efficiency, and flow range; (2) impeller; (3) housing; and (4) thrust balance system. Hydrodynamic, structural, and mechanical problems are addressed for the achievement of required pump performance within the constraints imposed by the engine/turbopump system. Materials and fabrication specifications are also discussed.

Informative genomic microsatellite markers for efficient genotyping applications in sugarcane.

PubMed

Parida, Swarup K; Kalia, Sanjay K; Kaul, Sunita; Dalal, Vivek; Hemaprabha, G; Selvi, Athiappan; Pandit, Awadhesh; Singh, Archana; Gaikwad, Kishor; Sharma, Tilak R; Srivastava, Prem Shankar; Singh, Nagendra K; Mohapatra, Trilochan

2009-01-01

Genomic microsatellite markers are capable of revealing high degree of polymorphism. Sugarcane (Saccharum sp.), having a complex polyploid genome requires more number of such informative markers for various applications in genetics and breeding. With the objective of generating a large set of microsatellite markers designated as Sugarcane Enriched Genomic MicroSatellite (SEGMS), 6,318 clones from genomic libraries of two hybrid sugarcane cultivars enriched with 18 different microsatellite repeat-motifs were sequenced to generate 4.16 Mb high-quality sequences. Microsatellites were identified in 1,261 of the 5,742 non-redundant clones that accounted for 22% enrichment of the libraries. Retro-transposon association was observed for 23.1% of the identified microsatellites. The utility of the microsatellite containing genomic sequences were demonstrated by higher primer designing potential (90%) and PCR amplification efficiency (87.4%). A total of 1,315 markers including 567 class I microsatellite markers were designed and placed in the public domain for unrestricted use. The level of polymorphism detected by these markers among sugarcane species, genera, and varieties was 88.6%, while cross-transferability rate was 93.2% within Saccharum complex and 25% to cereals. Cloning and sequencing of size variant amplicons revealed that the variation in the number of repeat-units was the main source of SEGMS fragment length polymorphism. High level of polymorphism and wide range of genetic diversity (0.16-0.82 with an average of 0.44) assayed with the SEGMS markers suggested their usefulness in various genotyping applications in sugarcane.

The effects of DNA supercoiling on G-quadruplex formation.

PubMed

Sekibo, Doreen A T; Fox, Keith R

2017-12-01

Guanine-rich DNAs can fold into four-stranded structures that contain stacks of G-quartets. Bioinformatics studies have revealed that G-rich sequences with the potential to adopt these structures are unevenly distributed throughout genomes, and are especially found in gene promoter regions. With the exception of the single-stranded telomeric DNA, all genomic G-rich sequences will always be present along with their C-rich complements, and quadruplex formation will be in competition with the corresponding Watson-Crick duplex. Quadruplex formation must therefore first require local dissociation (melting) of the duplex strands. Since negative supercoiling is known to facilitate the formation of alternative DNA structures, we have investigated G-quadruplex formation within negatively supercoiled DNA plasmids. Plasmids containing multiple copies of (G3T)n and (G3T4)n repeats, were probed with dimethylsulphate, potassium permanganate and S1 nuclease. While dimethylsulphate footprinting revealed some evidence for G-quadruplex formation in (G3T)n sequences, this was not affected by supercoiling, and permanganate failed to detect exposed thymines in the loop regions. (G3T4)n sequences were not protected from DMS and showed no reaction with permanganate. Similarly, both S1 nuclease and 2D gel electrophoresis of DNA topoisomers did not detect any supercoil-dependent structural transitions. These results suggest that negative supercoiling alone is not sufficient to drive G-quadruplex formation. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Interactions between the two surface proteins of rotavirus may alter the receptor-binding specificity of the virus.

PubMed Central

Méndez, E; Arias, C F; López, S

1996-01-01

The infection of target cells by most animal rotavirus strains requires the presence of sialic acids (SAs) on the cell surface. We recently isolated variants from simian rotavirus RRV whose infectivity is no longer dependent on SAs and showed that the mutant phenotype segregates with the gene coding for VP4, one of the two surface proteins of rotaviruses (the other one being VP7). The nucleotide sequence of the VP4 gene of four independently isolated variants showed three amino acid changes, at positions 37 (Leu to Pro), 187 (Lys to Arg), and 267 (Tyr to Cys), in all mutant VP4 proteins compared with RRV VP4. The characterization of revertant viruses from two independent mutants showed that the arginine residue at position 187 changed back to lysine, indicating that this amino acid is involved in the determination of the mutant phenotype. Surprisingly, sequence analysis of reassortant virus DS1XRRV, which depends on SAs to infect the cell, showed that its VP4 gene is identical to the VP4 gene of the variants. Since the only difference between DS1XRRV and the RRV variants is the parental origin of the VP7 gene (human rotavirus DS1 in the reassortant), these findings suggest that the receptor-binding specificity of rotaviruses, via VP4, may be influenced by the associated VP7 protein. PMID:8551583

G4RNA: an RNA G-quadruplex database

PubMed Central

Garant, Jean-Michel; Luce, Mikael J.; Scott, Michelle S.

2015-01-01

Abstract G-quadruplexes (G4) are tetrahelical structures formed from planar arrangement of guanines in nucleic acids. A simple, regular motif was originally proposed to describe G4-forming sequences. More recently, however, formation of G4 was discovered to depend, at least in part, on the contextual backdrop of neighboring sequences. Prediction of G4 folding is thus becoming more challenging as G4 outlier structures, not described by the originally proposed motif, are increasingly reported. Recent observations thus call for a comprehensive tool, capable of consolidating the expanding information on tested G4s, in order to conduct systematic comparative analyses of G4-promoting sequences. The G4RNA Database we propose was designed to help meet the need for easily-retrievable data on known RNA G4s. A user-friendly, flexible query system allows for data retrieval on experimentally tested sequences, from many separate genes, to assess G4-folding potential. Query output sorts data according to sequence position, G4 likelihood, experimental outcomes and associated bibliographical references. G4RNA also provides an ideal foundation to collect and store additional sequence and experimental data, considering the growing interest G4s currently generate. Database URL: scottgroup.med.usherbrooke.ca/G4RNA PMID:26200754

Characterization of Sarcocystis from four species of hawks from Georgia, USA.

PubMed

Yabsley, Michael J; Ellis, Angela E; Stallknecht, David E; Howerth, Elizabeth W

2009-02-01

During 2001 to 2004, 4 species of hawks (Buteo and Accipiter spp.) from Georgia were surveyed for Sarcocystis spp. infections by examining intestinal sections. In total, 159 of 238 (66.8%) hawks examined were infected with Sarcocystis spp. Samples from 10 birds were characterized by sequence analysis of a portion of the 18S rRNA gene (783 base pairs). Only 3 of the 10 sequences from the hawks were identical; the remainder differed by at least 1 nucleotide. Phylogenetic analysis failed to resolve the position of the hawk Sarcocystis species, but they were closely related several Sarcocystis species from raptors, rodents, and Sarcocystis neurona. The high genetic diversity of Sarcocystis suggests that more than 1 species infects these 4 hawk species; however, additional molecular or experimental work will be required to determine the speciation and diversity of parasites infecting these avian hosts. In addition to assisting with determining species richness of Sarcocystis in raptors, molecular analysis should be useful in the identification of potential intermediate hosts.

Automated sequence analysis and editing software for HIV drug resistance testing.

PubMed

Struck, Daniel; Wallis, Carole L; Denisov, Gennady; Lambert, Christine; Servais, Jean-Yves; Viana, Raquel V; Letsoalo, Esrom; Bronze, Michelle; Aitken, Sue C; Schuurman, Rob; Stevens, Wendy; Schmit, Jean Claude; Rinke de Wit, Tobias; Perez Bercoff, Danielle

2012-05-01

Access to antiretroviral treatment in resource-limited-settings is inevitably paralleled by the emergence of HIV drug resistance. Monitoring treatment efficacy and HIV drugs resistance testing are therefore of increasing importance in resource-limited settings. Yet low-cost technologies and procedures suited to the particular context and constraints of such settings are still lacking. The ART-A (Affordable Resistance Testing for Africa) consortium brought together public and private partners to address this issue. To develop an automated sequence analysis and editing software to support high throughput automated sequencing. The ART-A Software was designed to automatically process and edit ABI chromatograms or FASTA files from HIV-1 isolates. The ART-A Software performs the basecalling, assigns quality values, aligns query sequences against a set reference, infers a consensus sequence, identifies the HIV type and subtype, translates the nucleotide sequence to amino acids and reports insertions/deletions, premature stop codons, ambiguities and mixed calls. The results can be automatically exported to Excel to identify mutations. Automated analysis was compared to manual analysis using a panel of 1624 PR-RT sequences generated in 3 different laboratories. Discrepancies between manual and automated sequence analysis were 0.69% at the nucleotide level and 0.57% at the amino acid level (668,047 AA analyzed), and discordances at major resistance mutations were recorded in 62 cases (4.83% of differences, 0.04% of all AA) for PR and 171 (6.18% of differences, 0.03% of all AA) cases for RT. The ART-A Software is a time-sparing tool for pre-analyzing HIV and viral quasispecies sequences in high throughput laboratories and highlighting positions requiring attention. Copyright © 2012 Elsevier B.V. All rights reserved.

Observation of a Pharmacist-Conducted Group A Streptococcal Pharyngitis Point-of-Care Test: A Time and Motion Study.

PubMed

Corn, Carolyn E; Klepser, Donald G; Dering-Anderson, Allison M; Brown, Terrence G; Klepser, Michael E; Smith, Jaclyn K

2018-06-01

Acute pharyngitis is among the most common infectious diseases encountered in the United States, resulting in 13 million patient visits annually, with group A streptococcus (GAS) being a common causative pathogen. It is estimated that annual expenditures for the treatment of adult pharyngitis will exceed US$1.2 billion annually. This substantial projection reinforces the need to evaluate diagnosis and treatment of adult pharyngitis in nontraditional settings. The objective of this research is to quantify the amount of pharmacist time required to complete a point-of-care (POC) test for a patient presenting with pharyngitis symptoms. A standardized patient with pharyngitis symptoms visited 11 pharmacies for POC testing services for a total of 33 patient encounters. An observer was present at each encounter and recorded the total encounter time, divided into 9 categories. Pharmacists conducted POC testing in 1 of 2 ways: sequence 1-pharmacists performed all service-related tasks; sequence 2-both pharmacists and pharmacist interns performed service-related tasks. The average time for completion of a POC test for GAS pharyngitis was 25.3 ± 4.8 minutes. The average pharmacist participation time per encounter was 12.7 ± 3.0 minutes (sequence 1), which decreased to 2.6 ± 1.1 minutes when pharmacist interns were involved in the testing (sequence 2). Although additional studies are required to further assess service feasibility, this study indicates that a GAS POC testing service could be implemented in a community pharmacy with limited disruption or change to workflow and staff.

Productive mRNA stem loop-mediated transcriptional slippage: Crucial features in common with intrinsic terminators.

PubMed

Penno, Christophe; Sharma, Virag; Coakley, Arthur; O'Connell Motherway, Mary; van Sinderen, Douwe; Lubkowska, Lucyna; Kireeva, Maria L; Kashlev, Mikhail; Baranov, Pavel V; Atkins, John F

2015-04-21

Escherichia coli and yeast DNA-dependent RNA polymerases are shown to mediate efficient nascent transcript stem loop formation-dependent RNA-DNA hybrid realignment. The realignment was discovered on the heteropolymeric sequence T5C5 and yields transcripts lacking a C residue within a corresponding U5C4. The sequence studied is derived from a Roseiflexus insertion sequence (IS) element where the resulting transcriptional slippage is required for transposase synthesis. The stability of the RNA structure, the proximity of the stem loop to the slippage site, the length and composition of the slippage site motif, and the identity of its 3' adjacent nucleotides (nt) are crucial for transcripts lacking a single C. In many respects, the RNA structure requirements for this slippage resemble those for hairpin-dependent transcription termination. In a purified in vitro system, the slippage efficiency ranges from 5% to 75% depending on the concentration ratios of the nucleotides specified by the slippage sequence and the 3' nt context. The only previous proposal of stem loop mediated slippage, which was in Ebola virus expression, was based on incorrect data interpretation. We propose a mechanical slippage model involving the RNAP translocation state as the main motor in slippage directionality and efficiency. It is distinct from previously described models, including the one proposed for paramyxovirus, where following random movement efficiency is mainly dependent on the stability of the new realigned hybrid. In broadening the scope for utilization of transcription slippage for gene expression, the stimulatory structure provides parallels with programmed ribosomal frameshifting at the translation level.

Alignment efficiency and discomfort of three orthodontic archwire sequences: a randomized clinical trial.

PubMed

Ong, Emily; Ho, Christopher; Miles, Peter

2011-03-01

To compare the efficiency of orthodontic archwire sequences produced by three manufacturers. Prospective, randomized clinical trial with three parallel groups. Private orthodontic practice in Caloundra, QLD, Australia. One hundred and thirty-two consecutive patients were randomized to one of three archwire sequence groups: (i) 3M Unitek, 0·014 inch Nitinol, 0·017 inch × 0·017 inch heat activated Ni-Ti; (ii) GAC international, 0·014 inch Sentalloy, 0·016 × 0·022 inch Bioforce; and (iii) Ormco corporation, 0·014 inch Damon Copper Ni-Ti, 0·014 × 0·025 inch Damon Copper Ni-Ti. All patients received 0·018 × 0·025 inch slot Victory Series™ brackets. Mandibular impressions were taken before the insertion of each archwire. Patients completed discomfort surveys according to a seven-point Likert Scale at 4 h, 24 h, 3 days and 7 days after the insertion of each archwire. Efficiency was measured by time required to reach the working archwire, mandibular anterior alignment and level of discomfort. No significant differences were found in the reduction of irregularity between the archwire sequences at any time-point (T1: P = 0·12; T2: P = 0·06; T3: P = 0·21) or in the time to reach the working archwire (P = 0·28). No significant differences were found in the overall discomfort scores between the archwire sequences (4 h: P = 0·30; 24 h: P = 0·18; 3 days: P = 0·53; 7 days: P = 0·47). When the time-points were analysed individually, the 3M Unitek archwire sequence induced significantly less discomfort than GAC and Ormco archwires 24 h after the insertion of the third archwire (P = 0·02). This could possibly be attributed to the progression in archwire material and archform. The archwire sequences were similar in alignment efficiency and overall discomfort. Progression in archwire dimension and archform may contribute to discomfort levels. This study provides clinical justification for three common archwire sequences in 0·018 × 0·025 inch slot brackets.

Sustainable Design of EPA's Campus in Research Triangle Park, NC—Environmental Performance Specifications in Construction Contracts—Section 01450 Sequence of Finishes Installation

EPA Pesticide Factsheets

Learn more about the special construction scheduling/sequencing requirements and procedures necessary to assure achievement of designed Indoor Air Quality (IAQ) levels for the completed project required by the EPA IAQ Program.

A programmable CCD driver circuit for multiphase CCD operation

NASA Technical Reports Server (NTRS)

Ewin, Audrey J.; Reed, Kenneth V.

1989-01-01

A programmable CCD (charge-coupled device) driver circuit was designed to drive CCDs in multiphased modes. The purpose of the drive electronics is to operate developmental CCD imaging arrays for NASA's tiltable moderate resolution imaging spectrometer (MODIS-T). Five objectives for the driver were considered during its design: (1) the circuit drives CCD electrode voltages between 0 V and +30 V to produce reasonable potential wells, (2) the driving sequence is started with one input signal, (3) the driving sequence is started with one input signal, (4) the circuit allows programming of frame sequences required by arrays of any size, (5) it produces interfacing signals for the CCD and the DTF (detector test facility). Simulation of the driver verified its function with the master clock running up to 10 MHz. This suggests a maximum rate of 400,000 pixels/s. Timing and packaging parameters were verified. The design uses 54 TTL (transistor-transistor logic) chips. Two versions of hardware were fabricated: wirewrap and printed circuit board. Both were verified functionally with a logic analyzer.

Optimized invertase expression and secretion cassette for improving Yarrowia lipolytica growth on sucrose for industrial applications.

PubMed

Lazar, Zbigniew; Rossignol, Tristan; Verbeke, Jonathan; Crutz-Le Coq, Anne-Marie; Nicaud, Jean-Marc; Robak, Małgorzata

2013-11-01

Yarrowia lipolytica requires the expression of a heterologous invertase to grow on a sucrose-based substrate. This work reports the construction of an optimized invertase expression cassette composed of Saccharomyces cerevisiae Suc2p secretion signal sequence followed by the SUC2 sequence and under the control of the strong Y. lipolytica pTEF promoter. This new construction allows a fast and optimal cleavage of sucrose into glucose and fructose and allows cells to reach the maximum growth rate. Contrary to pre-existing constructions, the expression of SUC2 is not sensitive to medium composition in this context. The strain JMY2593, expressing this new cassette with an optimized secretion signal sequence and a strong promoter, produces 4,519 U/l of extracellular invertase in bioreactor experiments compared to 597 U/l in a strain expressing the former invertase construction. The expression of this cassette strongly improved production of invertase and is suitable for simultaneously high production level of citric acid from sucrose-based media.

GPU-Acceleration of Sequence Homology Searches with Database Subsequence Clustering

PubMed Central

Suzuki, Shuji; Kakuta, Masanori; Ishida, Takashi; Akiyama, Yutaka

2016-01-01

Sequence homology searches are used in various fields and require large amounts of computation time, especially for metagenomic analysis, owing to the large number of queries and the database size. To accelerate computing analyses, graphics processing units (GPUs) are widely used as a low-cost, high-performance computing platform. Therefore, we mapped the time-consuming steps involved in GHOSTZ, which is a state-of-the-art homology search algorithm for protein sequences, onto a GPU and implemented it as GHOSTZ-GPU. In addition, we optimized memory access for GPU calculations and for communication between the CPU and GPU. As per results of the evaluation test involving metagenomic data, GHOSTZ-GPU with 12 CPU threads and 1 GPU was approximately 3.0- to 4.1-fold faster than GHOSTZ with 12 CPU threads. Moreover, GHOSTZ-GPU with 12 CPU threads and 3 GPUs was approximately 5.8- to 7.7-fold faster than GHOSTZ with 12 CPU threads. PMID:27482905

Development of a candidate reference material for adventitious virus detection in vaccine and biologicals manufacturing by deep sequencing

PubMed Central

Mee, Edward T.; Preston, Mark D.; Minor, Philip D.; Schepelmann, Silke; Huang, Xuening; Nguyen, Jenny; Wall, David; Hargrove, Stacey; Fu, Thomas; Xu, George; Li, Li; Cote, Colette; Delwart, Eric; Li, Linlin; Hewlett, Indira; Simonyan, Vahan; Ragupathy, Viswanath; Alin, Voskanian-Kordi; Mermod, Nicolas; Hill, Christiane; Ottenwälder, Birgit; Richter, Daniel C.; Tehrani, Arman; Jacqueline, Weber-Lehmann; Cassart, Jean-Pol; Letellier, Carine; Vandeputte, Olivier; Ruelle, Jean-Louis; Deyati, Avisek; La Neve, Fabio; Modena, Chiara; Mee, Edward; Schepelmann, Silke; Preston, Mark; Minor, Philip; Eloit, Marc; Muth, Erika; Lamamy, Arnaud; Jagorel, Florence; Cheval, Justine; Anscombe, Catherine; Misra, Raju; Wooldridge, David; Gharbia, Saheer; Rose, Graham; Ng, Siemon H.S.; Charlebois, Robert L.; Gisonni-Lex, Lucy; Mallet, Laurent; Dorange, Fabien; Chiu, Charles; Naccache, Samia; Kellam, Paul; van der Hoek, Lia; Cotten, Matt; Mitchell, Christine; Baier, Brian S.; Sun, Wenping; Malicki, Heather D.

2016-01-01

Background Unbiased deep sequencing offers the potential for improved adventitious virus screening in vaccines and biotherapeutics. Successful implementation of such assays will require appropriate control materials to confirm assay performance and sensitivity. Methods A common reference material containing 25 target viruses was produced and 16 laboratories were invited to process it using their preferred adventitious virus detection assay. Results Fifteen laboratories returned results, obtained using a wide range of wet-lab and informatics methods. Six of 25 target viruses were detected by all laboratories, with the remaining viruses detected by 4–14 laboratories. Six non-target viruses were detected by three or more laboratories. Conclusion The study demonstrated that a wide range of methods are currently used for adventitious virus detection screening in biological products by deep sequencing and that they can yield significantly different results. This underscores the need for common reference materials to ensure satisfactory assay performance and enable comparisons between laboratories. PMID:26709640

Shuttle cryogenics supply system. Optimization study. Volume 5 B-4: Programmers manual for space shuttle orbit injection analysis (SOPSA)

NASA Technical Reports Server (NTRS)

1973-01-01

A computer program for space shuttle orbit injection propulsion system analysis (SOPSA) is described to show the operational characteristics and the computer system requirements. The program was developed as an analytical tool to aid in the preliminary design of propellant feed systems for the space shuttle orbiter main engines. The primary purpose of the program is to evaluate the propellant tank ullage pressure requirements imposed by the need to accelerate propellants rapidly during the engine start sequence. The SOPSA program will generate parametric feed system pressure histories and weight data for a range of nominal feedline sizes.

Purification and Molecular Characterization of the Tungsten-Containing Formaldehyde Ferredoxin Oxidoreductase from the Hyperthermophilic Archaeon Pyrococcus furiosus: the Third of a Putative Five-Member Tungstoenzyme Family

PubMed Central

Roy, Roopali; Mukund, Swarnalatha; Schut, Gerrit J.; Dunn, Dianne M.; Weiss, Robert; Adams, Michael W. W.

1999-01-01

Pyrococcus furiosus is a hyperthermophilic archaeon which grows optimally near 100°C by fermenting peptides and sugars to produce organic acids, CO2, and H2. Its growth requires tungsten, and two different tungsten-containing enzymes, aldehyde ferredoxin oxidoreductase (AOR) and glyceraldehyde-3-phosphate ferredoxin oxidoreductase (GAPOR), have been previously purified from P. furiosus. These two enzymes are thought to function in the metabolism of peptides and carbohydrates, respectively. A third type of tungsten-containing enzyme, formaldehyde ferredoxin oxidoreductase (FOR), has now been characterized. FOR is a homotetramer with a mass of 280 kDa and contains approximately 1 W atom, 4 Fe atoms, and 1 Ca atom per subunit, together with a pterin cofactor. The low recovery of FOR activity during purification was attributed to loss of sulfide, since the purified enzyme was activated up to fivefold by treatment with sulfide (HS−) under reducing conditions. FOR uses P. furiosus ferredoxin as an electron acceptor (Km = 100 μM) and oxidizes a range of aldehydes. Formaldehyde (Km = 15 mM for the sulfide-activated enzyme) was used in routine assays, but the physiological substrate is thought to be an aliphatic C5 semi- or dialdehyde, e.g., glutaric dialdehyde (Km = 1 mM). Based on its amino-terminal sequence, the gene encoding FOR (for) was identified in the genomic database, together with those encoding AOR and GAPOR. The amino acid sequence of FOR corresponded to a mass of 68.7 kDa and is highly similar to those of the subunits of AOR (61% similarity and 40% identity) and GAPOR (50% similarity and 23% identity). The three genes are not linked on the P. furiosus chromosome. Two additional (and nonlinked) genes (termed wor4 and wor5) that encode putative tungstoenzymes with 57% (WOR4) and 56% (WOR5) sequence similarity to FOR were also identified. Based on sequence motif similarities with FOR, both WOR4 and WOR5 are also proposed to contain a tungstobispterin site and one [4Fe-4S] cluster per subunit. PMID:9973343

The maize stripe virus major noncapsid protein messenger RNA transcripts contain heterogeneous leader sequences at their 5' termini.

PubMed

Huiet, L; Feldstein, P A; Tsai, J H; Falk, B W

1993-12-01

Primer extension analyses and a PCR-based cloning strategy were used to identify and characterize 5' nucleotide sequences on the maize stripe virus (MStV) RNA4 mRNA transcripts encoding the major noncapsid protein (NCP). Direct RNA sequence analysis by primer extension showed that the NCP mRNA transcripts had 10-15 nucleotides beyond the 5' terminus of the MStV RNA4 nucleotide sequence. MStV genomic RNAs isolated from ribonucleoprotein particles (RNPs) lacked the additional 5' nucleotides. cDNA clones representing the 5' region of the mRNA transcripts were constructed, and the nucleotide sequences of the 5' regions were determined for 16 clones. Each was found to have a distinct 10-15 nucleotide sequence immediately 5' of the MStV RNA4 sequence. Eleven of 16 clones had the correct MStV RNA4 5' nucleotide sequence, while five showed minor variations at or near the 5' most MStV RNA4 nucleotide. These characteristics show strong similarities to other viral mRNA transcripts which are synthesized by cap snatching.

Deletion mutants of Harvey ras p21 protein reveal the absolute requirement of at least two distant regions for GTP-binding and transforming activities.

PubMed Central

Lacal, J C; Anderson, P S; Aaronson, S A

1986-01-01

Deletions of small sequences from the viral Harvey ras gene have been generated, and resulting ras p21 mutants have been expressed in Escherichia coli. Purification of each deleted protein allowed the in vitro characterization of GTP-binding, GTPase and autokinase activity of the proteins. Microinjection of the highly purified proteins into quiescent NIH/3T3 cells, as well as transfection experiments utilizing a long terminal repeat (LTR)-containing vector, were utilized to analyze the biological activity of the deleted proteins. Two small regions located at 6-23 and 152-165 residues are shown to be absolutely required for in vitro and in vivo activities of the ras product. By contrast, the variable region comprising amino acids 165-184 was shown not to be necessary for either in vitro or in vivo activities. Thus, we demonstrate that: (i) amino acid sequences at positions 5-23 and 152-165 of ras p21 protein are probably directly involved in the GTP-binding activity; (ii) GTP-binding is required for the transforming activity of ras p21 and by extension for the normal function of the proto-oncogene product; and (iii) the variable region at the C-terminal end of the ras p21 molecule from amino acids 165 to 184 is not required for transformation. Images Fig.2. Fig.4. PMID:3011420

Supervised classification of brain tissues through local multi-scale texture analysis by coupling DIR and FLAIR MR sequences

NASA Astrophysics Data System (ADS)

Poletti, Enea; Veronese, Elisa; Calabrese, Massimiliano; Bertoldo, Alessandra; Grisan, Enrico

2012-02-01

The automatic segmentation of brain tissues in magnetic resonance (MR) is usually performed on T1-weighted images, due to their high spatial resolution. T1w sequence, however, has some major downsides when brain lesions are present: the altered appearance of diseased tissues causes errors in tissues classification. In order to overcome these drawbacks, we employed two different MR sequences: fluid attenuated inversion recovery (FLAIR) and double inversion recovery (DIR). The former highlights both gray matter (GM) and white matter (WM), the latter highlights GM alone. We propose here a supervised classification scheme that does not require any anatomical a priori information to identify the 3 classes, "GM", "WM", and "background". Features are extracted by means of a local multi-scale texture analysis, computed for each pixel of the DIR and FLAIR sequences. The 9 textures considered are average, standard deviation, kurtosis, entropy, contrast, correlation, energy, homogeneity, and skewness, evaluated on a neighborhood of 3x3, 5x5, and 7x7 pixels. Hence, the total number of features associated to a pixel is 56 (9 textures x3 scales x2 sequences +2 original pixel values). The classifier employed is a Support Vector Machine with Radial Basis Function as kernel. From each of the 4 brain volumes evaluated, a DIR and a FLAIR slice have been selected and manually segmented by 2 expert neurologists, providing 1st and 2nd human reference observations which agree with an average accuracy of 99.03%. SVM performances have been assessed with a 4-fold cross-validation, yielding an average classification accuracy of 98.79%.

The first whole genome sequence and pathogenicity characterization of a fowl adenovirus 4 isolated from ducks associated with inclusion body hepatitis and hydropericardium syndrome.

PubMed

Pan, Qing; Liu, Linlin; Wang, Yongqiang; Zhang, Yanping; Qi, Xiaole; Liu, Changjun; Gao, Yulong; Wang, Xiaomei; Cui, Hongyu

2017-10-01

In June 2015, an infectious disease with high prevalence causing severe hydropericardium syndrome (HPS) first appeared in duck farms of northeast China. The disease showed high morbidity of 35% and mortality of 15% in a commercial duck farm with 200,000 45-day-old ducks. One strain of hypervirulent fowl adenovirus serotype 4 was identified and designated as HLJDAd15. The whole genome of the duck isolate was sequenced and found to contain the same large deletions as a genotype that has become prevalent in chickens in China recently, indicating that this disease might be transmitted from chickens to ducks. The pathogenicity of HLJDAd15 was evaluated in SPF chickens and ducks. The results showed that chickens were more susceptible to this new genotype of fowl adenovirus, and it was more difficult to infect ducks than chickens with the duck origin virus. Thus, it appears that this severe HPS in ducks is far more likely to have been transmitted from chickens to ducks than from ducks to ducks. Therefore, transmission from chickens to ducks constitutes a threat to the duck farming industry, and this transmission route is a very important consideration for the prevention and control of the new genotype of fowl adenovirus. This is the first whole genome sequence of a FAdV-4 isolated from ducks, and this information is important for understanding the molecular characteristics and evolution of aviadenoviruses. The potential risks of infection with this new hypervirulent FAdV-4 genotype in chickens and ducks urgently require an effective vaccine.

Drinking from the Fire Hose: Why the Flight Management System Can Be Hard to Train and Difficult to Use

NASA Technical Reports Server (NTRS)

Sherry, Lance; Feary, Michael; Polson, Peter; Fennell, Karl

2003-01-01

The Flight Management Computer (FMC) and its interface, the Multi-function Control and Display Unit (MCDU) have been identified by researchers and airlines as difficult to train and use. Specifically, airline pilots have described the "drinking from the fire-hose" effect during training. Previous research has identified memorized action sequences as a major factor in a user s ability to learn and operate complex devices. This paper discusses the use of a method to examine the quantity of memorized action sequences required to perform a sample of 102 tasks, using features of the Boeing 777 Flight Management Computer Interface. The analysis identified a large number of memorized action sequences that must be learned during training and then recalled during line operations. Seventy-five percent of the tasks examined require recall of at least one memorized action sequence. Forty-five percent of the tasks require recall of a memorized action sequence and occur infrequently. The large number of memorized action sequences may provide an explanation for the difficulties in training and usage of the automation. Based on these findings, implications for training and the design of new user-interfaces are discussed.

Identification of an EMS-induced causal mutation in a gene required for boron-mediated root development by low-coverage genome re-sequencing in Arabidopsis

PubMed Central

Tabata, Ryo; Kamiya, Takehiro; Shigenobu, Shuji; Yamaguchi, Katsushi; Yamada, Masashi; Hasebe, Mitsuyasu; Fujiwara, Toru; Sawa, Shinichiro

2013-01-01

Next-generation sequencing (NGS) technologies enable the rapid production of an enormous quantity of sequence data. These powerful new technologies allow the identification of mutations by whole-genome sequencing. However, most reported NGS-based mapping methods, which are based on bulked segregant analysis, are costly and laborious. To address these limitations, we designed a versatile NGS-based mapping method that consists of a combination of low- to medium-coverage multiplex SOLiD (Sequencing by Oligonucleotide Ligation and Detection) and classical genetic rough mapping. Using only low to medium coverage reduces the SOLiD sequencing costs and, since just 10 to 20 mutant F2 plants are required for rough mapping, the operation is simple enough to handle in a laboratory with limited space and funding. As a proof of principle, we successfully applied this method to identify the CTR1, which is involved in boron-mediated root development, from among a population of high boron requiring Arabidopsis thaliana mutants. Our work demonstrates that this NGS-based mapping method is a moderately priced and versatile method that can readily be applied to other model organisms. PMID:23104114

NRPD4, a protein related to the RPB4 subunit of RNA polymerase II, is a component of RNA polymerases IV and V and is required for RNA-directed DNA methylation

PubMed Central

He, Xin-Jian; Hsu, Yi-Feng; Pontes, Olga; Zhu, Jianhua; Lu, Jian; Bressan, Ray A.; Pikaard, Craig; Wang, Co-Shine; Zhu, Jian-Kang

2009-01-01

RNA-directed DNA methylation (RdDM) is an RNAi-based mechanism for establishing transcriptional gene silencing in plants. The plant-specific RNA polymerases IV and V are required for the generation of 24-nucleotide (nt) siRNAs and for guiding sequence-specific DNA methylation by the siRNAs, respectively. However, unlike the extensively studied multisubunit Pol II, our current knowledge about Pol IV and Pol V is restricted to only the two largest subunits NRPD1a/NRPD1 and NRPD1b/NRPE1 and the one second-largest subunit NRPD2a. It is unclear whether other subunits may be required for the functioning of Pol IV and Pol V in RdDM. From a genetic screen for second-site suppressors of the DNA demethylase mutant ros1, we identified a new component (referred to as RDM2) as well as seven known components (NRPD1, NRPE1, NRPD2a, AGO4, HEN1, DRD1, and HDA6) of the RdDM pathway. The differential effects of the mutations on two mechanistically distinct transcriptional silencing reporters suggest that RDM2, NRPD1, NRPE1, NRPD2a, HEN1, and DRD1 function only in the siRNA-dependent pathway of transcriptional silencing, whereas HDA6 and AGO4 have roles in both siRNA-dependent and -independent pathways of transcriptional silencing. In the rdm2 mutants, DNA methylation and siRNA accumulation were reduced substantially at loci previously identified as endogenous targets of Pol IV and Pol V, including 5S rDNA, MEA-ISR, AtSN1, AtGP1, and AtMU1. The amino acid sequence of RDM2 is similar to that of RPB4 subunit of Pol II, but we show evidence that RDM2 has diverged significantly from RPB4 and cannot function in Pol II. An association of RDM2 with both NRPD1 and NRPE1 was observed by coimmunoprecipitation and coimmunolocalization assays. Our results show that RDM2/NRPD4/NRPE4 is a new component of the RdDM pathway in Arabidopsis and that it functions as part of Pol IV and Pol V. PMID:19204117

NRPD4, a Protein Related to the RPB4 Subunit of RNA Polymerase II, is a Component of RNA Polymerases IV and V and is Required for RNA-directed DNA methylation

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

He, Xin-Jian; Hsu, Yi-Feng; Pontes, Olga

2009-01-01

RNA-directed DNA methylation (RdDM) is an RNAi-based mechanism for establishing transcriptional gene silencing in plants. The plant-specific RNA polymerases IV and V are required for the generation of 24-nucleotide (nt) siRNAs and for guiding sequence-specific DNA methylation by the siRNAs, respectively. However, unlike the extensively studied multisubunit Pol II, our current knowledge about Pol IV and Pol V is restricted to only the two largest subunits NRPD1a/NRPD1 and NRPD1b/NRPE1 and the one second-largest subunit NRPD2a. It is unclear whether other subunits may be required for the functioning of Pol IV and Pol V in RdDM. From a genetic screen formore » second-site suppressors of the DNA demethylase mutant ros1, we identified a new component (referred to as RDM2) as well as seven known components (NRPD1, NRPE1, NRPD2a, AGO4, HEN1, DRD1, and HDA6) of the RdDM pathway. The differential effects of the mutations on two mechanistically distinct transcriptional silencing reporters suggest that RDM2, NRPD1, NRPE1, NRPD2a, HEN1, and DRD1 function only in the siRNA-dependent pathway of transcriptional silencing, whereas HDA6 and AGO4 have roles in both siRNA-dependent and -independent pathways of transcriptional silencing. In the rdm2 mutants, DNA methylation and siRNA accumulation were reduced substantially at loci previously identified as endogenous targets of Pol IV and Pol V, including 5S rDNA, MEA-ISR, AtSN1, AtGP1, and AtMU1. The amino acid sequence of RDM2 is similar to that of RPB4 subunit of Pol II, but we show evidence that RDM2 has diverged significantly from RPB4 and cannot function in Pol II. An association of RDM2 with both NRPD1 and NRPE1 was observed by coimmunoprecipitation and coimmunolocalization assays. Our results show that RDM2/NRPD4/NRPE4 is a new component of the RdDM pathway in Arabidopsis and that it functions as part of Pol IV and Pol V.« less

Measurement of nitrous acid (HONO) by external-cavity quantum cascade laser based quartz-enhanced photoacoustic absorption spectroscopy

NASA Astrophysics Data System (ADS)

Yi, Hongming; Maamary, Rabih; Gao, Xiaoming; Sigrist, Markus W.; Fertein, Eric; Chen, Weidong

2016-04-01

Spectroscopic detection of short-lived gaseous nitrous acid (HONO) at 1254.85 cm-1 was realized by off-beam coupled quartz-enhanced photoacoustic spectroscopy (QEPAS) in conjunction with an external cavity quantum cascade lasers (EC-QCL). High sensitivity monitoring of HONO was performed within a very small gas-sample volume (of ~40 mm3) allowing a significant reduction (of about 4 orders of magnitude) of air sampling residence time which is highly desired for accurate quantification of chemically reactive short-lived species. Calibration of the developed QEPAS-based HONO sensor was carried out by means of lab-generated HONO samples whose concentrations were determined by simultaneous measurements of direct HONO absorption spectra in a 109.5 m multipass cell using a distributed feedback (DBF) QCL. A minimum detection limit (MDL @ SNR=1) of 66 ppbv HONO was achieved at 70 mbar using a laser output power of 50 mW and 1 s integration time, which corresponded to a normalized noise equivalent absorption coefficient of 3.6×10-8 cm-1.W/Hz1/2. This MDL was down to 7 ppbv at the optimal integration time of 150 s. The corresponding minimum detected absorption coefficient (SNR=1) is ~1.1×10-7 cm-1 (MDL: ~3 ppbv) in 1 s and ~1.1×10-8 cm-1 (MDL~330 pptv) in 150 s, respectively, with 1 W laser power. Acknowledgements The authors acknowledge financial supports from the CaPPA project (ANR-10-LABX-005) and the CPER CLIMIBIO program. References H. Yi, R. Maamary, X. Gao, M. W. Sigrist, E. Fertein, W. Chen, "Short-lived species detection of nitrous acid by external-cavity quantum cascade laser based quartz-enhanced photoacoustic absorption spectroscopy", Appl. Phys. Lett. 106 (2015) 101109

40 CFR 92.124 - Test sequence; general requirements.

Code of Federal Regulations, 2012 CFR

2012-07-01

.... (e) Pre-test engine measurements (e.g., idle and throttle notch speeds, fuel flows, etc.), pre-test engine performance checks (e.g., verification of engine power, etc.) and pre-test system calibrations (e... 40 Protection of Environment 21 2012-07-01 2012-07-01 false Test sequence; general requirements...

40 CFR 92.124 - Test sequence; general requirements.

Code of Federal Regulations, 2014 CFR

2014-07-01

.... (e) Pre-test engine measurements (e.g., idle and throttle notch speeds, fuel flows, etc.), pre-test engine performance checks (e.g., verification of engine power, etc.) and pre-test system calibrations (e... 40 Protection of Environment 20 2014-07-01 2013-07-01 true Test sequence; general requirements. 92...

40 CFR 92.124 - Test sequence; general requirements.

Code of Federal Regulations, 2010 CFR

2010-07-01

.... (e) Pre-test engine measurements (e.g., idle and throttle notch speeds, fuel flows, etc.), pre-test engine performance checks (e.g., verification of engine power, etc.) and pre-test system calibrations (e... 40 Protection of Environment 20 2010-07-01 2010-07-01 false Test sequence; general requirements...

40 CFR 92.124 - Test sequence; general requirements.

Code of Federal Regulations, 2013 CFR

2013-07-01

.... (e) Pre-test engine measurements (e.g., idle and throttle notch speeds, fuel flows, etc.), pre-test engine performance checks (e.g., verification of engine power, etc.) and pre-test system calibrations (e... 40 Protection of Environment 21 2013-07-01 2013-07-01 false Test sequence; general requirements...

40 CFR 92.124 - Test sequence; general requirements.

Code of Federal Regulations, 2011 CFR

2011-07-01

.... (e) Pre-test engine measurements (e.g., idle and throttle notch speeds, fuel flows, etc.), pre-test engine performance checks (e.g., verification of engine power, etc.) and pre-test system calibrations (e... 40 Protection of Environment 20 2011-07-01 2011-07-01 false Test sequence; general requirements...

Genome analysis of Desulfotomaculum gibsoniae strain GrollT a highly versatile Gram-positive sulfate-reducing bacterium

PubMed Central

Kuever, Jan; Visser, Michael; Loeffler, Claudia; Boll, Matthias; Worm, Petra; Sousa, Diana Z.; Plugge, Caroline M.; Schaap, Peter J.; Muyzer, Gerard; Pereira, Ines A.C.; Parshina, Sofiya N.; Goodwin, Lynne A.; Kyrpides, Nikos C.; Detter, Janine; Woyke, Tanja; Chain, Patrick; Davenport, Karen W.; Rohde, Manfred; Spring, Stefan; Klenk, Hans-Peter; Stams, Alfons J.M.

2014-01-01

Desulfotomaculum gibsoniae is a mesophilic member of the polyphyletic spore-forming genus Desulfotomaculum within the family Peptococcaceae. This bacterium was isolated from a freshwater ditch and is of interest because it can grow with a large variety of organic substrates, in particular several aromatic compounds, short-chain and medium-chain fatty acids, which are degraded completely to carbon dioxide coupled to the reduction of sulfate. It can grow autotrophically with H2 + CO2 and sulfate and slowly acetogenically with H2 + CO2, formate or methoxylated aromatic compounds in the absence of sulfate. It does not require any vitamins for growth. Here, we describe the features of D. gibsoniae strain GrollT together with the genome sequence and annotation. The chromosome has 4,855,529 bp organized in one circular contig and is the largest genome of all sequenced Desulfotomaculum spp. to date. A total of 4,666 candidate protein-encoding genes and 96 RNA genes were identified. Genes of the acetyl-CoA pathway, possibly involved in heterotrophic growth and in CO2 fixation during autotrophic growth, are present. The genome contains a large set of genes for the anaerobic transformation and degradation of aromatic compounds, which are lacking in the other sequenced Desulfotomaculum genomes. PMID:25197466

[BIOINFORMATIC SEARCH AND PHYLOGENETIC ANALYSIS OF THE CELLULOSE SYNTHASE GENES OF FLAX (LINUM USITATISSIMUM)].

PubMed

Pydiura, N A; Bayer, G Ya; Galinousky, D V; Yemets, A I; Pirko, Ya V; Podvitski, T A; Anisimova, N V; Khotyleva, L V; Kilchevsky, A V; Blume, Ya B

2015-01-01

A bioinformatic search of sequences encoding cellulose synthase genes in the flax genome, and their comparison to dicots orthologs was carried out. The analysis revealed 32 cellulose synthase gene candidates, 16 of which are highly likely to encode cellulose synthases, and the remaining 16--cellulose synthase-like proteins (Csl). Phylogenetic analysis of gene products of cellulose synthase genes allowed distinguishing 6 groups of cellulose synthase genes of different classes: CesA1/10, CesA3, CesA4, CesA5/6/2/9, CesA7 and CesA8. Paralogous sequences within classes CesA1/10 and CesA5/6/2/9 which are associated with the primary cell wall formation are characterized by a greater similarity within these classes than orthologous sequences. Whereas the genes controlling the biosynthesis of secondary cell wall cellulose form distinct clades: CesA4, CesA7, and CesA8. The analysis of 16 identified flax cellulose synthase gene candidates shows the presence of at least 12 different cellulose synthase gene variants in flax genome which are represented in all six clades of cellulose synthase genes. Thus, at this point genes of all ten known cellulose synthase classes are identify in flax genome, but their correct classification requires additional research.

Defining Differential Genetic Signatures in CXCR4- and the CCR5-Utilizing HIV-1 Co-Linear Sequences

PubMed Central

Aiamkitsumrit, Benjamas; Dampier, Will; Martin-Garcia, Julio; Nonnemacher, Michael R.; Pirrone, Vanessa; Ivanova, Tatyana; Zhong, Wen; Kilareski, Evelyn; Aldigun, Hazeez; Frantz, Brian; Rimbey, Matthew; Wojno, Adam; Passic, Shendra; Williams, Jean W.; Shah, Sonia; Blakey, Brandon; Parikh, Nirzari; Jacobson, Jeffrey M.; Moldover, Brian; Wigdahl, Brian

2014-01-01

The adaptation of human immunodeficiency virus type-1 (HIV-1) to an array of physiologic niches is advantaged by the plasticity of the viral genome, encoded proteins, and promoter. CXCR4-utilizing (X4) viruses preferentially, but not universally, infect CD4+ T cells, generating high levels of virus within activated HIV-1-infected T cells that can be detected in regional lymph nodes and peripheral blood. By comparison, the CCR5-utilizing (R5) viruses have a greater preference for cells of the monocyte-macrophage lineage; however, while R5 viruses also display a propensity to enter and replicate in T cells, they infect a smaller percentage of CD4+ T cells in comparison to X4 viruses. Additionally, R5 viruses have been associated with viral transmission and CNS disease and are also more prevalent during HIV-1 disease. Specific adaptive changes associated with X4 and R5 viruses were identified in co-linear viral sequences beyond the Env-V3. The in silico position-specific scoring matrix (PSSM) algorithm was used to define distinct groups of X4 and R5 sequences based solely on sequences in Env-V3. Bioinformatic tools were used to identify genetic signatures involving specific protein domains or long terminal repeat (LTR) transcription factor sites within co-linear viral protein R (Vpr), trans-activator of transcription (Tat), or LTR sequences that were preferentially associated with X4 or R5 Env-V3 sequences. A number of differential amino acid and nucleotide changes were identified across the co-linear Vpr, Tat, and LTR sequences, suggesting the presence of specific genetic signatures that preferentially associate with X4 or R5 viruses. Investigation of the genetic relatedness between X4 and R5 viruses utilizing phylogenetic analyses of complete sequences could not be used to definitively and uniquely identify groups of R5 or X4 sequences; in contrast, differences in the genetic diversities between X4 and R5 were readily identified within these co-linear sequences in HIV-1-infected patients. PMID:25265194

Porcine insulin receptor substrate 4 (IRS4) gene: cloning, polymorphism and association study

USDA-ARS?s Scientific Manuscript database

Using PCR and IPCR techniques we obtained a 4498 bp nucleotide sequence FN424076 encompassing the complete coding sequence of the porcine IRS4 gene and its proximal promoter. The 1269-amino acid porcine protein deduced from the nucleotide sequence shares 92% identity with the human IRS4 and possesse...

The influence of tightening sequence and method on screw preload in implant superstructures.

PubMed

Al-Sahan, Maha M; Al Maflehi, Nassr S; Akeel, Riyadh F

2014-01-01

This study evaluated the effect of six screw-tightening sequences and two tightening methods on the screw preload in implant-supported superstructures. The preload was measured using strain gauges following the screw tightening of a metal framework connected to four implants. The experiment included six sequences ([1] 1-2-3-4, [2] 4-2-3-1, [3] 4-3-1-2, [4] 1-4-2-3, [5] 2-3-4-1, and [6] 3-2-4-1), two methods (onestep, three-step), and five replications. Significant differences were found between tightening sequences and methods. In the three-step method, a higher total preload was found in sequences 2 (312 ± 85 N), 3 (246 ± 54 N), and 4 (310 ± 96 N). In the one-step method, a higher total preload was found in sequences 1 (286 ± 94 N), 5 (764 ± 142 N), and 6 (350 ± 69 N). It is concluded that the highest total screw preload was achieved when anterior implants of the superstructure were first tightened in one step, followed by posterior implants.

Neural-network-designed pulse sequences for robust control of singlet-triplet qubits

NASA Astrophysics Data System (ADS)

Yang, Xu-Chen; Yung, Man-Hong; Wang, Xin

2018-04-01

Composite pulses are essential for universal manipulation of singlet-triplet spin qubits. In the absence of noise, they are required to perform arbitrary single-qubit operations due to the special control constraint of a singlet-triplet qubit, while in a noisy environment, more complicated sequences have been developed to dynamically correct the error. Tailoring these sequences typically requires numerically solving a set of nonlinear equations. Here we demonstrate that these pulse sequences can be generated by a well-trained, double-layer neural network. For sequences designed for the noise-free case, the trained neural network is capable of producing almost exactly the same pulses known in the literature. For more complicated noise-correcting sequences, the neural network produces pulses with slightly different line shapes, but the robustness against noises remains comparable. These results indicate that the neural network can be a judicious and powerful alternative to existing techniques in developing pulse sequences for universal fault-tolerant quantum computation.

Elimination sequence optimization for SPAR

NASA Technical Reports Server (NTRS)

Hogan, Harry A.

1986-01-01

SPAR is a large-scale computer program for finite element structural analysis. The program allows user specification of the order in which the joints of a structure are to be eliminated since this order can have significant influence over solution performance, in terms of both storage requirements and computer time. An efficient elimination sequence can improve performance by over 50% for some problems. Obtaining such sequences, however, requires the expertise of an experienced user and can take hours of tedious effort to affect. Thus, an automatic elimination sequence optimizer would enhance productivity by reducing the analysts' problem definition time and by lowering computer costs. Two possible methods for automating the elimination sequence specifications were examined. Several algorithms based on the graph theory representations of sparse matrices were studied with mixed results. Significant improvement in the program performance was achieved, but sequencing by an experienced user still yields substantially better results. The initial results provide encouraging evidence that the potential benefits of such an automatic sequencer would be well worth the effort.

Shotgun Protein Sequencing with Meta-contig Assembly*

PubMed Central

Guthals, Adrian; Clauser, Karl R.; Bandeira, Nuno

2012-01-01

Full-length de novo sequencing from tandem mass (MS/MS) spectra of unknown proteins such as antibodies or proteins from organisms with unsequenced genomes remains a challenging open problem. Conventional algorithms designed to individually sequence each MS/MS spectrum are limited by incomplete peptide fragmentation or low signal to noise ratios and tend to result in short de novo sequences at low sequencing accuracy. Our shotgun protein sequencing (SPS) approach was developed to ameliorate these limitations by first finding groups of unidentified spectra from the same peptides (contigs) and then deriving a consensus de novo sequence for each assembled set of spectra (contig sequences). But whereas SPS enables much more accurate reconstruction of de novo sequences longer than can be recovered from individual MS/MS spectra, it still requires error-tolerant matching to homologous proteins to group smaller contig sequences into full-length protein sequences, thus limiting its effectiveness on sequences from poorly annotated proteins. Using low and high resolution CID and high resolution HCD MS/MS spectra, we address this limitation with a Meta-SPS algorithm designed to overlap and further assemble SPS contigs into Meta-SPS de novo contig sequences extending as long as 100 amino acids at over 97% accuracy without requiring any knowledge of homologous protein sequences. We demonstrate Meta-SPS using distinct MS/MS data sets obtained with separate enzymatic digestions and discuss how the remaining de novo sequencing limitations relate to MS/MS acquisition settings. PMID:22798278

Shotgun protein sequencing with meta-contig assembly.

PubMed

Guthals, Adrian; Clauser, Karl R; Bandeira, Nuno

2012-10-01

Full-length de novo sequencing from tandem mass (MS/MS) spectra of unknown proteins such as antibodies or proteins from organisms with unsequenced genomes remains a challenging open problem. Conventional algorithms designed to individually sequence each MS/MS spectrum are limited by incomplete peptide fragmentation or low signal to noise ratios and tend to result in short de novo sequences at low sequencing accuracy. Our shotgun protein sequencing (SPS) approach was developed to ameliorate these limitations by first finding groups of unidentified spectra from the same peptides (contigs) and then deriving a consensus de novo sequence for each assembled set of spectra (contig sequences). But whereas SPS enables much more accurate reconstruction of de novo sequences longer than can be recovered from individual MS/MS spectra, it still requires error-tolerant matching to homologous proteins to group smaller contig sequences into full-length protein sequences, thus limiting its effectiveness on sequences from poorly annotated proteins. Using low and high resolution CID and high resolution HCD MS/MS spectra, we address this limitation with a Meta-SPS algorithm designed to overlap and further assemble SPS contigs into Meta-SPS de novo contig sequences extending as long as 100 amino acids at over 97% accuracy without requiring any knowledge of homologous protein sequences. We demonstrate Meta-SPS using distinct MS/MS data sets obtained with separate enzymatic digestions and discuss how the remaining de novo sequencing limitations relate to MS/MS acquisition settings.

Rapid phylogenetic dissection of prokaryotic community structure in tidal flat using pyrosequencing.

PubMed

Kim, Bong-Soo; Kim, Byung Kwon; Lee, Jae-Hak; Kim, Myungjin; Lim, Young Woon; Chun, Jongsik

2008-08-01

Dissection of prokaryotic community structure is prerequisite to understand their ecological roles. Various methods are available for such a purpose which amplification and sequencing of 16S rRNA genes gained its popularity. However, conventional methods based on Sanger sequencing technique require cloning process prior to sequencing, and are expensive and labor-intensive. We investigated prokaryotic community structure in tidal flat sediments, Korea, using pyrosequencing and a subsequent automated bioinformatic pipeline for the rapid and accurate taxonomic assignment of each amplicon. The combination of pyrosequencing and bioinformatic analysis showed that bacterial and archaeal communities were more diverse than previously reported in clone library studies. Pyrosequencing analysis revealed 21 bacterial divisions and 37 candidate divisions. Proteobacteria was the most abundant division in the bacterial community, of which Gamma-and Delta-Proteobacteria were the most abundant. Similarly, 4 archaeal divisions were found in tidal flat sediments. Euryarchaeota was the most abundant division in the archaeal sequences, which were further divided into 8 classes and 11 unclassified euryarchaeota groups. The system developed here provides a simple, in-depth and automated way of dissecting a prokaryotic community structure without extensive pretreatment such as cloning.

Sequence change in the HS2-LCR and Ggamma-globin gene promoter region of sickle cell anemia patients.

PubMed

Adorno, E V; Moura-Neto, J P; Lyra, I; Zanette, A; Santos, L F O; Seixas, M O; Reis, M G; Goncalves, M S

2008-02-01

The fetal hemoglobin (HbF) levels and betaS-globin gene haplotypes of 125 sickle cell anemia patients from Brazil were investigated. We sequenced the Ggamma- and Agamma-globin gene promoters and the DNase I-2 hypersensitive sites in the locus control regions (HS2-LCR) of patients with HbF level disparities as compared to their betaS haplotypes. Sixty-four (51.2%) patients had CAR/Ben genotype; 36 (28.8%) Ben/Ben; 18 (14.4%) CAR/CAR; 2 (1.6%) CAR/Atypical; 2 (1.6%) Ben/Cam; 1 (0.8%) CAR/Cam; 1 (0.8%) CAR/Arab-Indian, and 1 (0.8%) Sen/Atypical. The HS2-LCR sequence analyses demonstrated a c.-10.677G>A change in patients with the Ben haplotype and high HbF levels. The Gg gene promoter sequence analyses showed a c.-157T>C substitution shared by all patients, and a c.-222_-225del related to the Cam haplotype. These results identify new polymorphisms in the HS2-LCR and Gg-globin gene promoter. Further studies are required to determine the correlation between HbF synthesis and the clinical profile of sickle cell anemia patients.

Application of the MIDAS approach for analysis of lysine acetylation sites.

PubMed

Evans, Caroline A; Griffiths, John R; Unwin, Richard D; Whetton, Anthony D; Corfe, Bernard M

2013-01-01

Multiple Reaction Monitoring Initiated Detection and Sequencing (MIDAS™) is a mass spectrometry-based technique for the detection and characterization of specific post-translational modifications (Unwin et al. 4:1134-1144, 2005), for example acetylated lysine residues (Griffiths et al. 18:1423-1428, 2007). The MIDAS™ technique has application for discovery and analysis of acetylation sites. It is a hypothesis-driven approach that requires a priori knowledge of the primary sequence of the target protein and a proteolytic digest of this protein. MIDAS essentially performs a targeted search for the presence of modified, for example acetylated, peptides. The detection is based on the combination of the predicted molecular weight (measured as mass-charge ratio) of the acetylated proteolytic peptide and a diagnostic fragment (product ion of m/z 126.1), which is generated by specific fragmentation of acetylated peptides during collision induced dissociation performed in tandem mass spectrometry (MS) analysis. Sequence information is subsequently obtained which enables acetylation site assignment. The technique of MIDAS was later trademarked by ABSciex for targeted protein analysis where an MRM scan is combined with full MS/MS product ion scan to enable sequence confirmation.

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

Rahayu, Suparni Setyowati, E-mail: suparnirahayu@yahoo.co.id; Department of Mechanical Engineering, State Polytechnic of Semarang, Semarang Indonesia; Purwanto,, E-mail: p.purwanto@che.undip.ac.id

The small industry of tofu production process releases the waste water without being processed first, and the wastewater is directly discharged into water. In this study, Anaerobic Sequencing Batch Reactor in Pilot Scale for Treatment of Tofu Industry was developed through an anaerobic process to produce biogas as one kind of environmentally friendly renewable energy which can be developed into the countryside. The purpose of this study was to examine the fundamental characteristics of organic matter elimination of industrial wastewater with small tofu effective method and utilize anaerobic active sludge with Anaerobic Sequencing Bath Reactor (ASBR) to get rural biogasmore » as an energy source. The first factor is the amount of the active sludge concentration which functions as the decomposers of organic matter and controlling selectivity allowance to degrade organic matter. The second factor is that HRT is the average period required substrate to react with the bacteria in the Anaerobic Sequencing Bath Reactor (ASBR).The results of processing the waste of tofu production industry using ASBR reactor with active sludge additions as starter generates cumulative volume of 5814.4 mL at HRT 5 days so that in this study it is obtained the conversion 0.16 L of CH{sub 4}/g COD and produce biogas containing of CH{sub 4}: 81.23% and CO{sub 2}: 16.12%. The wastewater treatment of tofu production using ASBR reactor is able to produce renewable energy that has economic value as well as environmentally friendly by nature.« less

Comparative analysis of seven viral nuclear export signals (NESs) reveals the crucial role of nuclear export mediated by the third NES consensus sequence of nucleoprotein (NP) in influenza A virus replication.

PubMed

Chutiwitoonchai, Nopporn; Kakisaka, Michinori; Yamada, Kazunori; Aida, Yoko

2014-01-01

The assembly of influenza virus progeny virions requires machinery that exports viral genomic ribonucleoproteins from the cell nucleus. Currently, seven nuclear export signal (NES) consensus sequences have been identified in different viral proteins, including NS1, NS2, M1, and NP. The present study examined the roles of viral NES consensus sequences and their significance in terms of viral replication and nuclear export. Mutation of the NP-NES3 consensus sequence resulted in a failure to rescue viruses using a reverse genetics approach, whereas mutation of the NS2-NES1 and NS2-NES2 sequences led to a strong reduction in viral replication kinetics compared with the wild-type sequence. While the viral replication kinetics for other NES mutant viruses were also lower than those of the wild-type, the difference was not so marked. Immunofluorescence analysis after transient expression of NP-NES3, NS2-NES1, or NS2-NES2 proteins in host cells showed that they accumulated in the cell nucleus. These results suggest that the NP-NES3 consensus sequence is mostly required for viral replication. Therefore, each of the hydrophobic (Φ) residues within this NES consensus sequence (Φ1, Φ2, Φ3, or Φ4) was mutated, and its viral replication and nuclear export function were analyzed. No viruses harboring NP-NES3 Φ2 or Φ3 mutants could be rescued. Consistent with this, the NP-NES3 Φ2 and Φ3 mutants showed reduced binding affinity with CRM1 in a pull-down assay, and both accumulated in the cell nucleus. Indeed, a nuclear export assay revealed that these mutant proteins showed lower nuclear export activity than the wild-type protein. Moreover, the Φ2 and Φ3 residues (along with other Φ residues) within the NP-NES3 consensus were highly conserved among different influenza A viruses, including human, avian, and swine. Taken together, these results suggest that the Φ2 and Φ3 residues within the NP-NES3 protein are important for its nuclear export function during viral replication.

UFO: a web server for ultra-fast functional profiling of whole genome protein sequences.

PubMed

Meinicke, Peter

2009-09-02

Functional profiling is a key technique to characterize and compare the functional potential of entire genomes. The estimation of profiles according to an assignment of sequences to functional categories is a computationally expensive task because it requires the comparison of all protein sequences from a genome with a usually large database of annotated sequences or sequence families. Based on machine learning techniques for Pfam domain detection, the UFO web server for ultra-fast functional profiling allows researchers to process large protein sequence collections instantaneously. Besides the frequencies of Pfam and GO categories, the user also obtains the sequence specific assignments to Pfam domain families. In addition, a comparison with existing genomes provides dissimilarity scores with respect to 821 reference proteomes. Considering the underlying UFO domain detection, the results on 206 test genomes indicate a high sensitivity of the approach. In comparison with current state-of-the-art HMMs, the runtime measurements show a considerable speed up in the range of four orders of magnitude. For an average size prokaryotic genome, the computation of a functional profile together with its comparison typically requires about 10 seconds of processing time. For the first time the UFO web server makes it possible to get a quick overview on the functional inventory of newly sequenced organisms. The genome scale comparison with a large number of precomputed profiles allows a first guess about functionally related organisms. The service is freely available and does not require user registration or specification of a valid email address.

Hot Spots of Recombination in Fission Yeast: Inactivation of the M26 Hot Spot by Deletion of the Ade6 Promoter and the Novel Hotspot Ura4-Aim

PubMed Central

Zahn-Zabal, M.; Lehmann, E.; Kohli, J.

1995-01-01

The M26 mutation in the ade6 gene of Schizosaccharomyces pombe creates a hot spot of meiotic recombination. A single base substitution, the M26 mutation is situated within the open reading frame, near the 5' end. It has previously been shown that the heptanucleotide sequence 5' ATGACGT 3', which includes the M26 mutation, is required for hot spot activity. The 510-bp ade6-delXB deletion encompasses the promoter and the first 23 bp of the open reading frame, ending 112 bp upstream of M26. Deletion of the promoter in cis to M26 abolishes hot spot activity, while deletion in trans to M26 has no effect. Homozygous deletion of the promoter also eliminates M26 hot spot activity, indicating that the heterology created through deletion of the promoter per se is not responsible for the loss of hot spot activity. Thus, DNA sequences other than the heptanucleotide 5' ATGACGT 3', which must be located at the 5' end of the ade6 gene, appear to be required for hot spot activity. While the M26 hotspot stimulates crossovers associated with M26 conversion, it does not affect the crossover frequency in the intervals adjacent to ade6. The flanking marker ura4-aim, a heterology created by insertion of the ura4(+) gene upstream of ade6, turned out to be a hot spot itself. It shows disparity of conversion with preferential loss of the insertion. The frequency of conversion at ura4-aim is reduced when the M26 hot spot is active 15 kb away, indicating competition for recombination factors by hot spots in close proximity. PMID:7498729

Science Opportunity Analyzer (SOA): Not Just Another Pretty Face

NASA Technical Reports Server (NTRS)

Polanskey, Carol A.; Streiiffert, Barbara; O'Reilly, Taifun

2004-01-01

This viewgraph presentation reviews the Science Opportunity Analyzer (SOA). For the first time at JPL, the Cassini mission to Saturn is using distributed science operations for sequence generation. This means that scientist at other institutions has more responsibility to build the spacecraft sequence. Tools are required to support the sequence development. JPL tools required a complete configuration behind a firewall, and the tools that the user community had developed did not interface with the JPL tools. Therefore the SOA was created to bridge the gap between the remote scientists and the JPL operations teams. The presentation reviews the development of the SOA, and what was required of the system. The presentation reviews the functions that the SOA performed.

The Complete Sequence of a Human Parainfluenzavirus 4 Genome

PubMed Central

Yea, Carmen; Cheung, Rose; Collins, Carol; Adachi, Dena; Nishikawa, John; Tellier, Raymond

2009-01-01

Although the human parainfluenza virus 4 (HPIV4) has been known for a long time, its genome, alone among the human paramyxoviruses, has not been completely sequenced to date. In this study we obtained the first complete genomic sequence of HPIV4 from a clinical isolate named SKPIV4 obtained at the Hospital for Sick Children in Toronto (Ontario, Canada). The coding regions for the N, P/V, M, F and HN proteins show very high identities (95% to 97%) with previously available partial sequences for HPIV4B. The sequence for the L protein and the non-coding regions represent new information. A surprising feature of the genome is its length, more than 17 kb, making it the longest genome within the genus Rubulavirus, although the length is well within the known range of 15 kb to 19 kb for the subfamily Paramyxovirinae. The availability of a complete genomic sequence will facilitate investigations on a respiratory virus that is still not completely characterized. PMID:21994536

A Sequence-Independent, Unstructured Internal Ribosome Entry Site Is Responsible for Internal Expression of the Coat Protein of Turnip Crinkle Virus

PubMed Central

May, Jared; Johnson, Philip; Saleem, Huma

2017-01-01

ABSTRACT To maximize the coding potential of viral genomes, internal ribosome entry sites (IRES) can be used to bypass the traditional requirement of a 5′ cap and some/all of the associated translation initiation factors. Although viral IRES typically contain higher-order RNA structure, an unstructured sequence of about 84 nucleotides (nt) immediately upstream of the Turnip crinkle virus (TCV) coat protein (CP) open reading frame (ORF) has been found to promote internal expression of the CP from the genomic RNA (gRNA) both in vitro and in vivo. An absence of extensive RNA structure was predicted using RNA folding algorithms and confirmed by selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) RNA structure probing. Analysis of the IRES region in vitro by use of both the TCV gRNA and reporter constructs did not reveal any sequence-specific elements but rather suggested that an overall lack of structure was an important feature for IRES activity. The CP IRES is A-rich, independent of orientation, and strongly conserved among viruses in the same genus. The IRES was dependent on eIF4G, but not eIF4E, for activity. Low levels of CP accumulated in vivo in the absence of detectable TCV subgenomic RNAs, strongly suggesting that the IRES was active in the gRNA in vivo. Since the TCV CP also serves as the viral silencing suppressor, early translation of the CP from the viral gRNA is likely important for countering host defenses. Cellular mRNA IRES also lack extensive RNA structures or sequence conservation, suggesting that this viral IRES and cellular IRES may have similar strategies for internal translation initiation. IMPORTANCE Cap-independent translation is a common strategy among positive-sense, single-stranded RNA viruses for bypassing the host cell requirement of a 5′ cap structure. Viral IRES, in general, contain extensive secondary structure that is critical for activity. In contrast, we demonstrate that a region of viral RNA devoid of extensive secondary structure has IRES activity and produces low levels of viral coat protein in vitro and in vivo. Our findings may be applicable to cellular mRNA IRES that also have little or no sequences/structures in common. PMID:28179526

Activation of RNA polymerase III transcription of human Alu repetitive elements by adenovirus type 5: Requirement for the E1b 58-Kilodalton protein and the products of E4 open reading frames 3 and 6

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

Panning, B.; Smiley, J.R.

1993-06-01

Alu elements are the single most abundant class of dispersed repeated sequences in the human genome, comprising 5-10% of the mass of human DNA. This report demonstrates that Ad5 infection strongly stimulates Pol III transcription of human Alu elements in HeLa and 293 cells. In contrast to the cases of Ad5-induced Pol III transcriptional activation, this process requires the E1b 58-kDa protein and the products of E4 open reading frames (ORFs) 3 and 6 in addition to the E1a 289-residue product. These findings suggest novel regulatory properties of the Ad5 E1b and E4 proteins and raise the possibility that analogousmore » cellular trans-acting factors serve to modulate Alu expression in vivo.« less

"The devil's in the detail": Release of an expanded, enhanced and dynamically revised forensic STR Sequence Guide.

PubMed

Phillips, C; Gettings, K Butler; King, J L; Ballard, D; Bodner, M; Borsuk, L; Parson, W

2018-05-01

The STR sequence template file published in 2016 as part of the considerations from the DNA Commission of the International Society for Forensic Genetics on minimal STR sequence nomenclature requirements, has been comprehensively revised and audited using the latest GRCh38 genome assembly. The list of forensic STRs characterized was expanded by including supplementary autosomal, X- and Y-chromosome microsatellites in less common use for routine DNA profiling, but some likely to be adopted in future massively parallel sequencing (MPS) STR panels. We outline several aspects of sequence alignment and annotation that required care and attention to detail when comparing sequences to GRCh37 and GRCh38 assemblies, as well as the necessary matching of MPS-based allele descriptions to previously established repeat region structures described in initial sequencing studies of the less well known forensic STRs. The revised sequence guide is now available in a dynamically updated FTP format from the STRidER website with a date-stamped change log to allow users to explore their own MPS data with the most up-to-date forensic STR sequence information compiled in a simple guide. Copyright © 2018 Elsevier B.V. All rights reserved.

Rational design of DNA sequences for nanotechnology, microarrays and molecular computers using Eulerian graphs.

PubMed

Pancoska, Petr; Moravek, Zdenek; Moll, Ute M

2004-01-01

Nucleic acids are molecules of choice for both established and emerging nanoscale technologies. These technologies benefit from large functional densities of 'DNA processing elements' that can be readily manufactured. To achieve the desired functionality, polynucleotide sequences are currently designed by a process that involves tedious and laborious filtering of potential candidates against a series of requirements and parameters. Here, we present a complete novel methodology for the rapid rational design of large sets of DNA sequences. This method allows for the direct implementation of very complex and detailed requirements for the generated sequences, thus avoiding 'brute force' filtering. At the same time, these sequences have narrow distributions of melting temperatures. The molecular part of the design process can be done without computer assistance, using an efficient 'human engineering' approach by drawing a single blueprint graph that represents all generated sequences. Moreover, the method eliminates the necessity for extensive thermodynamic calculations. Melting temperature can be calculated only once (or not at all). In addition, the isostability of the sequences is independent of the selection of a particular set of thermodynamic parameters. Applications are presented for DNA sequence designs for microarrays, universal microarray zip sequences and electron transfer experiments.

Analysis of Pre-Analytic Factors Affecting the Success of Clinical Next-Generation Sequencing of Solid Organ Malignancies.

PubMed

Chen, Hui; Luthra, Rajyalakshmi; Goswami, Rashmi S; Singh, Rajesh R; Roy-Chowdhuri, Sinchita

2015-08-28

Application of next-generation sequencing (NGS) technology to routine clinical practice has enabled characterization of personalized cancer genomes to identify patients likely to have a response to targeted therapy. The proper selection of tumor sample for downstream NGS based mutational analysis is critical to generate accurate results and to guide therapeutic intervention. However, multiple pre-analytic factors come into play in determining the success of NGS testing. In this review, we discuss pre-analytic requirements for AmpliSeq PCR-based sequencing using Ion Torrent Personal Genome Machine (PGM) (Life Technologies), a NGS sequencing platform that is often used by clinical laboratories for sequencing solid tumors because of its low input DNA requirement from formalin fixed and paraffin embedded tissue. The success of NGS mutational analysis is affected not only by the input DNA quantity but also by several other factors, including the specimen type, the DNA quality, and the tumor cellularity. Here, we review tissue requirements for solid tumor NGS based mutational analysis, including procedure types, tissue types, tumor volume and fraction, decalcification, and treatment effects.

A rapid and cost-effective method for sequencing pooled cDNA clones by using a combination of transposon insertion and Gateway technology.

PubMed

Morozumi, Takeya; Toki, Daisuke; Eguchi-Ogawa, Tomoko; Uenishi, Hirohide

2011-09-01

Large-scale cDNA-sequencing projects require an efficient strategy for mass sequencing. Here we describe a method for sequencing pooled cDNA clones using a combination of transposon insertion and Gateway technology. Our method reduces the number of shotgun clones that are unsuitable for reconstruction of cDNA sequences, and has the advantage of reducing the total costs of the sequencing project.

A novel, privacy-preserving cryptographic approach for sharing sequencing data

PubMed Central

Cassa, Christopher A; Miller, Rachel A; Mandl, Kenneth D

2013-01-01

Objective DNA samples are often processed and sequenced in facilities external to the point of collection. These samples are routinely labeled with patient identifiers or pseudonyms, allowing for potential linkage to identity and private clinical information if intercepted during transmission. We present a cryptographic scheme to securely transmit externally generated sequence data which does not require any patient identifiers, public key infrastructure, or the transmission of passwords. Materials and methods This novel encryption scheme cryptographically protects participant sequence data using a shared secret key that is derived from a unique subset of an individual’s genetic sequence. This scheme requires access to a subset of an individual’s genetic sequence to acquire full access to the transmitted sequence data, which helps to prevent sample mismatch. Results We validate that the proposed encryption scheme is robust to sequencing errors, population uniqueness, and sibling disambiguation, and provides sufficient cryptographic key space. Discussion Access to a set of an individual’s genotypes and a mutually agreed cryptographic seed is needed to unlock the full sequence, which provides additional sample authentication and authorization security. We present modest fixed and marginal costs to implement this transmission architecture. Conclusions It is possible for genomics researchers who sequence participant samples externally to protect the transmission of sequence data using unique features of an individual’s genetic sequence. PMID:23125421

In-space propellant logistics. Volume 4: Project planning data

NASA Technical Reports Server (NTRS)

1972-01-01

The prephase A conceptual project planning data as it pertains to the development of the selected logistics module configuration transported into earth orbit by the space shuttle orbiter. The data represents the test, implementation, and supporting research and technology requirements for attaining the propellant transfer operational capability for early 1985. The plan is based on a propellant module designed to support the space-based tug with cryogenic oxygen-hydrogen propellants. A logical sequence of activities that is required to define, design, develop, fabricate, test, launch, and flight test the propellant logistics module is described. Included are the facility and ground support equipment requirements. The schedule of activities are based on the evolution and relationship between the R and T, the development issues, and the resultant test program.

GIGYF1/2 proteins use auxiliary sequences to selectively bind to 4EHP and repress target mRNA expression

PubMed Central

Peter, Daniel; Weber, Ramona; Sandmeir, Felix; Wohlbold, Lara; Helms, Sigrun; Bawankar, Praveen; Valkov, Eugene; Igreja, Cátia; Izaurralde, Elisa

2017-01-01

The eIF4E homologous protein (4EHP) is thought to repress translation by competing with eIF4E for binding to the 5′ cap structure of specific mRNAs to which it is recruited through interactions with various proteins, including the GRB10-interacting GYF (glycine–tyrosine–phenylalanine domain) proteins 1 and 2 (GIGYF1/2). Despite its similarity to eIF4E, 4EHP does not interact with eIF4G and therefore fails to initiate translation. In contrast to eIF4G, GIGYF1/2 bind selectively to 4EHP but not eIF4E. Here, we present crystal structures of the 4EHP-binding regions of GIGYF1 and GIGYF2 in complex with 4EHP, which reveal the molecular basis for the selectivity of the GIGYF1/2 proteins for 4EHP. Complementation assays in a GIGYF1/2-null cell line using structure-based mutants indicate that 4EHP requires interactions with GIGYF1/2 to down-regulate target mRNA expression. Our studies provide structural insights into the assembly of 4EHP–GIGYF1/2 repressor complexes and reveal that rather than merely facilitating 4EHP recruitment to transcripts, GIGYF1/2 proteins are required for repressive activity. PMID:28698298

Complete genome sequence analysis of novel human bocavirus reveals genetic recombination between human bocavirus 2 and human bocavirus 4.

PubMed

Khamrin, Pattara; Okitsu, Shoko; Ushijima, Hiroshi; Maneekarn, Niwat

2013-07-01

Epidemiological surveillance of human bocavirus (HBoV) was conducted on fecal specimens collected from hospitalized children with diarrhea in Chiang Mai, Thailand in 2011. By partial sequence analysis of VP1 gene, an unusual strain of HBoV (CMH-S011-11), was initially identified as HBoV4. The complete genome sequence of CMH-S011-11 was performed and analyzed further to clarify whether it was a recombinant strain or a new HBoV variant. Analysis of complete genome sequence revealed that the coding sequence starting from NS1, NP1 to VP1/VP2 was 4795 nucleotides long. Interestingly, the nucleotide sequence of NS1 gene of CMH-S011-11 was most closely related to the HBoV2 reference strains detected in Pakistan, which contradicted to the initial genotyping result of the partial VP1 region in the previous study. In addition, comparison of NP1 nucleotide sequence of CMH-S011-11 with those of other HBoV1-4 reference strains also revealed a high level of sequence identity with HBoV2. On the other hand, nucleotide sequence of VP1/VP2 gene of CMH-S011-11 was most closely related to those of HBoV4 reference strains detected in Nigeria. The overall full-length sequence analysis revealed that this CMH-S011-11 was grouped within HBoV4 species, but located in a separate branch from other HBoV4 prototype strains. Recombination analysis revealed that CMH-S011-11 was the result of recombination between HBoV2 and HBoV4 strains with the break point located near the start codon of VP2. Copyright © 2013 Elsevier B.V. All rights reserved.

Probing DNA in nanopores via tunneling: from sequencing to ``quantum'' analogies

NASA Astrophysics Data System (ADS)

di Ventra, Massimiliano

2012-02-01

Fast and low-cost DNA sequencing methods would revolutionize medicine: a person could have his/her full genome sequenced so that drugs could be tailored to his/her specific illnesses; doctors could know in advance patients' likelihood to develop a given ailment; cures to major diseases could be found faster [1]. However, this goal of ``personalized medicine'' is hampered today by the high cost and slow speed of DNA sequencing methods. In this talk, I will discuss the sequencing protocol we suggest which requires the measurement of the distributions of transverse currents during the translocation of single-stranded DNA into nanopores [2-5]. I will support our conclusions with a combination of molecular dynamics simulations coupled to quantum mechanical calculations of electrical current in experimentally realizable systems [2-5]. I will also discuss recent experiments that support these theoretical predictions. In addition, I will show how this relatively unexplored area of research at the interface between solids, liquids, and biomolecules at the nanometer length scale is a fertile ground to study quantum phenomena that have a classical counterpart, such as ionic quasi-particles, ionic ``quantized'' conductance [6,7] and Coulomb blockade [8]. Work supported in part by NIH. [4pt] [1] M. Zwolak, M. Di Ventra, Physical Approaches to DNA Sequencing and Detection, Rev. Mod. Phys. 80, 141 (2008).[0pt] [2] M. Zwolak and M. Di Ventra, Electronic signature of DNA nucleotides via transverse transport, Nano Lett. 5, 421 (2005).[0pt] [3] J. Lagerqvist, M. Zwolak, and M. Di Ventra, Fast DNA sequencing via transverse electronic transport, Nano Lett. 6, 779 (2006).[0pt] [4] J. Lagerqvist, M. Zwolak, and M. Di Ventra, Influence of the environment and probes on rapid DNA sequencing via transverse electronic transport, Biophys. J. 93, 2384 (2007).[0pt] [5] M. Krems, M. Zwolak, Y.V. Pershin, and M. Di Ventra, Effect of noise on DNA sequencing via transverse electronic transport, Biophys. J. 97, 1990, (2009).[0pt] [6] M. Zwolak, J. Lagerqvist, and M. Di Ventra, Ionic conductance quantization in nanopores, Phys. Rev.Lett. 103, 128102 (2009).[0pt] [7] M. Zwolak, J. Wilson, and M. Di Ventra, Dehydration and ionic conductance quantization in nanopores, J. Phys. Cond. Matt. 22 454126 (2011). [0pt] [8] M. Krems and M. Di Ventra, Ionic Coulomb blockade in nanopores arXiv:1103.2749.

An ethylene-responsive enhancer element is involved in the senescence-related expression of the carnation glutathione-S-transferase (GST1) gene.

PubMed

Itzhaki, H; Maxson, J M; Woodson, W R

1994-09-13

The increased production of ethylene during carnation petal senescence regulates the transcription of the GST1 gene encoding a subunit of glutathione-S-transferase. We have investigated the molecular basis for this ethylene-responsive transcription by examining the cis elements and trans-acting factors involved in the expression of the GST1 gene. Transient expression assays following delivery of GST1 5' flanking DNA fused to a beta-glucuronidase receptor gene were used to functionally define sequences responsible for ethylene-responsive expression. Deletion analysis of the 5' flanking sequences of GST1 identified a single positive regulatory element of 197 bp between -667 and -470 necessary for ethylene-responsive expression. The sequences within this ethylene-responsive region were further localized to 126 bp between -596 and -470. The ethylene-responsive element (ERE) within this region conferred ethylene-regulated expression upon a minimal cauliflower mosaic virus-35S TATA-box promoter in an orientation-independent manner. Gel electrophoresis mobility-shift assays and DNase I footprinting were used to identify proteins that bind to sequences within the ERE. Nuclear proteins from carnation petals were shown to specifically interact with the 126-bp ERE and the presence and binding of these proteins were independent of ethylene or petal senescence. DNase I footprinting defined DNA sequences between -510 and -488 within the ERE specifically protected by bound protein. An 8-bp sequence (ATTTCAAA) within the protected region shares significant homology with promoter sequences required for ethylene responsiveness from the tomato fruit-ripening E4 gene.

A compact, in vivo screen of all 6-mers reveals drivers of tissue-specific expression and guides synthetic regulatory element design.

PubMed

Smith, Robin P; Riesenfeld, Samantha J; Holloway, Alisha K; Li, Qiang; Murphy, Karl K; Feliciano, Natalie M; Orecchia, Lorenzo; Oksenberg, Nir; Pollard, Katherine S; Ahituv, Nadav

2013-07-18

Large-scale annotation efforts have improved our ability to coarsely predict regulatory elements throughout vertebrate genomes. However, it is unclear how complex spatiotemporal patterns of gene expression driven by these elements emerge from the activity of short, transcription factor binding sequences. We describe a comprehensive promoter extension assay in which the regulatory potential of all 6 base-pair (bp) sequences was tested in the context of a minimal promoter. To enable this large-scale screen, we developed algorithms that use a reverse-complement aware decomposition of the de Bruijn graph to design a library of DNA oligomers incorporating every 6-bp sequence exactly once. Our library multiplexes all 4,096 unique 6-mers into 184 double-stranded 15-bp oligomers, which is sufficiently compact for in vivo testing. We injected each multiplexed construct into zebrafish embryos and scored GFP expression in 15 tissues at two developmental time points. Twenty-seven constructs produced consistent expression patterns, with the majority doing so in only one tissue. Functional sequences are enriched near biologically relevant genes, match motifs for developmental transcription factors, and are required for enhancer activity. By concatenating tissue-specific functional sequences, we generated completely synthetic enhancers for the notochord, epidermis, spinal cord, forebrain and otic lateral line, and show that short regulatory sequences do not always function modularly. This work introduces a unique in vivo catalog of short, functional regulatory sequences and demonstrates several important principles of regulatory element organization. Furthermore, we provide resources for designing compact, reverse-complement aware k-mer libraries.

The transition to increased automaticity during finger sequence learning in adult males who stutter.

PubMed

Smits-Bandstra, Sarah; De Nil, Luc; Rochon, Elizabeth

2006-01-01

The present study compared the automaticity levels of persons who stutter (PWS) and persons who do not stutter (PNS) on a practiced finger sequencing task under dual task conditions. Automaticity was defined as the amount of attention required for task performance. Twelve PWS and 12 control subjects practiced finger tapping sequences under single and then dual task conditions. Control subjects performed the sequencing task significantly faster and less variably under single versus dual task conditions while PWS' performance was consistently slow and variable (comparable to the dual task performance of control subjects) under both conditions. Control subjects were significantly more accurate on a colour recognition distracter task than PWS under dual task conditions. These results suggested that control subjects transitioned to quick, accurate and increasingly automatic performance on the sequencing task after practice, while PWS did not. Because most stuttering treatment programs for adults include practice and automatization of new motor speech skills, findings of this finger sequencing study and future studies of speech sequence learning may have important implications for how to maximize stuttering treatment effectiveness. As a result of this activity, the participant will be able to: (1) Define automaticity and explain the importance of dual task paradigms to investigate automaticity; (2) Relate the proposed relationship between motor learning and automaticity as stated by the authors; (3) Summarize the reviewed literature concerning the performance of PWS on dual tasks; and (4) Explain why the ability to transition to automaticity during motor learning may have important clinical implications for stuttering treatment effectiveness.

Highly Precise and Developmentally Programmed Genome Assembly in Paramecium Requires Ligase IV–Dependent End Joining

PubMed Central

Marmignon, Antoine; Ku, Michael; Silve, Aude; Meyer, Eric; Forney, James D.; Malinsky, Sophie; Bétermier, Mireille

2011-01-01

During the sexual cycle of the ciliate Paramecium, assembly of the somatic genome includes the precise excision of tens of thousands of short, non-coding germline sequences (Internal Eliminated Sequences or IESs), each one flanked by two TA dinucleotides. It has been reported previously that these genome rearrangements are initiated by the introduction of developmentally programmed DNA double-strand breaks (DSBs), which depend on the domesticated transposase PiggyMac. These DSBs all exhibit a characteristic geometry, with 4-base 5′ overhangs centered on the conserved TA, and may readily align and undergo ligation with minimal processing. However, the molecular steps and actors involved in the final and precise assembly of somatic genes have remained unknown. We demonstrate here that Ligase IV and Xrcc4p, core components of the non-homologous end-joining pathway (NHEJ), are required both for the repair of IES excision sites and for the circularization of excised IESs. The transcription of LIG4 and XRCC4 is induced early during the sexual cycle and a Lig4p-GFP fusion protein accumulates in the developing somatic nucleus by the time IES excision takes place. RNAi–mediated silencing of either gene results in the persistence of free broken DNA ends, apparently protected against extensive resection. At the nucleotide level, controlled removal of the 5′-terminal nucleotide occurs normally in LIG4-silenced cells, while nucleotide addition to the 3′ ends of the breaks is blocked, together with the final joining step, indicative of a coupling between NHEJ polymerase and ligase activities. Taken together, our data indicate that IES excision is a “cut-and-close” mechanism, which involves the introduction of initiating double-strand cleavages at both ends of each IES, followed by DSB repair via highly precise end joining. This work broadens our current view on how the cellular NHEJ pathway has cooperated with domesticated transposases for the emergence of new mechanisms involved in genome dynamics. PMID:21533177

Highly precise and developmentally programmed genome assembly in Paramecium requires ligase IV-dependent end joining.

PubMed

Kapusta, Aurélie; Matsuda, Atsushi; Marmignon, Antoine; Ku, Michael; Silve, Aude; Meyer, Eric; Forney, James D; Malinsky, Sophie; Bétermier, Mireille

2011-04-01

During the sexual cycle of the ciliate Paramecium, assembly of the somatic genome includes the precise excision of tens of thousands of short, non-coding germline sequences (Internal Eliminated Sequences or IESs), each one flanked by two TA dinucleotides. It has been reported previously that these genome rearrangements are initiated by the introduction of developmentally programmed DNA double-strand breaks (DSBs), which depend on the domesticated transposase PiggyMac. These DSBs all exhibit a characteristic geometry, with 4-base 5' overhangs centered on the conserved TA, and may readily align and undergo ligation with minimal processing. However, the molecular steps and actors involved in the final and precise assembly of somatic genes have remained unknown. We demonstrate here that Ligase IV and Xrcc4p, core components of the non-homologous end-joining pathway (NHEJ), are required both for the repair of IES excision sites and for the circularization of excised IESs. The transcription of LIG4 and XRCC4 is induced early during the sexual cycle and a Lig4p-GFP fusion protein accumulates in the developing somatic nucleus by the time IES excision takes place. RNAi-mediated silencing of either gene results in the persistence of free broken DNA ends, apparently protected against extensive resection. At the nucleotide level, controlled removal of the 5'-terminal nucleotide occurs normally in LIG4-silenced cells, while nucleotide addition to the 3' ends of the breaks is blocked, together with the final joining step, indicative of a coupling between NHEJ polymerase and ligase activities. Taken together, our data indicate that IES excision is a "cut-and-close" mechanism, which involves the introduction of initiating double-strand cleavages at both ends of each IES, followed by DSB repair via highly precise end joining. This work broadens our current view on how the cellular NHEJ pathway has cooperated with domesticated transposases for the emergence of new mechanisms involved in genome dynamics.

Structural requirements for recognition of the HLA-Dw14 class II epitope: A key HLA determinant associated with rheumatoid arthritis

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

Hiraiwa, Akikazu; Yamanaka, Katsuo; Kwok, W.W.

Although HLA genes have been shown to be associated with certain diseases, the basis for this association is unknown. Recent studies, however, have documented patterns of nucleotide sequence variation among some HLA genes associated with a particular disease. For rheumatoid arthritis, HLA genes in most patients have a shared nucleotide sequence encoding a key structural element of an HLA class II polypeptide; this sequence element is critical for the interaction of the HLA molecule with antigenic peptides and with responding T cells, suggestive of a direct role for this sequence element in disease susceptibility. The authors describe the serological andmore » cellular immunologic characteristics encoded by this rheumatoid arthritis-associated sequence element. Site-directed mutagenesis of the DRB1 gene was used to define amino acids critical for antibody and T-cell recognition of this structural element, focusing on residues that distinguish the rheumatoid arthritis-associated alleles Dw4 and Dw14 from a closely related allele, Dw10, not associated with disease. Both the gain and loss of rheumatoid arthritis-associated epitopes were highly dependent on three residues within a discrete domain of the HLA-DR molecule. Recognition was most strongly influenced by the following amino acids (in order): 70 > 71 > 67. Some alloreactive T-cell clones were also influenced by amino acid variation in portions of the DR molecule lying outside the shared sequence element.« less

Evolution of Sphingomonad Gene Clusters Related to Pesticide Catabolism Revealed by Genome Sequence and Mobilomics of Sphingobium herbicidovorans MH.

PubMed

Nielsen, Tue Kjærgaard; Rasmussen, Morten; Demanèche, Sandrine; Cecillon, Sébastien; Vogel, Timothy M; Hansen, Lars Hestbjerg

2017-09-01

Bacterial degraders of chlorophenoxy herbicides have been isolated from various ecosystems, including pristine environments. Among these degraders, the sphingomonads constitute a prominent group that displays versatile xenobiotic-degradation capabilities. Four separate sequencing strategies were required to provide the complete sequence of the complex and plastic genome of the canonical chlorophenoxy herbicide-degrading Sphingobium herbicidovorans MH. The genome has an intricate organization of the chlorophenoxy-herbicide catabolic genes sdpA, rdpA, and cadABCD that encode the (R)- and (S)-enantiomer-specific 2,4-dichlorophenoxypropionate dioxygenases and four subunits of a Rieske non-heme iron oxygenase involved in 2-methyl-chlorophenoxyacetic acid degradation, respectively. Several major genomic rearrangements are proposed to help understand the evolution and mobility of these important genes and their genetic context. Single-strain mobilomic sequence analysis uncovered plasmids and insertion sequence-associated circular intermediates in this environmentally important bacterium and enabled the description of evolutionary models for pesticide degradation in strain MH and related organisms. The mobilome presented a complex mosaic of mobile genetic elements including four plasmids and several circular intermediate DNA molecules of insertion-sequence elements and transposons that are central to the evolution of xenobiotics degradation. Furthermore, two individual chromosomally integrated prophages were shown to excise and form free circular DNA molecules. This approach holds great potential for improving the understanding of genome plasticity, evolution, and microbial ecology. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Airway and Feeding Outcomes of Mandibular Distraction, Tongue-Lip Adhesion, and Conservative Management in Pierre Robin Sequence: A Prospective Study.

PubMed

Khansa, Ibrahim; Hall, Courtney; Madhoun, Lauren L; Splaingard, Mark; Baylis, Adriane; Kirschner, Richard E; Pearson, Gregory D

2017-04-01

Pierre Robin sequence is characterized by mandibular retrognathia and glossoptosis resulting in airway obstruction and feeding difficulties. When conservative management fails, mandibular distraction osteogenesis or tongue-lip adhesion may be required to avoid tracheostomy. The authors' goal was to prospectively evaluate the airway and feeding outcomes of their comprehensive approach to Pierre Robin sequence, which includes conservative management, mandibular distraction osteogenesis, and tongue-lip adhesion. A longitudinal study of newborns with Pierre Robin sequence treated at a pediatric academic medical center between 2010 and 2015 was performed. Baseline feeding and respiratory data were collected. Patients underwent conservative management if they demonstrated sustainable weight gain without tube feeds, and if their airway was stable with positioning alone. Patients who required surgery underwent tongue-lip adhesion or mandibular distraction osteogenesis based on family and surgeon preference. Postoperative airway and feeding data were collected. Twenty-eight patients with Pierre Robin sequence were followed prospectively. Thirty-two percent had a syndrome. Ten underwent mandibular distraction osteogenesis, eight underwent tongue-lip adhesion, and 10 were treated conservatively. There were no differences in days to extubation or discharge, change in weight percentile, requirement for gastrostomy tube, or residual obstructive sleep apnea between the three groups. No patients required tracheostomy. The greatest reduction in apnea-hypopnea index occurred with mandibular distraction osteogenesis, followed by tongue-lip adhesion and conservative management. Careful selection of which patients with Pierre Robin sequence need surgery, and of the most appropriate surgical procedure for each patient, can minimize the need for postprocedure tracheostomy. A comprehensive approach to Pierre Robin sequence that includes conservative management, mandibular distraction osteogenesis, and tongue-lip adhesion can result in excellent airway and feeding outcomes. Therapeutic, II.

Affordable hands-on DNA sequencing and genotyping: an exercise for teaching DNA analysis to undergraduates.

PubMed

Shah, Kushani; Thomas, Shelby; Stein, Arnold

2013-01-01

In this report, we describe a 5-week laboratory exercise for undergraduate biology and biochemistry students in which students learn to sequence DNA and to genotype their DNA for selected single nucleotide polymorphisms (SNPs). Students use miniaturized DNA sequencing gels that require approximately 8 min to run. The students perform G, A, T, C Sanger sequencing reactions. They prepare and run the gels, perform Southern blots (which require only 10 min), and detect sequencing ladders using a colorimetric detection system. Students enlarge their sequencing ladders from digital images of their small nylon membranes, and read the sequence manually. They compare their reads with the actual DNA sequence using BLAST2. After mastering the DNA sequencing system, students prepare their own DNA from a cheek swab, polymerase chain reaction-amplify a region of their DNA that encompasses a SNP of interest, and perform sequencing to determine their genotype at the SNP position. A family pedigree can also be constructed. The SNP chosen by the instructor was rs17822931, which is in the ABCC11 gene and is the determinant of human earwax type. Genotypes at the rs178229931 site vary in different ethnic populations. © 2013 by The International Union of Biochemistry and Molecular Biology.

Autogen Version 2.0

NASA Technical Reports Server (NTRS)

Gladden, Roy

2007-01-01

Version 2.0 of the autogen software has been released. "Autogen" (automated sequence generation) signifies both a process and software used to implement the process of automated generation of sequences of commands in a standard format for uplink to spacecraft. Autogen requires fewer workers than are needed for older manual sequence-generation processes and reduces sequence-generation times from weeks to minutes.

Detection of malignant hepatic tumors with ferumoxides-enhanced MRI: comparison of five gradient-recalled echo sequences with different TEs.

PubMed

Matsuo, Masayuki; Kanematsu, Masayuki; Itoh, Kyo; Murakami, Takamichi; Maetani, Yoji; Kondo, Hiroshi; Goshima, Satoshi; Kako, Nobuo; Hoshi, Hiroaki; Konishi, Junji; Moriyama, Noriyuki; Nakamura, Hironobu

2004-01-01

The purpose of our study was to compare the detectability of malignant hepatic tumors on ferumoxides-enhanced MRI using five gradient-recalled echo sequences at different TEs. Ferumoxides-enhanced MRIs obtained in 31 patients with 50 malignant hepatic tumors (33 hepatocellular carcinomas, 17 metastases) were reviewed retrospectively by three independent offsite radiologists. T1-weighted gradient-recalled echo images with TEs of 1.4 and 4.2 msec; T2*-weighted gradient-recalled echo images with TEs of 6, 8, and 10 msec; and T2-weighted fast spin-echo images of livers were randomly reviewed on a segment-by-segment basis. Observer performance was tested using the McNemar test and receiver operating characteristic analysis for the clustered data. Lesion-to-liver contrast-to-noise ratio was also assessed. Mean lesion-to-liver contrast-to-noise ratios were negative and lower with gradient-recalled echo at 1.4 msec than with the other sequences. Sensitivity was higher (p < 0.05) with gradient-recalled echo at 6, 8, and 10 msec and fast spin-echo sequences (75-83%) than with gradient-recalled echo sequences at 1.4 and 4.2 msec (46-48%), and was higher (p < 0.05) with gradient-recalled echo sequence at 8 msec (83%) than with gradient-recalled echo at 6 msec and fast spin-echo sequences (75-78%). Specificity was comparably high with all sequences (95-98%). The area under the receiver operating characteristic curve (A(z)) was greater (p < 0.05) with gradient-recalled echo at 6, 8, and 10 msec and fast spin-echo sequences (A(z) = 0.91-0.93) than with gradient-recalled echo sequences at 1.4 and 4.2 msec (A(z) = 0.82-0.85). In the detection of malignant hepatic tumors, gradient-recalled echo sequences at 8 msec showed the highest sensitivity and had an A(z) value and lesion-to-liver contrast-to-noise ratio comparable with values from gradient-recalled echo sequences at 6 and 10 msec and fast spin-echo sequences.

Inhibition of coxsackievirus B3 replication by small interfering RNAs requires perfect sequence match in the central region of the viral positive strand.

PubMed

Yuan, Ji; Cheung, Paul K M; Zhang, Huifang M; Chau, David; Yang, Decheng

2005-02-01

Coxsackievirus B3 (CVB3) is the most common causal agent of viral myocarditis, but existing drug therapies are of limited value. Application of small interfering RNA (siRNA) in knockdown of gene expression is an emerging technology in antiviral gene therapy. To investigate whether RNA interference (RNAi) can protect against CVB3 infection, we evaluated the effects of RNAi on viral replication in HeLa cells and murine cardiomyocytes by using five CVB3-specific siRNAs targeting distinct regions of the viral genome. The most effective one is siRNA-4, targeting the viral protease 2A, achieving a 92% inhibition of CVB3 replication. The specific RNAi effects could last at least 48 h, and cell viability assay revealed that 90% of siRNA-4-pretreated cells were still alive and lacked detectable viral protein expression 48 h postinfection. Moreover, administration of siRNAs after viral infection could also effectively inhibit viral replication, indicating its therapeutic potential. Further evaluation by combination found that no enhanced inhibitory effects were observed when siRNA-4 was cotransfected with each of the other four candidates. In mutational analysis of the mechanisms of siRNA action, we found that siRNA functions by targeting the positive strand of virus and requires a perfect sequence match in the central region of the target, but mismatches were more tolerated near the 3' end than the 5' end of the antisense strand. These findings reveal an effective target for CVB3 silencing and provide a new possibility for antiviral intervention.

A nucleotide sequence comparison of coxsackievirus B4 isolates from aquatic samples and clinical specimens.

PubMed Central

Hughes, M. S.; Hoey, E. M.; Coyle, P. V.

1993-01-01

Ten coxsackievirus B4 (CVB4) strains isolated from clinical and environmental sources in Northern Ireland in 1985-7, were compared at the nucleotide sequence level. Dideoxynucleotide sequencing of a polymerase chain reaction (PCR) amplified fragment, spanning the VP1/P2A genomic region, classified the isolates into two distinct groups or genotypes as defined by Rico-Hesse and colleagues for poliovirus type 1. Isolates within each group shared approximately 99% sequence identity at the nucleotide level whereas < or = 86% sequence identity was shared between groups. One isolate derived from a clinical specimen in 1987 was grouped with six CVB4 isolates recovered from the aquatic environment in 1986-7. The second group comprised CVB4 isolates from clinical specimens in 1985-6. Both groups were different at the nucleotide level from the prototype strain isolated in 1950. It was concluded that the method could be used to sub-type CVB4 isolates and would be of value in epidemiological studies of CVB4. Predicted amino acid sequences revealed non-conservation of the tyrosine residue at the VP1/P2A cleavage site but were of little value in distinguishing CVB4 variants. PMID:8386098

3-Hydroxylaminophenol Mutase from Ralstonia eutropha JMP134 Catalyzes a Bamberger Rearrangement

PubMed Central

Schenzle, Andreas; Lenke, Hiltrud; Spain, Jim C.; Knackmuss, Hans-Joachim

1999-01-01

3-Hydroxylaminophenol mutase from Ralstonia eutropha JMP134 is involved in the degradative pathway of 3-nitrophenol, in which it catalyzes the conversion of 3-hydroxylaminophenol to aminohydroquinone. To show that the reaction was really catalyzed by a single enzyme without the release of intermediates, the corresponding protein was purified to apparent homogeneity from an extract of cells grown on 3-nitrophenol as the nitrogen source and succinate as the carbon and energy source. 3-Hydroxylaminophenol mutase appears to be a relatively hydrophobic but soluble and colorless protein consisting of a single 62-kDa polypeptide. The pI was determined to be at pH 4.5. In a database search, the NH2-terminal amino acid sequence of the undigested protein and of two internal sequences of 3-hydroxylaminophenol mutase were found to be most similar to those of glutamine synthetases from different species. Hydroxylaminobenzene, 4-hydroxylaminotoluene, and 2-chloro-5-hydroxylaminophenol, but not 4-hydroxylaminobenzoate, can also serve as substrates for the enzyme. The enzyme requires no oxygen or added cofactors for its reaction, which suggests an enzymatic mechanism analogous to the acid-catalyzed Bamberger rearrangement. PMID:10049374

Genetic analyses of bone morphogenetic protein 2, 4 and 7 in congenital combined pituitary hormone deficiency.

PubMed

Breitfeld, Jana; Martens, Susanne; Klammt, Jürgen; Schlicke, Marina; Pfäffle, Roland; Krause, Kerstin; Weidle, Kerstin; Schleinitz, Dorit; Stumvoll, Michael; Führer, Dagmar; Kovacs, Peter; Tönjes, Anke

2013-12-01

The complex process of development of the pituitary gland is regulated by a number of signalling molecules and transcription factors. Mutations in these factors have been identified in rare cases of congenital hypopituitarism but for most subjects with combined pituitary hormone deficiency (CPHD) genetic causes are unknown. Bone morphogenetic proteins (BMPs) affect induction and growth of the pituitary primordium and thus represent plausible candidates for mutational screening of patients with CPHD. We sequenced BMP2, 4 and 7 in 19 subjects with CPHD. For validation purposes, novel genetic variants were genotyped in 1046 healthy subjects. Additionally, potential functional relevance for most promising variants has been assessed by phylogenetic analyses and prediction of effects on protein structure. Sequencing revealed two novel variants and confirmed 30 previously known polymorphisms and mutations in BMP2, 4 and 7. Although phylogenetic analyses indicated that these variants map within strongly conserved gene regions, there was no direct support for their impact on protein structure when applying predictive bioinformatics tools. A mutation in the BMP4 coding region resulting in an amino acid exchange (p.Arg300Pro) appeared most interesting among the identified variants. Further functional analyses are required to ultimately map the relevance of these novel variants in CPHD.

Genetic analyses of bone morphogenetic protein 2, 4 and 7 in congenital combined pituitary hormone deficiency

PubMed Central

2013-01-01

Background The complex process of development of the pituitary gland is regulated by a number of signalling molecules and transcription factors. Mutations in these factors have been identified in rare cases of congenital hypopituitarism but for most subjects with combined pituitary hormone deficiency (CPHD) genetic causes are unknown. Bone morphogenetic proteins (BMPs) affect induction and growth of the pituitary primordium and thus represent plausible candidates for mutational screening of patients with CPHD. Methods We sequenced BMP2, 4 and 7 in 19 subjects with CPHD. For validation purposes, novel genetic variants were genotyped in 1046 healthy subjects. Additionally, potential functional relevance for most promising variants has been assessed by phylogenetic analyses and prediction of effects on protein structure. Results Sequencing revealed two novel variants and confirmed 30 previously known polymorphisms and mutations in BMP2, 4 and 7. Although phylogenetic analyses indicated that these variants map within strongly conserved gene regions, there was no direct support for their impact on protein structure when applying predictive bioinformatics tools. Conclusions A mutation in the BMP4 coding region resulting in an amino acid exchange (p.Arg300Pro) appeared most interesting among the identified variants. Further functional analyses are required to ultimately map the relevance of these novel variants in CPHD. PMID:24289245

Characteristics and management of Enterobacteriaceae harboring IMP-4 or IMP-8 carbapenemase in a tertiary hospital.

PubMed

Pang, Feng; Jia, Xiu-Qin; Song, Zhen-Zhu; Li, Yan-Hua; Wang, Bin; Zhao, Qi-Gang; Wang, Chuan-Xin; Zhang, Yi; Wang, Le-Xin

2016-03-01

The emergence of Enterobacteriaceae harboring IMP-4 or IMP-8 carbapenemases is rare. We report an occurrence of Enterobacteriaceae harboring IMP-4 or IMP-8 carbapenemases in a Chinese tertiary care hospital from November 2010 to December 2012. The clinical characteristics of 30 patients were described. The genetic relationship of isolates was determined by pulsed-field gel electrophoresis (PFGE). Carbapenemases were detected by modified Hodge test (MHT) and polymerase chain reactions (PCRs). Amplicons were sequenced and blasted to determine the genotype. Most infected patients were from intensive care unit and had complex and serious underlying illnesses requiring mechanical ventilation. PFGE revealed that Klebsiella pneumoniae showed two major PFGE types. Two Klebsiella oxytoca had an indistinguishable PFGE pattern, while four Enterobacter cloacae were different strains. The sequencing studies showed Enterobacteriaceae harboring IMP-4 or IMP-8 carbapenemase in the 23 infected patients. The majority of patients had infections with the carbapenemase-producing Enterobacteriaceae (CPE) strain, most were successfully treated with a range of antibiotics and discharged. It is important to maintain a high index of suspicion to screen for carbapenemase-producing Enterobacteriaceae strains. Rapid identification of these strains and implementation of stringent procedures are the key to prevent major outbreaks in a hospital setting.

Rapid amplification of 5' complementary DNA ends (5' RACE).

PubMed

2005-08-01

This method is used to extend partial cDNA clones by amplifying the 5' sequences of the corresponding mRNAs 1-3. The technique requires knowledge of only a small region of sequence within the partial cDNA clone. During PCR, the thermostable DNA polymerase is directed to the appropriate target RNA by a single primer derived from the region of known sequence; the second primer required for PCR is complementary to a general feature of the target-in the case of 5' RACE, to a homopolymeric tail added (via terminal transferase) to the 3' termini of cDNAs transcribed from a preparation of mRNA. This synthetic tail provides a primer-binding site upstream of the unknown 5' sequence of the target mRNA. The products of the amplification reaction are cloned into a plasmid vector for sequencing and subsequent manipulation.

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

Wang, Y.; Tobias, B.; Chang, Y. -T.

Electron cyclotron emission (ECE) imaging is a passive radiometric technique that measures electron temperature fluctuations; and microwave imaging reflectometry (MIR) is an active radar imaging technique that measures electron density fluctuations. The microwave imaging diagnostic instruments employing these techniques have made important contributions to fusion science and have been adopted at major fusion facilities worldwide including DIII-D, EAST, ASDEX Upgrade, HL-2A, KSTAR, LHD, and J-TEXT. In this paper, we describe the development status of three major technological advancements: custom mm-wave integrated circuits (ICs), digital beamforming (DBF), and synthetic diagnostic modeling (SDM). These also have the potential to greatly advance microwavemore » fusion plasma imaging, enabling compact and low-noise transceiver systems with real-time, fast tracking ability to address critical fusion physics issues, including ELM suppression and disruptions in the ITER baseline scenario, naturally ELM-free states such as QH-mode, and energetic particle confinement (i.e. Alfven eigenmode stability) in high-performance regimes that include steady-state and advanced tokamak scenarios. Furthermore, these systems are fully compatible with today's most challenging non-inductive heating and current drive systems and capable of operating in harsh environments, making them the ideal approach for diagnosing long-pulse and steady-state tokamaks.« less

Multi-Objective Optimization of Spacecraft Trajectories for Small-Body Coverage Missions

NASA Technical Reports Server (NTRS)

Hinckley, David, Jr.; Englander, Jacob; Hitt, Darren

2017-01-01

Visual coverage of surface elements of a small-body object requires multiple images to be taken that meet many requirements on their viewing angles, illumination angles, times of day, and combinations thereof. Designing trajectories capable of maximizing total possible coverage may not be useful since the image target sequence and the feasibility of said sequence given the rotation-rate limitations of the spacecraft are not taken into account. This work presents a means of optimizing, in a multi-objective manner, surface target sequences that account for such limitations.

Efficient strategy for the molecular diagnosis of intellectual disability using targeted high-throughput sequencing.

PubMed

Redin, Claire; Gérard, Bénédicte; Lauer, Julia; Herenger, Yvan; Muller, Jean; Quartier, Angélique; Masurel-Paulet, Alice; Willems, Marjolaine; Lesca, Gaétan; El-Chehadeh, Salima; Le Gras, Stéphanie; Vicaire, Serge; Philipps, Muriel; Dumas, Michaël; Geoffroy, Véronique; Feger, Claire; Haumesser, Nicolas; Alembik, Yves; Barth, Magalie; Bonneau, Dominique; Colin, Estelle; Dollfus, Hélène; Doray, Bérénice; Delrue, Marie-Ange; Drouin-Garraud, Valérie; Flori, Elisabeth; Fradin, Mélanie; Francannet, Christine; Goldenberg, Alice; Lumbroso, Serge; Mathieu-Dramard, Michèle; Martin-Coignard, Dominique; Lacombe, Didier; Morin, Gilles; Polge, Anne; Sukno, Sylvie; Thauvin-Robinet, Christel; Thevenon, Julien; Doco-Fenzy, Martine; Genevieve, David; Sarda, Pierre; Edery, Patrick; Isidor, Bertrand; Jost, Bernard; Olivier-Faivre, Laurence; Mandel, Jean-Louis; Piton, Amélie

2014-11-01

Intellectual disability (ID) is characterised by an extreme genetic heterogeneity. Several hundred genes have been associated to monogenic forms of ID, considerably complicating molecular diagnostics. Trio-exome sequencing was recently proposed as a diagnostic approach, yet remains costly for a general implementation. We report the alternative strategy of targeted high-throughput sequencing of 217 genes in which mutations had been reported in patients with ID or autism as the major clinical concern. We analysed 106 patients with ID of unknown aetiology following array-CGH analysis and other genetic investigations. Ninety per cent of these patients were males, and 75% sporadic cases. We identified 26 causative mutations: 16 in X-linked genes (ATRX, CUL4B, DMD, FMR1, HCFC1, IL1RAPL1, IQSEC2, KDM5C, MAOA, MECP2, SLC9A6, SLC16A2, PHF8) and 10 de novo in autosomal-dominant genes (DYRK1A, GRIN1, MED13L, TCF4, RAI1, SHANK3, SLC2A1, SYNGAP1). We also detected four possibly causative mutations (eg, in NLGN3) requiring further investigations. We present detailed reasoning for assigning causality for each mutation, and associated patients' clinical information. Some genes were hit more than once in our cohort, suggesting they correspond to more frequent ID-associated conditions (KDM5C, MECP2, DYRK1A, TCF4). We highlight some unexpected genotype to phenotype correlations, with causative mutations being identified in genes associated to defined syndromes in patients deviating from the classic phenotype (DMD, TCF4, MECP2). We also bring additional supportive (HCFC1, MED13L) or unsupportive (SHROOM4, SRPX2) evidences for the implication of previous candidate genes or mutations in cognitive disorders. With a diagnostic yield of 25% targeted sequencing appears relevant as a first intention test for the diagnosis of ID, but importantly will also contribute to a better understanding regarding the specific contribution of the many genes implicated in ID and autism. 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.

Efficient strategy for the molecular diagnosis of intellectual disability using targeted high-throughput sequencing

PubMed Central

Redin, Claire; Gérard, Bénédicte; Lauer, Julia; Herenger, Yvan; Muller, Jean; Quartier, Angélique; Masurel-Paulet, Alice; Willems, Marjolaine; Lesca, Gaétan; El-Chehadeh, Salima; Le Gras, Stéphanie; Vicaire, Serge; Philipps, Muriel; Dumas, Michaël; Geoffroy, Véronique; Feger, Claire; Haumesser, Nicolas; Alembik, Yves; Barth, Magalie; Bonneau, Dominique; Colin, Estelle; Dollfus, Hélène; Doray, Bérénice; Delrue, Marie-Ange; Drouin-Garraud, Valérie; Flori, Elisabeth; Fradin, Mélanie; Francannet, Christine; Goldenberg, Alice; Lumbroso, Serge; Mathieu-Dramard, Michèle; Martin-Coignard, Dominique; Lacombe, Didier; Morin, Gilles; Polge, Anne; Sukno, Sylvie; Thauvin-Robinet, Christel; Thevenon, Julien; Doco-Fenzy, Martine; Genevieve, David; Sarda, Pierre; Edery, Patrick; Isidor, Bertrand; Jost, Bernard; Olivier-Faivre, Laurence; Mandel, Jean-Louis; Piton, Amélie

2014-01-01

Background Intellectual disability (ID) is characterised by an extreme genetic heterogeneity. Several hundred genes have been associated to monogenic forms of ID, considerably complicating molecular diagnostics. Trio-exome sequencing was recently proposed as a diagnostic approach, yet remains costly for a general implementation. Methods We report the alternative strategy of targeted high-throughput sequencing of 217 genes in which mutations had been reported in patients with ID or autism as the major clinical concern. We analysed 106 patients with ID of unknown aetiology following array-CGH analysis and other genetic investigations. Ninety per cent of these patients were males, and 75% sporadic cases. Results We identified 26 causative mutations: 16 in X-linked genes (ATRX, CUL4B, DMD, FMR1, HCFC1, IL1RAPL1, IQSEC2, KDM5C, MAOA, MECP2, SLC9A6, SLC16A2, PHF8) and 10 de novo in autosomal-dominant genes (DYRK1A, GRIN1, MED13L, TCF4, RAI1, SHANK3, SLC2A1, SYNGAP1). We also detected four possibly causative mutations (eg, in NLGN3) requiring further investigations. We present detailed reasoning for assigning causality for each mutation, and associated patients’ clinical information. Some genes were hit more than once in our cohort, suggesting they correspond to more frequent ID-associated conditions (KDM5C, MECP2, DYRK1A, TCF4). We highlight some unexpected genotype to phenotype correlations, with causative mutations being identified in genes associated to defined syndromes in patients deviating from the classic phenotype (DMD, TCF4, MECP2). We also bring additional supportive (HCFC1, MED13L) or unsupportive (SHROOM4, SRPX2) evidences for the implication of previous candidate genes or mutations in cognitive disorders. Conclusions With a diagnostic yield of 25% targeted sequencing appears relevant as a first intention test for the diagnosis of ID, but importantly will also contribute to a better understanding regarding the specific contribution of the many genes implicated in ID and autism. PMID:25167861

SU-E-J-232: Feasibility of MRI-Based Preplan On Low Dose Rate Prostate Brachytherapy

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

Huang, Y; Tward, J; Rassiah-Szegedi, P

Purpose: To investigate the feasibility of using MRI-based preplan for low dose rate prostate brachytherapy. Methods: 12 patients who received transrectal ultrasound (TRUS) guided prostate brachytherapy with Pd-103 were retrospectively studied. Our care-standard of the TRUS-based preplan served as the control. One or more prostate T2-weighted wide and/or narrow-field of view MRIs obtained within the 3 months prior to the implant were imported into the MIM Symphony software v6.3 (MIM Software Inc., Cleveland, OH) for each patient. In total, 37 MRI preplans (10 different image sequences with average thickness of 4.8mm) were generated. The contoured prostate volume and the seedmore » counts required to achieve adequate dosimetric coverage from TRUS and MRI preplans were compared for each patient. The effects of different MRI sequences and image thicknesses were also investigated statistically using Student’s t-test. Lastly, the nomogram from the MRI preplan and TRUS preplan from our historical treatment data were compared. Results: The average prostate volume contoured on the TRUS and MRI were 26.6cc (range: 12.6∼41.3cc), and 27.4 cc (range: 14.3∼50.0cc), respectively. Axial MRI thicknesses (range: 3.5∼8.1mm) did not significantly affect the contoured volume or the number of seeds required on the preplan (R2 = 0.0002 and 0.0012, respectively). Four of the MRI sequences (AX-T2, AX-T2-Whole-Pelvis, AX-T2-FSE, and AXIALT2- Hi-Res) showed statistically significant better prostate volume agreement with TRUS than the other seven sequences (P <0.01). Nomogram overlay between the MRI and TRUS preplans showed good agreement; indicating volumes contoured on MRI preplan scan reliably predict how many seeds are needed for implant. Conclusion: Although MRI does not allow for determination of the actual implant geometry, it can give reliable volumes for seed ordering purposes. Our future work will investigate if MRI is sufficient to reliably replace TRUS preplanning in patients where preplan TRUS may be technically challenging.« less

Equally parsimonious pathways through an RNA sequence space are not equally likely

NASA Technical Reports Server (NTRS)

Lee, Y. H.; DSouza, L. M.; Fox, G. E.

1997-01-01

An experimental system for determining the potential ability of sequences resembling 5S ribosomal RNA (rRNA) to perform as functional 5S rRNAs in vivo in the Escherichia coli cellular environment was devised previously. Presumably, the only 5S rRNA sequences that would have been fixed by ancestral populations are ones that were functionally valid, and hence the actual historical paths taken through RNA sequence space during 5S rRNA evolution would have most likely utilized valid sequences. Herein, we examine the potential validity of all sequence intermediates along alternative equally parsimonious trajectories through RNA sequence space which connect two pairs of sequences that had previously been shown to behave as valid 5S rRNAs in E. coli. The first trajectory requires a total of four changes. The 14 sequence intermediates provide 24 apparently equally parsimonious paths by which the transition could occur. The second trajectory involves three changes, six intermediate sequences, and six potentially equally parsimonious paths. In total, only eight of the 20 sequence intermediates were found to be clearly invalid. As a consequence of the position of these invalid intermediates in the sequence space, seven of the 30 possible paths consisted of exclusively valid sequences. In several cases, the apparent validity/invalidity of the intermediate sequences could not be anticipated on the basis of current knowledge of the 5S rRNA structure. This suggests that the interdependencies in RNA sequence space may be more complex than currently appreciated. If ancestral sequences predicted by parsimony are to be regarded as actual historical sequences, then the present results would suggest that they should also satisfy a validity requirement and that, in at least limited cases, this conjecture can be tested experimentally.

Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of proteinase-activated receptor 4 (PAR4).

PubMed

Cunningham, Margaret R; McIntosh, Kathryn A; Pediani, John D; Robben, Joris; Cooke, Alexandra E; Nilsson, Mary; Gould, Gwyn W; Mundell, Stuart; Milligan, Graeme; Plevin, Robin

2012-05-11

Proteinase-activated receptors 4 (PAR(4)) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) to the plasma membrane. PAR(4) was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit β-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)AR → A(183)AA), mutation of which allowed efficient membrane delivery of PAR(4). Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signaling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) was shown to both facilitate and abrogate protein interactions with PAR(4), impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR(4) in normal physiology and disease.

Lessons for livestock genomics from genome and transcriptome sequencing in cattle and other mammals.

PubMed

Taylor, Jeremy F; Whitacre, Lynsey K; Hoff, Jesse L; Tizioto, Polyana C; Kim, JaeWoo; Decker, Jared E; Schnabel, Robert D

2016-08-17

Decreasing sequencing costs and development of new protocols for characterizing global methylation, gene expression patterns and regulatory regions have stimulated the generation of large livestock datasets. Here, we discuss experiences in the analysis of whole-genome and transcriptome sequence data. We analyzed whole-genome sequence (WGS) data from 132 individuals from five canid species (Canis familiaris, C. latrans, C. dingo, C. aureus and C. lupus) and 61 breeds, three bison (Bison bison), 64 water buffalo (Bubalus bubalis) and 297 bovines from 17 breeds. By individual, data vary in extent of reference genome depth of coverage from 4.9X to 64.0X. We have also analyzed RNA-seq data for 580 samples representing 159 Bos taurus and Rattus norvegicus animals and 98 tissues. By aligning reads to a reference assembly and calling variants, we assessed effects of average depth of coverage on the actual coverage and on the number of called variants. We examined the identity of unmapped reads by assembling them and querying produced contigs against the non-redundant nucleic acids database. By imputing high-density single nucleotide polymorphism data on 4010 US registered Angus animals to WGS using Run4 of the 1000 Bull Genomes Project and assessing the accuracy of imputation, we identified misassembled reference sequence regions. We estimate that a 24X depth of coverage is required to achieve 99.5 % coverage of the reference assembly and identify 95 % of the variants within an individual's genome. Genomes sequenced to low average coverage (e.g., <10X) may fail to cover 10 % of the reference genome and identify <75 % of variants. About 10 % of genomic DNA or transcriptome sequence reads fail to align to the reference assembly. These reads include loci missing from the reference assembly and misassembled genes and interesting symbionts, commensal and pathogenic organisms. Assembly errors and a lack of annotation of functional elements significantly limit the utility of the current draft livestock reference assemblies. The Functional Annotation of Animal Genomes initiative seeks to annotate functional elements, while a 70X Pac-Bio assembly for cow is underway and may result in a significantly improved reference assembly.

Characterization of the genetic elements required for site-specific integration of plasmid pSE211 in Saccharopolyspora erythraea.

PubMed Central

Brown, D P; Idler, K B; Katz, L

1990-01-01

The 18.1-kilobase plasmid pSE211 integrates into the chromosome of Saccharopolyspora erythraea at a specific attB site. Restriction analysis of the integrated plasmid, pSE211int, and adjacent chromosomal sequences allowed identification of attP, the plasmid attachment site. Nucleotide sequencing of attP, attB, attL, and attR revealed a 57-base-pair sequence common to all sites with no duplications of adjacent plasmid or chromosomal sequences in the integrated state, indicating that integration takes place through conservative, reciprocal strand exchange. An analysis of the sequences indicated the presence of a putative gene for Phe-tRNA at attB which is preserved at attL after integration has occurred. A comparison of the attB site for a number of actinomycete plasmids is presented. Integration at attB was also observed when a 2.4-kilobase segment of pSE211 containing attP and the adjacent plasmid sequence was used to transform a pSE211- host. Nucleotide sequencing of this segment revealed the presence of two complete open reading frames (ORFs) and a segment of a third ORF. The ORF adjacent to attP encodes a putative polypeptide 437 amino acids in length that shows similarity, at its C-terminal domain, to sequences of site-specific recombinases of the integrase family. The adjacent ORF encodes a putative 98-amino-acid basic polypeptide that contains a helix-turn-helix motif at its N terminus which corresponds to domains in the Xis proteins of a number of bacteriophages. A proposal for the function of this polypeptide is presented. The deduced amino acid sequence of the third ORF did not reveal similarities to polypeptide sequences in the current data banks. Images FIG. 2 FIG. 3 PMID:2180909

Using deep RNA sequencing for the structural annotation of the laccaria bicolor mycorrhizal transcriptome.

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

Larsen, P. E.; Trivedi, G.; Sreedasyam, A.

2010-07-06

Accurate structural annotation is important for prediction of function and required for in vitro approaches to characterize or validate the gene expression products. Despite significant efforts in the field, determination of the gene structure from genomic data alone is a challenging and inaccurate process. The ease of acquisition of transcriptomic sequence provides a direct route to identify expressed sequences and determine the correct gene structure. We developed methods to utilize RNA-seq data to correct errors in the structural annotation and extend the boundaries of current gene models using assembly approaches. The methods were validated with a transcriptomic data set derivedmore » from the fungus Laccaria bicolor, which develops a mycorrhizal symbiotic association with the roots of many tree species. Our analysis focused on the subset of 1501 gene models that are differentially expressed in the free living vs. mycorrhizal transcriptome and are expected to be important elements related to carbon metabolism, membrane permeability and transport, and intracellular signaling. Of the set of 1501 gene models, 1439 (96%) successfully generated modified gene models in which all error flags were successfully resolved and the sequences aligned to the genomic sequence. The remaining 4% (62 gene models) either had deviations from transcriptomic data that could not be spanned or generated sequence that did not align to genomic sequence. The outcome of this process is a set of high confidence gene models that can be reliably used for experimental characterization of protein function. 69% of expressed mycorrhizal JGI 'best' gene models deviated from the transcript sequence derived by this method. The transcriptomic sequence enabled correction of a majority of the structural inconsistencies and resulted in a set of validated models for 96% of the mycorrhizal genes. The method described here can be applied to improve gene structural annotation in other species, provided that there is a sequenced genome and a set of gene models.« less

The conserved CAAGAAAGA spacer sequence is an essential element for the formation of 3' termini of the sea urchin H3 histone mRNA by RNA processing.

PubMed Central

Georgiev, O; Birnstiel, M L

1985-01-01

Analysis of cDNA sequences obtained from the small nuclear RNA U7 has previously suggested specific contacts, by base pairing, between the conserved stem-loop structure and CAAGAAAGA sequence of the histone pre-mRNA and the 5'-terminal sequence of the U7 RNA during RNA processing. In order to test some aspects of the model we have created a series of linker scan, deletion and insertion mutants of the 3' terminus of a sea urchin H3 histone gene and have injected mutant DNAs or in vitro synthesized precursors into frog oocyte nuclei for interpretation. We find that, in addition to the stem-loop structure of the mRNA, the CAAGAAAGA spacer transcript within the histone pre-mRNA is required absolutely for RNA processing, as predicted from our model. Spacer sequences immediately downstream of the CAAGAAAGA motif are not complementary to U7 RNA. Nevertheless, they are necessary for obtaining a maximal rate of RNA processing, as is the ACCA sequence coding for the 3' terminus of the mature mRNA. An increase of distance between the mRNA palindrome and the CAAGAAAGA by as little as six nucleotides abolishes all processing. It may, therefore, be useful to regard both these sequence motifs as part of one and the same RNA processing signal with narrowly defined topologies. Interestingly, U7 RNA-dependent 3' processing of histone pre-mRNA can occur in RNA injection experiments only when the in vitro synthesized pre-mRNA contains sequence extensions well beyond the region of sequence complementarities to the U7 RNA. In addition to directing 3' processing the terminal mRNA sequences may have a role in histone mRNA stabilization in the cytoplasmic compartment. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:2410259

Pulseq: A rapid and hardware-independent pulse sequence prototyping framework.

PubMed

Layton, Kelvin J; Kroboth, Stefan; Jia, Feng; Littin, Sebastian; Yu, Huijun; Leupold, Jochen; Nielsen, Jon-Fredrik; Stöcker, Tony; Zaitsev, Maxim

2017-04-01

Implementing new magnetic resonance experiments, or sequences, often involves extensive programming on vendor-specific platforms, which can be time consuming and costly. This situation is exacerbated when research sequences need to be implemented on several platforms simultaneously, for example, at different field strengths. This work presents an alternative programming environment that is hardware-independent, open-source, and promotes rapid sequence prototyping. A novel file format is described to efficiently store the hardware events and timing information required for an MR pulse sequence. Platform-dependent interpreter modules convert the file to appropriate instructions to run the sequence on MR hardware. Sequences can be designed in high-level languages, such as MATLAB, or with a graphical interface. Spin physics simulation tools are incorporated into the framework, allowing for comparison between real and virtual experiments. Minimal effort is required to implement relatively advanced sequences using the tools provided. Sequences are executed on three different MR platforms, demonstrating the flexibility of the approach. A high-level, flexible and hardware-independent approach to sequence programming is ideal for the rapid development of new sequences. The framework is currently not suitable for large patient studies or routine scanning although this would be possible with deeper integration into existing workflows. Magn Reson Med 77:1544-1552, 2017. © 2016 International Society for Magnetic Resonance in Medicine. © 2016 International Society for Magnetic Resonance in Medicine.

Rapid Identification and Differentiation of Trichophyton Species, Based on Sequence Polymorphisms of the Ribosomal Internal Transcribed Spacer Regions, by Rolling-Circle Amplification▿

PubMed Central

Kong, Fanrong; Tong, Zhongsheng; Chen, Xiaoyou; Sorrell, Tania; Wang, Bin; Wu, Qixuan; Ellis, David; Chen, Sharon

2008-01-01

DNA sequencing analyses have demonstrated relatively limited polymorphisms within the fungal internal transcribed spacer (ITS) regions among Trichophyton spp. We sequenced the ITS region (ITS1, 5.8S, and ITS2) for 42 dermatophytes belonging to seven species (Trichophyton rubrum, T. mentagrophytes, T. soudanense, T. tonsurans, Epidermophyton floccosum, Microsporum canis, and M. gypseum) and developed a novel padlock probe and rolling-circle amplification (RCA)-based method for identification of single nucleotide polymorphisms (SNPs) that could be exploited to differentiate between Trichophyton spp. Sequencing results demonstrated intraspecies genetic variation for T. tonsurans, T. mentagrophytes, and T. soudanense but not T. rubrum. Signature sets of SNPs between T. rubrum and T. soudanense (4-bp difference) and T. violaceum and T. soudanense (3-bp difference) were identified. The RCA assay correctly identified five Trichophyton species. Although the use of two “group-specific” probes targeting both the ITS1 and the ITS2 regions were required to identify T. soudanense, the other species were identified by single ITS1- or ITS2-targeted species-specific probes. There was good agreement between ITS sequencing and the RCA assay. Despite limited genetic variation between Trichophyton spp., the sensitive, specific RCA-based SNP detection assay showed potential as a simple, reproducible method for the rapid (2-h) identification of Trichophyton spp. PMID:18234865

Terminal Duplex Stability and Nucleotide Identity Differentially Control siRNA Loading and Activity in RNA Interference

PubMed Central

Angart, Phillip A.; Carlson, Rebecca J.; Adu-Berchie, Kwasi

2016-01-01

Efficient short interfering RNA (siRNA)-mediated gene silencing requires selection of a sequence that is complementary to the intended target and possesses sequence and structural features that encourage favorable functional interactions with the RNA interference (RNAi) pathway proteins. In this study, we investigated how terminal sequence and structural characteristics of siRNAs contribute to siRNA strand loading and silencing activity and how these characteristics ultimately result in a functionally asymmetric duplex in cultured HeLa cells. Our results reiterate that the most important characteristic in determining siRNA activity is the 5′ terminal nucleotide identity. Our findings further suggest that siRNA loading is controlled principally by the hybridization stability of the 5′ terminus (Nucleotides: 1–2) of each siRNA strand, independent of the opposing terminus. Postloading, RNA-induced silencing complex (RISC)–specific activity was found to be improved by lower hybridization stability in the 5′ terminus (Nucleotides: 3–4) of the loaded siRNA strand and greater hybridization stability toward the 3′ terminus (Nucleotides: 17–18). Concomitantly, specific recognition of the 5′ terminal nucleotide sequence by human Argonaute 2 (Ago2) improves RISC half-life. These findings indicate that careful selection of siRNA sequences can maximize both the loading and the specific activity of the intended guide strand. PMID:27399870

Modeling participation duration, with application to the North American Breeding Bird Survey

USGS Publications Warehouse

Link, William; Sauer, John

2014-01-01

We consider “participation histories,” binary sequences consisting of alternating finite sequences of 1s and 0s, ending with an infinite sequence of 0s. Our work is motivated by a study of observer tenure in the North American Breeding Bird Survey (BBS). In our analysis, j indexes an observer’s years of service and Xj is an indicator of participation in the survey; 0s interspersed among 1s correspond to years when observers did not participate, but subsequently returned to service. Of interest is the observer’s duration D = max {j: Xj = 1}. Because observed records X = (X1, X2,..., Xn)1 are of finite length, all that we can directly infer about duration is that D ⩾ max {j ⩽n: Xj = 1}; model-based analysis is required for inference about D. We propose models in which lengths of 0s and 1s sequences have distributions determined by the index j at which they begin; 0s sequences are infinite with positive probability, an estimable parameter. We found that BBS observers’ lengths of service vary greatly, with 25.3% participating for only a single year, 49.5% serving for 4 or fewer years, and an average duration of 8.7 years, producing an average of 7.7 counts.

The histidine permease gene (HIP1) of Saccharomyces cerevisiae.

PubMed

Tanaka, J; Fink, G R

1985-01-01

The histidine-specific permease gene (HIP1) of Saccharomyces cerevisiae has been mapped, cloned, and sequenced. The HIP1 gene maps to the right arm of chromosome VII, approx. 11 cM distal to the ADE3 gene. The gene was isolated as an 8.6-kb BamHI-Sau3A fragment by complementation of the histidine-specific permease deficiency in recipient yeast cells. We sequenced a 2.4-kb subfragment of this BamHI-Sau3A fragment containing the HIP1 gene and identified a 1596-bp open reading frame (ORF). We confirmed the assignment of the 1596-bp ORF as the HIP1 coding sequence by sequencing a hip1 nonsense mutation. Analysis of the amino acid (aa) sequence of the HIP1 gene reveals several hydrophobic stretches, but shows no obvious N-terminal signal peptide. We have constructed a deletion of the HIP1 gene in vitro and replaced the wild-type copy of the gene with this deletion. The hip1 deletion mutant can grow when it is supplemented with 30 mM histidine, 50 times the amount required for the growth of HIP1 cells. Revertants of this deletion mutant able to grow on a normal level of histidine arise by mutation in unlinked genes. Both these observations suggest that there are additional, low-affinity pathways for histidine uptake.

The sequence specificity of UV-induced DNA damage in a systematically altered DNA sequence.

PubMed

Khoe, Clairine V; Chung, Long H; Murray, Vincent

2018-06-01

The sequence specificity of UV-induced DNA damage was investigated in a specifically designed DNA plasmid using two procedures: end-labelling and linear amplification. Absorption of UV photons by DNA leads to dimerisation of pyrimidine bases and produces two major photoproducts, cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (6-4PPs). A previous study had determined that two hexanucleotide sequences, 5'-GCTC*AC and 5'-TATT*AA, were high intensity UV-induced DNA damage sites. The UV clone plasmid was constructed by systematically altering each nucleotide of these two hexanucleotide sequences. One of the main goals of this study was to determine the influence of single nucleotide alterations on the intensity of UV-induced DNA damage. The sequence 5'-GCTC*AC was designed to examine the sequence specificity of 6-4PPs and the highest intensity 6-4PP damage sites were found at 5'-GTTC*CC nucleotides. The sequence 5'-TATT*AA was devised to investigate the sequence specificity of CPDs and the highest intensity CPD damage sites were found at 5'-TTTT*CG nucleotides. It was proposed that the tetranucleotide DNA sequence, 5'-YTC*Y (where Y is T or C), was the consensus sequence for the highest intensity UV-induced 6-4PP adduct sites; while it was 5'-YTT*C for the highest intensity UV-induced CPD damage sites. These consensus tetranucleotides are composed entirely of consecutive pyrimidines and must have a DNA conformation that is highly productive for the absorption of UV photons. Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.

AfterQC: automatic filtering, trimming, error removing and quality control for fastq data.

PubMed

Chen, Shifu; Huang, Tanxiao; Zhou, Yanqing; Han, Yue; Xu, Mingyan; Gu, Jia

2017-03-14

Some applications, especially those clinical applications requiring high accuracy of sequencing data, usually have to face the troubles caused by unavoidable sequencing errors. Several tools have been proposed to profile the sequencing quality, but few of them can quantify or correct the sequencing errors. This unmet requirement motivated us to develop AfterQC, a tool with functions to profile sequencing errors and correct most of them, plus highly automated quality control and data filtering features. Different from most tools, AfterQC analyses the overlapping of paired sequences for pair-end sequencing data. Based on overlapping analysis, AfterQC can detect and cut adapters, and furthermore it gives a novel function to correct wrong bases in the overlapping regions. Another new feature is to detect and visualise sequencing bubbles, which can be commonly found on the flowcell lanes and may raise sequencing errors. Besides normal per cycle quality and base content plotting, AfterQC also provides features like polyX (a long sub-sequence of a same base X) filtering, automatic trimming and K-MER based strand bias profiling. For each single or pair of FastQ files, AfterQC filters out bad reads, detects and eliminates sequencer's bubble effects, trims reads at front and tail, detects the sequencing errors and corrects part of them, and finally outputs clean data and generates HTML reports with interactive figures. AfterQC can run in batch mode with multiprocess support, it can run with a single FastQ file, a single pair of FastQ files (for pair-end sequencing), or a folder for all included FastQ files to be processed automatically. Based on overlapping analysis, AfterQC can estimate the sequencing error rate and profile the error transform distribution. The results of our error profiling tests show that the error distribution is highly platform dependent. Much more than just another new quality control (QC) tool, AfterQC is able to perform quality control, data filtering, error profiling and base correction automatically. Experimental results show that AfterQC can help to eliminate the sequencing errors for pair-end sequencing data to provide much cleaner outputs, and consequently help to reduce the false-positive variants, especially for the low-frequency somatic mutations. While providing rich configurable options, AfterQC can detect and set all the options automatically and require no argument in most cases.

A simple grid implementation with Berkeley Open Infrastructure for Network Computing using BLAST as a model

PubMed Central

Pinthong, Watthanai; Muangruen, Panya

2016-01-01

Development of high-throughput technologies, such as Next-generation sequencing, allows thousands of experiments to be performed simultaneously while reducing resource requirement. Consequently, a massive amount of experiment data is now rapidly generated. Nevertheless, the data are not readily usable or meaningful until they are further analysed and interpreted. Due to the size of the data, a high performance computer (HPC) is required for the analysis and interpretation. However, the HPC is expensive and difficult to access. Other means were developed to allow researchers to acquire the power of HPC without a need to purchase and maintain one such as cloud computing services and grid computing system. In this study, we implemented grid computing in a computer training center environment using Berkeley Open Infrastructure for Network Computing (BOINC) as a job distributor and data manager combining all desktop computers to virtualize the HPC. Fifty desktop computers were used for setting up a grid system during the off-hours. In order to test the performance of the grid system, we adapted the Basic Local Alignment Search Tools (BLAST) to the BOINC system. Sequencing results from Illumina platform were aligned to the human genome database by BLAST on the grid system. The result and processing time were compared to those from a single desktop computer and HPC. The estimated durations of BLAST analysis for 4 million sequence reads on a desktop PC, HPC and the grid system were 568, 24 and 5 days, respectively. Thus, the grid implementation of BLAST by BOINC is an efficient alternative to the HPC for sequence alignment. The grid implementation by BOINC also helped tap unused computing resources during the off-hours and could be easily modified for other available bioinformatics software. PMID:27547555

Not all order memory is equal: Test demands reveal dissociations in memory for sequence information.

PubMed

Jonker, Tanya R; MacLeod, Colin M

2017-02-01

Remembering the order of a sequence of events is a fundamental feature of episodic memory. Indeed, a number of formal models represent temporal context as part of the memory system, and memory for order has been researched extensively. Yet, the nature of the code(s) underlying sequence memory is still relatively unknown. Across 4 experiments that manipulated encoding task, we found evidence for 3 dissociable facets of order memory. Experiment 1 introduced a test requiring a judgment of which of 2 alternatives had immediately followed a word during encoding. This measure revealed better retention of interitem associations following relational encoding (silent reading) than relatively item-specific encoding (judging referent size), a pattern consistent with that observed in previous research using order reconstruction tests. In sharp contrast, Experiment 2 demonstrated the reverse pattern: Memory for the studied order of 2 sequentially presented items was actually better following item-specific encoding than following relational encoding. Experiment 3 reproduced this dissociation in a single experiment using both tests. Experiment 4 extended these findings by further dissociating the roles of relational encoding and item strength in the 2 tests. Taken together, these results indicate that memory for event sequence is influenced by (a) interitem associations, (b) the emphasized directionality of an association, and (c) an item's strength independent of other items. Memory for order is more complicated than has been portrayed in theories of memory and its nuances should be carefully considered when designing tests and models of temporal and relational memory. (PsycINFO Database Record (c) 2017 APA, all rights reserved).