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Sample records for cleavage site analysis

  1. Identification of succinimide sites in proteins by N-terminal sequence analysis after alkaline hydroxylamine cleavage.

    Kwong, M. Y.; Harris, R. J.

    1994-01-01

    Under favorable conditions, Asp or Asn residues can undergo rearrangement to a succinimide (cyclic imide), which may also serve as an intermediate for deamidation and/or isoaspartate formation. Direct identification of such succinimides by peptide mapping is hampered by their lability at neutral and alkaline pH. We determined that incubation in 2 M hydroxylamine, 0.2 M Tris buffer, pH 9, for 2 h at 45 degrees C will specifically cleave on the C-terminal side of succinimides without cleavage at Asn-Gly bonds; yields are typically approximately 50%. N-terminal sequence analysis can then be used to identify an internal sequence generated by cleavage of the succinimide, hence identifying the succinimide site. PMID:8142891

  2. Analysis of the Proteolytic Processing of ABCA3: Identification of Cleavage Site and Involved Proteases.

    Nicole Hofmann

    Full Text Available ABCA3 is a lipid transporter in the limiting membrane of lamellar bodies in alveolar type II cells. Mutations in the ABCA3 gene cause respiratory distress syndrome in new-borns and childhood interstitial lung disease. ABCA3 is N-terminally cleaved by an as yet unknown protease, a process believed to regulate ABCA3 activity.The exact site where ABCA3 is cleaved was localized using mass spectrometry (MS. Proteases involved in ABCA3 processing were identified using small molecule inhibitors and siRNA mediated gene knockdown. Results were verified by in vitro digestion of a synthetic peptide substrate mimicking ABCA3's cleavage region, followed by MS analysis.We found that cleavage of ABCA3 occurs after Lys174 which is located in the proteins' first luminal loop. Inhibition of cathepsin L and, to a lesser extent, cathepsin B resulted in attenuation of ABCA3 cleavage. Both enzymes showed activity against the ABCA3 peptide in vitro with cathepsin L being more active.We show here that, like some other proteins of the lysosomal membrane, ABCA3 is a substrate of cathepsin L. Therefore, cathepsin L may represent a potential target to therapeutically influence ABCA3 activity in ABCA3-associated lung disease.

  3. Dataset of cocoa aspartic protease cleavage sites

    Katharina Janek

    2016-09-01

    Full Text Available The data provide information in support of the research article, “The cleavage specificity of the aspartic protease of cocoa beans involved in the generation of the cocoa-specific aroma precursors” (Janek et al., 2016 [1]. Three different protein substrates were partially digested with the aspartic protease isolated from cocoa beans and commercial pepsin, respectively. The obtained peptide fragments were analyzed by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS/MS and identified using the MASCOT server. The N- and C-terminal ends of the peptide fragments were used to identify the corresponding in-vitro cleavage sites by comparison with the amino acid sequences of the substrate proteins. The same procedure was applied to identify the cleavage sites used by the cocoa aspartic protease during cocoa fermentation starting from the published amino acid sequences of oligopeptides isolated from fermented cocoa beans. Keywords: Aspartic protease, Cleavage sites, Cocoa, In-vitro proteolysis, Mass spectrometry, Peptides

  4. In vivo analysis of the Notch receptor S1 cleavage.

    Robert J Lake

    2009-08-01

    Full Text Available A ligand-independent cleavage (S1 in the extracellular domain of the mammalian Notch receptor results in what is considered to be the canonical heterodimeric form of Notch on the cell surface. The in vivo consequences and significance of this cleavage on Drosophila Notch signaling remain unclear and contradictory. We determined the cleavage site in Drosophila and examined its in vivo function by a transgenic analysis of receptors that cannot be cleaved. Our results demonstrate a correlation between loss of cleavage and loss of in vivo function of the Notch receptor, supporting the notion that S1 cleavage is an in vivo mechanism of Notch signal control.

  5. Pripper: prediction of caspase cleavage sites from whole proteomes

    Salmi Jussi

    2010-06-01

    Full Text Available Abstract Background Caspases are a family of proteases that have central functions in programmed cell death (apoptosis and inflammation. Caspases mediate their effects through aspartate-specific cleavage of their target proteins, and at present almost 400 caspase substrates are known. There are several methods developed to predict caspase cleavage sites from individual proteins, but currently none of them can be used to predict caspase cleavage sites from multiple proteins or entire proteomes, or to use several classifiers in combination. The possibility to create a database from predicted caspase cleavage products for the whole genome could significantly aid in identifying novel caspase targets from tandem mass spectrometry based proteomic experiments. Results Three different pattern recognition classifiers were developed for predicting caspase cleavage sites from protein sequences. Evaluation of the classifiers with quality measures indicated that all of the three classifiers performed well in predicting caspase cleavage sites, and when combining different classifiers the accuracy increased further. A new tool, Pripper, was developed to utilize the classifiers and predict the caspase cut sites from an arbitrary number of input sequences. A database was constructed with the developed tool, and it was used to identify caspase target proteins from tandem mass spectrometry data from two different proteomic experiments. Both known caspase cleavage products as well as novel cleavage products were identified using the database demonstrating the usefulness of the tool. Pripper is not restricted to predicting only caspase cut sites, but it gives the possibility to scan protein sequences for any given motif(s and predict cut sites once a suitable cut site prediction model for any other protease has been developed. Pripper is freely available and can be downloaded from http://users.utu.fi/mijopi/Pripper. Conclusions We have developed Pripper, a tool for

  6. Variable context Markov chains for HIV protease cleavage site prediction.

    Oğul, Hasan

    2009-06-01

    Deciphering the knowledge of HIV protease specificity and developing computational tools for detecting its cleavage sites in protein polypeptide chain are very desirable for designing efficient and specific chemical inhibitors to prevent acquired immunodeficiency syndrome. In this study, we developed a generative model based on a generalization of variable order Markov chains (VOMC) for peptide sequences and adapted the model for prediction of their cleavability by certain proteases. The new method, called variable context Markov chains (VCMC), attempts to identify the context equivalence based on the evolutionary similarities between individual amino acids. It was applied for HIV-1 protease cleavage site prediction problem and shown to outperform existing methods in terms of prediction accuracy on a common dataset. In general, the method is a promising tool for prediction of cleavage sites of all proteases and encouraged to be used for any kind of peptide classification problem as well.

  7. Possible cleavage sites of glutelin partial degradation confirmed by immunological analysis in globulin-less mutants of rice (Oryza sativa L.).

    Khan, Nadar; Yamaguchi, Satoru; Katsube-Tanaka, Tomoyuki

    2017-10-01

    Proteolytic cleavage or partial degradation of proteins is one of the important post-translational modifications for various biological processes, but it is difficult to analyze. Previously, we demonstrated that some subunits of the major rice (Oryza sativa L.) seed storage protein glutelin are partially degraded to produce newly identified polypeptides X1-X5 in mutants in which another major seed storage protein globulin is absent. In this study, the new polypeptides X3 and X4/X5 were immunologically confirmed to be derived from GluA3 and GluA1/GluA2 subunits, respectively. Additionally, the new polypeptides X1 and X2 were at least in part the α polypeptides of the GluB4 subunit partially degraded at the C-terminus. Simulated 2D-PAGE migration patterns of intact and partially degraded α polypeptides based on the calculation of their MWs and pIs enabled us to narrow or predict the possible locations of cleavage sites. The predicted cleavage sites were also verified by the comparison of 2D-PAGE patterns between seed-extracted and E. coli-expressed proteins of the intact and truncated α polypeptides. The results and methodologies demonstrated here would be useful for analyses of partial degradation of proteins and the structure-function relationships of rice seed protein bodies. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Cleavage sites within the poliovirus capsid protein precursors

    Larsen, G.R.; Anderson, C.W.; Dorner, A.J.; Semler, B.L.; Wimmer, E.

    1982-01-01

    Partial amino-terminal sequence analysis was performed on radiolabeled poliovirus capsid proteins VP1, VP2, and VP3. A computer-assisted comparison of the amino acid sequences obtained with that predicted by the nucleotide sequence of the poliovirus genome allows assignment of the amino terminus of each capsid protein to a unique position within the virus polyprotein. Sequence analysis of trypsin-digested VP4, which has a blocked amino terminus, demonstrates that VP4 is encoded at or very near to the amino terminus of the polyprotein. The gene order of the capsid proteins is VP4-VP2-VP3-VP1. Cleavage of VP0 to VP4 and VP2 is shown to occur between asparagine and serine, whereas the cleavages that separate VP2/VP3 and VP3/VP1 occur between glutamine and glycine residues. This finding supports the hypothesis that the cleavage of VP0, which occurs during virion morphogenesis, is distinct from the cleavages that separate functional regions of the polyprotein

  9. Active site mutations change the cleavage specificity of neprilysin.

    Travis Sexton

    Full Text Available Neprilysin (NEP, a member of the M13 subgroup of the zinc-dependent endopeptidase family is a membrane bound peptidase capable of cleaving a variety of physiological peptides. We have generated a series of neprilysin variants containing mutations at either one of two active site residues, Phe(563 and Ser(546. Among the mutants studied in detail we observed changes in their activity towards leucine(5-enkephalin, insulin B chain, and amyloid β(1-40. For example, NEP(F563I displayed an increase in preference towards cleaving leucine(5-enkephalin relative to insulin B chain, while mutant NEP(S546E was less discriminating than neprilysin. Mutants NEP(F563L and NEP(S546E exhibit different cleavage site preferences than neprilysin with insulin B chain and amyloid ß(1-40 as substrates. These data indicate that it is possible to alter the cleavage site specificity of neprilysin opening the way for the development of substrate specific or substrate exclusive forms of the enzyme with enhanced therapeutic potential.

  10. PROSPER: an integrated feature-based tool for predicting protease substrate cleavage sites.

    Jiangning Song

    Full Text Available The ability to catalytically cleave protein substrates after synthesis is fundamental for all forms of life. Accordingly, site-specific proteolysis is one of the most important post-translational modifications. The key to understanding the physiological role of a protease is to identify its natural substrate(s. Knowledge of the substrate specificity of a protease can dramatically improve our ability to predict its target protein substrates, but this information must be utilized in an effective manner in order to efficiently identify protein substrates by in silico approaches. To address this problem, we present PROSPER, an integrated feature-based server for in silico identification of protease substrates and their cleavage sites for twenty-four different proteases. PROSPER utilizes established specificity information for these proteases (derived from the MEROPS database with a machine learning approach to predict protease cleavage sites by using different, but complementary sequence and structure characteristics. Features used by PROSPER include local amino acid sequence profile, predicted secondary structure, solvent accessibility and predicted native disorder. Thus, for proteases with known amino acid specificity, PROSPER provides a convenient, pre-prepared tool for use in identifying protein substrates for the enzymes. Systematic prediction analysis for the twenty-four proteases thus far included in the database revealed that the features we have included in the tool strongly improve performance in terms of cleavage site prediction, as evidenced by their contribution to performance improvement in terms of identifying known cleavage sites in substrates for these enzymes. In comparison with two state-of-the-art prediction tools, PoPS and SitePrediction, PROSPER achieves greater accuracy and coverage. To our knowledge, PROSPER is the first comprehensive server capable of predicting cleavage sites of multiple proteases within a single substrate

  11. [Prokaryotic expression and immunogenicity analysis of the chimeric HBcAg containing APP beta cleavage site peptide and Aβ(1-15);].

    Feng, Gai-feng; Wang, Jun-yang; Jin, Hui; Wang, Wei-xi; Qian, Yi-hua; Yang, Wei-na; Wang, Quan-ying; Yang, Guang-xiao

    2011-11-01

    To construct the recombinant prokaryotic expression plasmid pET/c-ABCSP-Aβ(15-c);, and evaluate the immunogenicity of the fusion protein expressed in E.coli. The gene fragment HBc88-144 was amplified by PCR and subcloned to pUC19. The APP beta cleavage site peptide(ABCSP) and Aβ(1-15); gene(ABCSP-Aβ(15);) was amplified by PCR and inserted downstream of HBc1-71 in pGEMEX/c1-71. After restriction enzyme digestion, c1-17-ABCSP-Aβ(15); were connected with HBc88-144, yielding the recombinant gene c-ABCSP-Aβ(15-c);. c-ABCSP-Aβ(15-c); gene was subcloned into pET-28a(+).The fusion protein expressed in transformed E.coli BL21 was induced with IPTG and analyzed by SDS-PAGE. The virus-like particles (VLP) formed by fusion protein was observed with Transmission Electron Microscope (TEM). 4 Kunming (KM) mice received intraperitoneal injection (i.p) of fusion protein VLP. The antibody was detected by indirect ELISA. The recombinant gene was confirmed by restriction enzyme digestion and DNA sequencing. After IPTG induction, fusion protein was expressed and mainly existed in the sediment of the bacterial lysate. The expression level was 40% of all the proteins in the sediment. The fusion protein could form VLP. After 5 times of immunization, the titer of anti-ABCSP and anti-Aβantibody in sera of KM mice reached up to 1:5 000 and 1:10 000 respectively, while the anti-HBc antibody was undetectable. Recombinant c-ABCSP-Aβ(15-c); gene can be expressed in E.coli. The expressed protein could form VLP and has a strong immunogenicity. This study lays the foundation for the study of AD genetic engineering vaccine.

  12. Signal peptide discrimination and cleavage site identification using SVM and NN.

    Kazemian, H B; Yusuf, S A; White, K

    2014-02-01

    About 15% of all proteins in a genome contain a signal peptide (SP) sequence, at the N-terminus, that targets the protein to intracellular secretory pathways. Once the protein is targeted correctly in the cell, the SP is cleaved, releasing the mature protein. Accurate prediction of the presence of these short amino-acid SP chains is crucial for modelling the topology of membrane proteins, since SP sequences can be confused with transmembrane domains due to similar composition of hydrophobic amino acids. This paper presents a cascaded Support Vector Machine (SVM)-Neural Network (NN) classification methodology for SP discrimination and cleavage site identification. The proposed method utilises a dual phase classification approach using SVM as a primary classifier to discriminate SP sequences from Non-SP. The methodology further employs NNs to predict the most suitable cleavage site candidates. In phase one, a SVM classification utilises hydrophobic propensities as a primary feature vector extraction using symmetric sliding window amino-acid sequence analysis for discrimination of SP and Non-SP. In phase two, a NN classification uses asymmetric sliding window sequence analysis for prediction of cleavage site identification. The proposed SVM-NN method was tested using Uni-Prot non-redundant datasets of eukaryotic and prokaryotic proteins with SP and Non-SP N-termini. Computer simulation results demonstrate an overall accuracy of 0.90 for SP and Non-SP discrimination based on Matthews Correlation Coefficient (MCC) tests using SVM. For SP cleavage site prediction, the overall accuracy is 91.5% based on cross-validation tests using the novel SVM-NN model. © 2013 Published by Elsevier Ltd.

  13. ChloroP, a neural network-based method for predicting chloroplast transitpeptides and their cleavage sites

    Emanuelsson, O.; Nielsen, Henrik; von Heijne, Gunnar

    1999-01-01

    the cleavage sites given in SWISS-PROT. An analysis of 715 Arabidopsis thaliana sequences from SWISS-PROT suggests that the ChloroP method should be useful for the identification of putative transit peptides in genome-wide sequence data. The ChloroP predictor is available as a web-server at http......We present a neural network based method (ChloroP) for identifying chloroplast transit peptides and their cleavage sites. Using cross-validation, 88% of the sequences in our homology reduced training set were correctly classified as transit peptides or nontransit peptides. This performance level...

  14. Snake venom serine proteinases specificity mapping by proteomic identification of cleavage sites.

    Zelanis, André; Huesgen, Pitter F; Oliveira, Ana Karina; Tashima, Alexandre K; Serrano, Solange M T; Overall, Christopher M

    2015-01-15

    Many snake venom toxins are serine proteases but their specific in vivo targets are mostly unknown. Various act on components of the coagulation cascade, and fibrinolytic and kallikrein-kinin systems to trigger various pathological effects observed in the envenomation. Despite showing high similarity in terms of primary structure snake venom serine proteinases (SVSPs) show exquisite specificity towards macromolecular substrates. Therefore, the characterization of their peptide bond specificity is important for understanding the active site preference associated with effective proteolysis as well as for the design of peptide substrates and inhibitors. Bothrops jararaca contains various SVSPs among which Bothrops protease A is a specific fibrinogenolytic agent and PA-BJ is a platelet-activating enzyme. In this study we used proteome derived peptide libraries in the Proteomic Identification of protease Cleavage Sites (PICS) approach to explore the peptide bond specificity of Bothrops protease A and PA-BJ in order to determine their individual peptide cleavage sequences. A total of 371 cleavage sites (208 for Bothrops protease A and 163 for PA-BJ) were detected and both proteinases displayed a clear preference for arginine at the P1 position. Moreover, the analysis of the specificity profiles of Bothrops protease A and PA-BJ revealed subtle differences in the preferences along P6-P6', despite a common yet unusual preference for Pro at P2. Taken together, these results map the subsite specificity of both SVSPs and shed light in the functional differences between these proteinases. Proteolysis is key to various pathological effects observed upon envenomation by viperid snakes. The use of the Proteomic Identification of protease Cleavage Sites (PICS) approach for the easy mapping of proteinase subsite preferences at both the prime- and non-prime sides concurrently gives rise to a fresh understanding of the interaction of the snake venom serine proteinases with peptide and

  15. Coronavirus 3CLpro proteinase cleavage sites: Possible relevance to SARS virus pathology

    Blom Nikolaj

    2004-06-01

    Full Text Available Abstract Background Despite the passing of more than a year since the first outbreak of Severe Acute Respiratory Syndrome (SARS, efficient counter-measures are still few and many believe that reappearance of SARS, or a similar disease caused by a coronavirus, is not unlikely. For other virus families like the picornaviruses it is known that pathology is related to proteolytic cleavage of host proteins by viral proteinases. Furthermore, several studies indicate that virus proliferation can be arrested using specific proteinase inhibitors supporting the belief that proteinases are indeed important during infection. Prompted by this, we set out to analyse and predict cleavage by the coronavirus main proteinase using computational methods. Results We retrieved sequence data on seven fully sequenced coronaviruses and identified the main 3CL proteinase cleavage sites in polyproteins using alignments. A neural network was trained to recognise the cleavage sites in the genomes obtaining a sensitivity of 87.0% and a specificity of 99.0%. Several proteins known to be cleaved by other viruses were submitted to prediction as well as proteins suspected relevant in coronavirus pathology. Cleavage sites were predicted in proteins such as the cystic fibrosis transmembrane conductance regulator (CFTR, transcription factors CREB-RP and OCT-1, and components of the ubiquitin pathway. Conclusions Our prediction method NetCorona predicts coronavirus cleavage sites with high specificity and several potential cleavage candidates were identified which might be important to elucidate coronavirus pathology. Furthermore, the method might assist in design of proteinase inhibitors for treatment of SARS and possible future diseases caused by coronaviruses. It is made available for public use at our website: http://www.cbs.dtu.dk/services/NetCorona/.

  16. Degradation of tropoelastin by matrix metalloproteinases--cleavage site specificities and release of matrikines

    Heinz, Andrea; Jung, Michael C; Duca, Laurent

    2010-01-01

    To provide a basis for the development of approaches to treat elastin-degrading diseases, the aim of this study was to investigate the degradation of the natural substrate tropoelastin by the elastinolytic matrix metalloproteinases MMP-7, MMP-9, and MMP-12 and to compare the cleavage site...

  17. Rendering one autolysis site in Bacillus subtilis neutral protease resistant to cleavage reveals a new fission

    Van den Burg, B; Eijsink, VGH; Vriend, G; Veltman, OR; Venema, G

    Autolytic degradation of the thermolysin-like proteinase of Bacillus subtilis (TLP-sub) is responsible for the irreversible inactivation of the enzyme at elevated temperatures. Previously we have reported five cleavage sites in Tip-sub [Van den Burg et al, (1990) Biochem. J. 272, 93-97]. In an

  18. Characterization of a Non-Canonical Signal Peptidase Cleavage Site in a Replication Protein from Tomato Ringspot Virus.

    Ting Wei

    Full Text Available The NTB-VPg polyprotein from tomato ringspot virus is an integral membrane replication protein associated with endoplasmic reticulum membranes. A signal peptidase (SPase cleavage was previously detected in the C-terminal region of NTB-VPg downstream of a 14 amino acid (aa-long hydrophobic region (termed TM2. However, the exact location of the cleavage site was not determined. Using in vitro translation assays, we show that the SPase cleavage site is conserved in the NTB-VPg protein from various ToRSV isolates, although the rate of cleavage varies from one isolate to another. Systematic site-directed mutagenesis of the NTB-VPg SPase cleavage sites of two ToRSV isolates allowed the identification of sequences that affect cleavage efficiency. We also present evidence that SPase cleavage in the ToRSV-Rasp2 isolate occurs within a GAAGG sequence likely after the AAG (GAAG/G. Mutation of a downstream MAAV sequence to AAAV resulted in SPase cleavage at both the natural GAAG/G and the mutated AAA/V sequences. Given that there is a distance of seven aa between the two cleavage sites, this indicates that there is flexibility in the positioning of the cleavage sites relative to the inner surface of the membrane and the SPase active site. SPase cleavage sites are typically located 3-7 aa downstream of the hydrophobic region. However, the NTB-VPg GAAG/G cleavage site is located 17 aa downstream of the TM2 hydrophobic region, highlighting unusual features of the NTB-VPg SPase cleavage site. A putative 11 aa-long amphipathic helix was identified immediately downstream of the TM2 region and five aa upstream of the GAAG/G cleavage site. Based on these results, we present an updated topology model in which the hydrophobic and amphipathic domains form a long tilted helix or a bent helix in the membrane lipid bilayer, with the downstream cleavage site(s oriented parallel to the membrane inner surface.

  19. Hemoglobin cleavage site-specificity of the Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3.

    Shoba Subramanian

    Full Text Available The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 degrade host hemoglobin to provide free amino acids for parasite protein synthesis. Hemoglobin hydrolysis has been described as an ordered process initiated by aspartic proteases, but cysteine protease inhibitors completely block the process, suggesting that cysteine proteases can also initiate hemoglobin hydrolysis. To characterize the specific roles of falcipains, we used three approaches. First, using random P(1 - P(4 amino acid substrate libraries, falcipain-2 and falcipain-3 demonstrated strong preference for cleavage sites with Leu at the P(2 position. Second, with overlapping peptides spanning alpha and beta globin and proteolysis-dependent (18O labeling, hydrolysis was seen at many cleavage sites. Third, with intact hemoglobin, numerous cleavage products were identified. Our results suggest that hemoglobin hydrolysis by malaria parasites is not a highly ordered process, but rather proceeds with rapid cleavage by falcipains at multiple sites. However, falcipain-2 and falcipain-3 show strong specificity for P(2 Leu in small peptide substrates, in agreement with the specificity in optimized small molecule inhibitors that was identified previously. These results are consistent with a principal role of falcipain-2 and falcipain-3 in the hydrolysis of hemoglobin by P. falciparum and with the possibility of developing small molecule inhibitors with optimized specificity as antimalarial agents.

  20. Cleavage specificity analysis of six type II transmembrane serine proteases (TTSPs using PICS with proteome-derived peptide libraries.

    Olivier Barré

    Full Text Available Type II transmembrane serine proteases (TTSPs are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors.To profile the active site specificities of the TTSPs, we applied Proteomic Identification of protease Cleavage Sites (PICS. Human proteome-derived database searchable peptide libraries were assayed with six human TTSPs (matriptase, matriptase-2, matriptase-3, HAT, DESC and hepsin to simultaneously determine sequence preferences on the N-terminal non-prime (P and C-terminal prime (P' sides of the scissile bond. Prime-side cleavage products were isolated following biotinylation and identified by tandem mass spectrometry. The corresponding non-prime side sequences were derived from human proteome databases using bioinformatics. Sequencing of 2,405 individual cleaved peptides allowed for the development of the family consensus protease cleavage site specificity revealing a strong specificity for arginine in the P1 position and surprisingly a lysine in P1' position. TTSP cleavage between R↓K was confirmed using synthetic peptides. By parsing through known substrates and known structures of TTSP catalytic domains, and by modeling the remainder, structural explanations for this strong specificity were derived.Degradomics analysis of 2,405 cleavage sites revealed a similar and characteristic TTSP family specificity at the P1 and P1' positions for arginine and lysine in unfolded peptides. The prime side is important for cleavage specificity, thus making these proteases unusual within the tryptic-enzyme class that generally has overriding non-prime side specificity.

  1. Conservation of a proteinase cleavage site between an insect retrovirus (gypsy) Env protein and a baculovirus envelope fusion protein

    Pearson, Margot N.; Rohrmann, George F.

    2004-01-01

    The predicted Env protein of insect retroviruses (errantiviruses) is related to the envelope fusion protein of a major division of the Baculoviridae. The highest degree of homology is found in a region that contains a furin cleavage site in the baculovirus proteins and an adjacent sequence that has the properties of a fusion peptide. In this investigation, the homologous region in the Env protein of the gypsy retrovirus of Drosophila melanogaster (DmegypV) was investigated. Alteration of the predicted DmegypV Env proteinase cleavage site from RIAR to AIAR significantly reduced cleavage of Env in both Spodoptera frugiperda (Sf-9) and D. melanogaster (S2) cell lines. When the predicted DmegypV Env cleavage site RIAR was substituted for the cleavage sequence RRKR in the Lymantria dispar nucleopolyhedrovirus fusion protein (LD130) sequence, cleavage of the hybrid LD130 molecules still occurred, although at a reduced level. The conserved 21-amino acid sequence just downstream of the cleavage site, which is thought to be the fusion peptide in LD130, was also characterized. When this sequence from DmegypV Env was substituted for the homologous sequence in LD130, cleavage still occurred, but no fusion was observed in either cell type. In addition, although a DmegypV-Env-green fluorescent protein construct localized to cell membranes, no cell fusion was observed

  2. ADAM13 cleavage of cadherin-11 promotes CNC migration independently of the homophilic binding site.

    Abbruzzese, Genevieve; Becker, Sarah F; Kashef, Jubin; Alfandari, Dominique

    2016-07-15

    The cranial neural crest (CNC) is a highly motile population of cells that is responsible for forming the face and jaw in all vertebrates and perturbing their migration can lead to craniofacial birth defects. Cell motility requires a dynamic modification of cell-cell and cell-matrix adhesion. In the CNC, cleavage of the cell adhesion molecule cadherin-11 by ADAM13 is essential for cell migration. This cleavage generates a shed extracellular fragment of cadherin-11 (EC1-3) that possesses pro-migratory activity via an unknown mechanism. Cadherin-11 plays an important role in modulating contact inhibition of locomotion (CIL) in the CNC to regulate directional cell migration. Here, we show that while the integral cadherin-11 requires the homophilic binding site to promote CNC migration in vivo, the EC1-3 fragment does not. In addition, we show that increased ADAM13 activity or expression of the EC1-3 fragment increases CNC invasiveness in vitro and blocks the repulsive CIL response in colliding cells. This activity requires the presence of an intact homophilic binding site on the EC1-3 suggesting that the cleavage fragment may function as a competitive inhibitor of cadherin-11 adhesion in CIL but not to promote cell migration in vivo. Copyright © 2015. Published by Elsevier Inc.

  3. Single-Molecule Analysis for RISC Assembly and Target Cleavage.

    Sasaki, Hiroshi M; Tadakuma, Hisashi; Tomari, Yukihide

    2018-01-01

    RNA-induced silencing complex (RISC) is a small RNA-protein complex that mediates silencing of complementary target RNAs. Biochemistry has been successfully used to characterize the molecular mechanism of RISC assembly and function for nearly two decades. However, further dissection of intermediate states during the reactions has been warranted to fill in the gaps in our understanding of RNA silencing mechanisms. Single-molecule analysis with total internal reflection fluorescence (TIRF) microscopy is a powerful imaging-based approach to interrogate complex formation and dynamics at the individual molecule level with high sensitivity. Combining this technique with our recently established in vitro reconstitution system of fly Ago2-RISC, we have developed a single-molecule observation system for RISC assembly. In this chapter, we summarize the detailed protocol for single-molecule analysis of chaperone-assisted assembly of fly Ago2-RISC as well as its target cleavage reaction.

  4. Mutation in Spike Protein Cleavage Site and Pathogenesis of Feline Coronavirus

    Licitra, Beth N.; Millet, Jean K.; Regan, Andrew D.; Hamilton, Brian S.; Rinaldi, Vera D.; Duhamel, Gerald E.

    2013-01-01

    Feline coronaviruses (FCoV) exist as 2 biotypes: feline enteric coronavirus (FECV) and feline infectious peritonitis virus (FIPV). FECV causes subclinical infections; FIPV causes feline infectious peritonitis (FIP), a systemic and fatal disease. It is thought that mutations in FECV enable infection of macrophages, causing FIP. However, the molecular basis for this biotype switch is unknown. We examined a furin cleavage site in the region between receptor-binding (S1) and fusion (S2) domains of the spike of serotype 1 FCoV. FECV sequences were compared with FIPV sequences. All FECVs had a conserved furin cleavage motif. For FIPV, there was a correlation with the disease and >1 substitution in the S1/S2 motif. Fluorogenic peptide assays confirmed that the substitutions modulate furin cleavage. We document a functionally relevant S1/S2 mutation that arises when FIP develops in a cat. These insights into FIP pathogenesis may be useful in development of diagnostic, prevention, and treatment measures against coronaviruses. PMID:23763835

  5. Base substitutions at scissile bond sites are sufficient to alter RNA-binding and cleavage activity of RNase III.

    Kim, Kyungsub; Sim, Se-Hoon; Jeon, Che Ok; Lee, Younghoon; Lee, Kangseok

    2011-02-01

    RNase III, a double-stranded RNA-specific endoribonuclease, degrades bdm mRNA via cleavage at specific sites. To better understand the mechanism of cleavage site selection by RNase III, we performed a genetic screen for sequences containing mutations at the bdm RNA cleavage sites that resulted in altered mRNA stability using a transcriptional bdm'-'cat fusion construct. While most of the isolated mutants showed the increased bdm'-'cat mRNA stability that resulted from the inability of RNase III to cleave the mutated sequences, one mutant sequence (wt-L) displayed in vivo RNA stability similar to that of the wild-type sequence. In vivo and in vitro analyses of the wt-L RNA substrate showed that it was cut only once on the RNA strand to the 5'-terminus by RNase III, while the binding constant of RNase III to this mutant substrate was moderately increased. A base substitution at the uncleaved RNase III cleavage site in wt-L mutant RNA found in another mutant lowered the RNA-binding affinity by 11-fold and abolished the hydrolysis of scissile bonds by RNase III. Our results show that base substitutions at sites forming the scissile bonds are sufficient to alter RNA cleavage as well as the binding activity of RNase III. © 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  6. Effects of mutations in the VP2/VP4 cleavage site of Swine vesicular disease virus on RNA encapsidation and viral infectivity

    Rebel, J.M.J.; Leendertse, C.H.; Dekker, A.; Moormann, R.J.M.

    2003-01-01

    We studied VP0 cleavage of Swine vesicular disease virus (SVDV), a member of the Picornaviridae using a full-length cDNA copy of the Dutch SVDV isolate. The influences of mutations, introduced at the cleavage site of SVDV, on VP0 cleavage, RNA encapsidation and viral infection were studied. Double

  7. Cleavage sites in the polypeptide precursors of poliovirus protein P2-X

    Selmer, B.L.; Hanecak, R.; Anderson, C.W.; Wimmer, E.

    1981-01-01

    Partial amino-terminal sequence analysis has been performed on the three major polypeptide products (P2-3b, P2-5b, and P2-X) from the central region (P2) of the poliovirus polyprotein, and this analysis precisely locates the amino termini of these products with respect to the nucleotide sequence of the poliovirus RNA genome. Like most of the products of the replicase region (P3), the amino termini of P2-5b and P2-X are generated by cleavage between glutamine and glycine residues. Thus, P2-5b and P2-X are probably both produced by the action of a singly (virus-encoded.) proteinase. The amino terminus of P2-3b, on the other hand, is produced by a cleavage between the carboxy-terminal tyrosine of VP1 and the glycine encoded by nucleotides 3381-3383. This result may suggest that more than one proteolytic activity is required for the complete processing of the poliovirus polyprotein

  8. Factor VIII S373L: mutation at P1' site confers thrombin cleavage resistance, causing mild haemophilia A.

    Johnson, D J; Pemberton, S; Acquila, M; Mori, P G; Tuddenham, E G; O'Brien, D P

    1994-04-01

    A novel CRM+ mutation, factor VIII position 373 serine to leucine substitution (FVIII 373-Leu) was identified during a survey of Factor VIII (FVIII) mutations. We have purified the variant protein from the patient's plasma in order to allow further characterisation of the molecule. The CRM+ plasma contained 120% Factor VIII antigen (FVIII:Ag) and 6% Factor VIII coagulant activity (FVIII:C). After purification the mutant FVIII was subjected to thrombin proteolysis, and was thereby activated 5.6-fold compared with 7-fold for wild type molecule. Subsequently, spontaneous inactivation of the mutant was much slower than noted for wild type FVIII. Western blot analysis using monoclonal antibodies demonstrated that thrombin cleavage of FVIII 373-Leu at positions 740 and 1689 were normal but that cleavage at position 372 was completely absent. Crystallographic coordinates of the active site of thrombin complexed to fibrinopeptide A were used to explore possible mechanistic reasons for the failure of thrombin to cleave the mutant FVIII at position 372. Steric hindrance between the mutant side chain and the side chain of the P1 residue was apparent. We conclude that the functional defect of FVIII 373-Leu results from the inability of thrombin to cleave the mutant at position 372-373, and propose that this is due to steric hindrance by the side chain of leucine 373, preventing correct formation of the enzyme substrate complex.

  9. Disclosure of key stereoelectronic factors for efficient H2 binding and cleavage in the active site of [NiFe]-hydrogenases.

    Bruschi, Maurizio; Tiberti, Matteo; Guerra, Alessandro; De Gioia, Luca

    2014-02-05

    A comparative analysis of a series of DFT models of [NiFe]-hydrogenases, ranging from minimal NiFe clusters to very large systems including both the first and second coordination sphere of the bimetallic cofactor, was carried out with the aim of unraveling which stereoelectronic properties of the active site of [NiFe]-hydrogenases are crucial for efficient H2 binding and cleavage. H2 binding to the Ni-SIa redox state is energetically favored (by 4.0 kcal mol(-1)) only when H2 binds to Ni, the NiFe metal cluster is in a low spin state, and the Ni cysteine ligands have a peculiar seesaw coordination geometry, which in the enzyme is stabilized by the protein environment. The influence of the Ni coordination geometry on the H2 binding affinity was then quantitatively evaluated and rationalized analyzing frontier molecular orbitals and populations. Several plausible reaction pathways leading to H2 cleavage were also studied. It turned out that a two-step pathway, where H2 cleavage takes place on the Ni-SIa redox state of the enzyme, is characterized by very low reaction barriers and favorable reaction energies. More importantly, the seesaw coordination geometry of Ni was found to be a key feature for facile H2 cleavage. The discovery of the crucial influence of the Ni coordination geometry on H2 binding and activation in the active site of [NiFe]-hydrogenases could be exploited in the design of novel biomimetic synthetic catalysts.

  10. Isolation of recombinant phage antibodies targeting the hemagglutinin cleavage site of highly pathogenic avian influenza virus.

    Jinhua Dong

    Full Text Available Highly pathogenic avian influenza (HPAI H5N1 viruses, which have emerged in poultry and other wildlife worldwide, contain a characteristic multi-basic cleavage site (CS in the hemagglutinin protein (HA. Because this arginine-rich CS is unique among influenza virus subtypes, antibodies against this site have the potential to specifically diagnose pathogenic H5N1. By immunizing mice with the CS peptide and screening a phage display library, we isolated four antibody Fab fragment clones that specifically bind the antigen peptide and several HPAI H5N1 HA proteins in different clades. The soluble Fab fragments expressed in Escherichia coli bound the CS peptide and the H5N1 HA protein with nanomolar affinity. In an immunofluorescence assay, these Fab fragments stained cells infected with HPAI H5N1 but not those infected with a less virulent strain. Lastly, all the Fab clones could detect the CS peptide and H5N1 HA protein by open sandwich ELISA. Thus, these recombinant Fab fragments will be useful novel reagents for the rapid and specific detection of HPAI H5N1 virus.

  11. Model for how type I restriction enzymes select cleavage sites in DNA

    Studier, F.W.; Bandyopadhyay, P.K.

    1988-01-01

    Under appropriate conditions, digestion of phage T7 DNA by the type I restriction enzyme EcoK produces an orderly progression of discrete DNA fragments. All details of the fragmentation pattern can be explained on the basis of the known properties of type I enzymes, together with two further assumptions: (i) in the ATP-stimulated translocation reaction, the enzyme bound at the recognition sequence translocates DNA toward itself from both directions simultaneously; and (ii) when translocation causes neighboring enzymes to meet, they cut the DNA between them. The kinetics of digestion at 37 degree C indicates that the rate of translocation of DNA from each side of a bound enzyme is about 200 base pairs per second, and the cuts are completed within 15-25 sec of the time neighboring enzymes meet. The resulting DNA fragments each contain a single recognition site with an enzyme (or subunit) remaining bound to it. At high enzyme concentrations, such fragments can bu further degraded, apparently by cooperation between the specifically bound and excess enzymes. This model is consistent with a substantial body of previous work on the nuclease activity of EcoB and EcoK, and it explains in a simple way how cleavage sites are selected

  12. Identification of the protease cleavage sites in a reconstituted Gag polyprotein of an HERV-K(HML-2 element

    Kurth Reinhard

    2011-05-01

    Full Text Available Abstract Background The human genome harbors several largely preserved HERV-K(HML-2 elements. Although this retroviral family comes closest of all known HERVs to producing replication competent virions, mutations acquired during their chromosomal residence have rendered them incapable of expressing infectious particles. This also holds true for the HERV-K113 element that has conserved open reading frames (ORFs for all its proteins in addition to a functional LTR promoter. Uncertainty concerning the localization and impact of post-insertional mutations has greatly hampered the functional characterization of these ancient retroviruses and their proteins. However, analogous to other betaretroviruses, it is known that HERV-K(HML-2 virions undergo a maturation process during or shortly after release from the host cell. During this process, the subdomains of the Gag polyproteins are released by proteolytic cleavage, although the nature of the mature HERV-K(HML-2 Gag proteins and the exact position of the cleavage sites have until now remained unknown. Results By aligning the amino acid sequences encoded by the gag-pro-pol ORFs of HERV-K113 with the corresponding segments from 10 other well-preserved human specific elements we identified non-synonymous post-insertional mutations that have occurred in this region of the provirus. Reversion of these mutations and a partial codon optimization facilitated the large-scale production of maturation-competent HERV-K113 virus-like particles (VLPs. The Gag subdomains of purified mature VLPs were separated by reversed-phase high-pressure liquid chromatography and initially characterized using specific antibodies. Cleavage sites were identified by mass spectrometry and N-terminal sequencing and confirmed by mutagenesis. Our results indicate that the gag gene product Pr74Gag of HERV-K(HML-2 is processed to yield p15-MA (matrix, SP1 (spacer peptide of 14 amino acids, p15, p27-CA (capsid, p10-NC (nucleocapsid and two

  13. Identification of the protease cleavage sites in a reconstituted Gag polyprotein of an HERV-K(HML-2) element.

    George, Maja; Schwecke, Torsten; Beimforde, Nadine; Hohn, Oliver; Chudak, Claudia; Zimmermann, Anja; Kurth, Reinhard; Naumann, Dieter; Bannert, Norbert

    2011-05-09

    The human genome harbors several largely preserved HERV-K(HML-2) elements. Although this retroviral family comes closest of all known HERVs to producing replication competent virions, mutations acquired during their chromosomal residence have rendered them incapable of expressing infectious particles. This also holds true for the HERV-K113 element that has conserved open reading frames (ORFs) for all its proteins in addition to a functional LTR promoter. Uncertainty concerning the localization and impact of post-insertional mutations has greatly hampered the functional characterization of these ancient retroviruses and their proteins. However, analogous to other betaretroviruses, it is known that HERV-K(HML-2) virions undergo a maturation process during or shortly after release from the host cell. During this process, the subdomains of the Gag polyproteins are released by proteolytic cleavage, although the nature of the mature HERV-K(HML-2) Gag proteins and the exact position of the cleavage sites have until now remained unknown. By aligning the amino acid sequences encoded by the gag-pro-pol ORFs of HERV-K113 with the corresponding segments from 10 other well-preserved human specific elements we identified non-synonymous post-insertional mutations that have occurred in this region of the provirus. Reversion of these mutations and a partial codon optimization facilitated the large-scale production of maturation-competent HERV-K113 virus-like particles (VLPs). The Gag subdomains of purified mature VLPs were separated by reversed-phase high-pressure liquid chromatography and initially characterized using specific antibodies. Cleavage sites were identified by mass spectrometry and N-terminal sequencing and confirmed by mutagenesis. Our results indicate that the gag gene product Pr74Gag of HERV-K(HML-2) is processed to yield p15-MA (matrix), SP1 (spacer peptide of 14 amino acids), p15, p27-CA (capsid), p10-NC (nucleocapsid) and two C-terminally encoded glutamine- and

  14. A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites

    Nielsen, Henrik; Engelbrecht, Jacob; Brunak, Søren

    1997-01-01

    We have developed a new method for the identication of signal peptides and their cleavage sites based on neural networks trained on separate sets of prokaryotic and eukaryotic sequences. The method performs signicantly better than previous prediction schemes, and can easily be applied to genome...

  15. Acquisition of a novel eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site confers intracellular cleavage of an H7N7 influenza virus hemagglutinin

    Hamilton, Brian S.; Sun, Xiangjie; Chung, Changik; Whittaker, Gary R.

    2012-01-01

    A critical feature of highly pathogenic avian influenza viruses (H5N1 and H7N7) is the efficient intracellular cleavage of the hemagglutinin (HA) protein. H7N7 viruses also exist in equine species, and a unique feature of the equine H7N7 HA is the presence of an eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site. Here, we show that three histidine residues within the unique insertion of the equine H7N7 HA are essential for intracellular cleavage. An asparagine residue within the insertion-derived glycosylation site was also found to be essential for intracellular cleavage. The presence of the histidine residues also appear to be involved in triggering fusion, since mutation of the histidine residues resulted in a destabilizing effect. Importantly, the addition of a tetrabasic site and the eleven amino acid insertion conferred efficient intracellular cleavage to the HA of an H7N3 low pathogenicity avian influenza virus. Our studies show that acquisition of the eleven amino acid insertion offers an alternative mechanism for intracellular cleavage of influenza HA.

  16. Acquisition of a novel eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site confers intracellular cleavage of an H7N7 influenza virus hemagglutinin

    Hamilton, Brian S.; Sun, Xiangjie; Chung, Changik [Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca NY 14853 (United States); New York Center of Excellence for Influenza Research and Surveillance, University of Rochester Medical Center, Rochester NY 14627 (United States); Whittaker, Gary R., E-mail: grw7@cornell.edu [Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca NY 14853 (United States); New York Center of Excellence for Influenza Research and Surveillance, University of Rochester Medical Center, Rochester NY 14627 (United States)

    2012-12-05

    A critical feature of highly pathogenic avian influenza viruses (H5N1 and H7N7) is the efficient intracellular cleavage of the hemagglutinin (HA) protein. H7N7 viruses also exist in equine species, and a unique feature of the equine H7N7 HA is the presence of an eleven amino acid insertion directly N-terminal to a tetrabasic cleavage site. Here, we show that three histidine residues within the unique insertion of the equine H7N7 HA are essential for intracellular cleavage. An asparagine residue within the insertion-derived glycosylation site was also found to be essential for intracellular cleavage. The presence of the histidine residues also appear to be involved in triggering fusion, since mutation of the histidine residues resulted in a destabilizing effect. Importantly, the addition of a tetrabasic site and the eleven amino acid insertion conferred efficient intracellular cleavage to the HA of an H7N3 low pathogenicity avian influenza virus. Our studies show that acquisition of the eleven amino acid insertion offers an alternative mechanism for intracellular cleavage of influenza HA.

  17. A camel-derived MERS-CoV with a variant spike protein cleavage site and distinct fusion activation properties

    Millet, Jean Kaoru; Goldstein, Monty E; Labitt, Rachael N; Hsu, Hung-Lun; Daniel, Susan; Whittaker, Gary R

    2016-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) continues to circulate in both humans and camels, and the origin and evolution of the virus remain unclear. Here we characterize the spike protein of a camel-derived MERS-CoV (NRCE-HKU205) identified in 2013, early in the MERS outbreak. NRCE-HKU205 spike protein has a variant cleavage motif with regard to the S2′ fusion activation site—notably, a novel substitution of isoleucine for the otherwise invariant serine at the critical P1′ cleavage site position. The substitutions resulted in a loss of furin-mediated cleavage, as shown by fluorogenic peptide cleavage and western blot assays. Cell–cell fusion and pseudotyped virus infectivity assays demonstrated that the S2′ substitutions decreased spike-mediated fusion and viral entry. However, cathepsin and trypsin-like protease activation were retained, albeit with much reduced efficiency compared with the prototypical EMC/2012 human strain. We show that NRCE-HKU205 has more limited fusion activation properties possibly resulting in more restricted viral tropism and may represent an intermediate in the complex pattern of MERS-CoV ecology and evolution. PMID:27999426

  18. Protein cleavage strategies for an improved analysis of the membrane proteome

    Poetsch Ansgar

    2006-03-01

    Full Text Available Abstract Background Membrane proteins still remain elusive in proteomic studies. This is in part due to the distribution of the amino acids lysine and arginine, which are less frequent in integral membrane proteins and almost absent in transmembrane helices. As these amino acids are cleavage targets for the commonly used protease trypsin, alternative cleavage conditions, which should improve membrane protein analysis, were tested by in silico digestion for the three organisms Saccharomyces cerevisiae, Halobacterium sp. NRC-1, and Corynebacterium glutamicum as hallmarks for eukaryotes, archea and eubacteria. Results For the membrane proteomes from all three analyzed organisms, we identified cleavage conditions that achieve better sequence and proteome coverage than trypsin. Greater improvement was obtained for bacteria than for yeast, which was attributed to differences in protein size and GRAVY. It was demonstrated for bacteriorhodopsin that the in silico predictions agree well with the experimental observations. Conclusion For all three examined organisms, it was found that a combination of chymotrypsin and staphylococcal peptidase I gave significantly better results than trypsin. As some of the improved cleavage conditions are not more elaborate than trypsin digestion and have been proven useful in practice, we suppose that the cleavage at both hydrophilic and hydrophobic amino acids should facilitate in general the analysis of membrane proteins for all organisms.

  19. Computational analysis and modeling of cleavage by the immunoproteasome and the constitutive proteasome

    Lafuente Esther M

    2010-09-01

    Full Text Available Abstract Background Proteasomes play a central role in the major histocompatibility class I (MHCI antigen processing pathway. They conduct the proteolytic degradation of proteins in the cytosol, generating the C-terminus of CD8 T cell epitopes and MHCI-peptide ligands (P1 residue of cleavage site. There are two types of proteasomes, the constitutive form, expressed in most cell types, and the immunoproteasome, which is constitutively expressed in mature dendritic cells. Protective CD8 T cell epitopes are likely generated by the immunoproteasome and the constitutive proteasome, and here we have modeled and analyzed the cleavage by these two proteases. Results We have modeled the immunoproteasome and proteasome cleavage sites upon two non-overlapping sets of peptides consisting of 553 CD8 T cell epitopes, naturally processed and restricted by human MHCI molecules, and 382 peptides eluted from human MHCI molecules, respectively, using N-grams. Cleavage models were generated considering different epitope and MHCI-eluted fragment lengths and the same number of C-terminal flanking residues. Models were evaluated in 5-fold cross-validation. Judging by the Mathew's Correlation Coefficient (MCC, optimal cleavage models for the proteasome (MCC = 0.43 ± 0.07 and the immunoproteasome (MCC = 0.36 ± 0.06 were obtained from 12-residue peptide fragments. Using an independent dataset consisting of 137 HIV1-specific CD8 T cell epitopes, the immunoproteasome and proteasome cleavage models achieved MCC values of 0.30 and 0.18, respectively, comparatively better than those achieved by related methods. Using ROC analyses, we have also shown that, combined with MHCI-peptide binding predictions, cleavage predictions by the immunoproteasome and proteasome models significantly increase the discovery rate of CD8 T cell epitopes restricted by different MHCI molecules, including A*0201, A*0301, A*2402, B*0702, B*2705. Conclusions We have developed models that are specific

  20. Coronavirus 3CL(pro) proteinase cleavage sites: Possible relevance to SARS virus pathology

    Kiemer, Lars; Lund, Ole; Brunak, Søren

    2004-01-01

    the picornaviruses it is known that pathology is related to proteolytic cleavage of host proteins by viral proteinases. Furthermore, several studies indicate that virus proliferation can be arrested using specific proteinase inhibitors supporting the belief that proteinases are indeed important during infection...

  1. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites

    Nielsen, Henrik; Engelbrecht, Jacob; Brunak, Søren

    1997-01-01

    We have developed a new method for the identification of signal peptides and their cleavage based on neural networks trained on separate sets of prokaryotic and eukaryotic sequence. The method performs significantly better than previous prediction schemes and can easily be applied on genome...

  2. Sequence motif upstream of the Hendra virus fusion protein cleavage site is not sufficient to promote efficient proteolytic processing

    Craft, Willie Warren; Dutch, Rebecca Ellis

    2005-01-01

    The Hendra virus fusion (HeV F) protein is synthesized as a precursor, F 0 , and proteolytically cleaved into the mature F 1 and F 2 heterodimer, following an HDLVDGVK 109 motif. This cleavage event is required for fusogenic activity. To determine the amino acid requirements for processing of the HeV F protein, we constructed multiple mutants. Individual and simultaneous alanine substitutions of the eight residues immediately upstream of the cleavage site did not eliminate processing. A chimeric SV5 F protein in which the furin site was substituted for the VDGVK 109 motif of the HeV F protein was not processed but was expressed on the cell surface. Another chimeric SV5 F protein containing the HDLVDGVK 109 motif of the HeV F protein underwent partial cleavage. These data indicate that the upstream region can play a role in protease recognition, but is neither absolutely required nor sufficient for efficient processing of the HeV F protein

  3. Molecular pathogenesis of H5 highly pathogenic avian influenza: the role of the haemagglutinin cleavage site motif

    Luczo, Jasmina M.; Stambas, John; Durr, Peter A.; Michalski, Wojtek P.

    2015-01-01

    Summary The emergence of H5N1 highly pathogenic avian influenza has caused a heavy socio‐economic burden through culling of poultry to minimise human and livestock infection. Although human infections with H5N1 have to date been limited, concerns for the pandemic potential of this zoonotic virus have been greatly intensified following experimental evidence of aerosol transmission of H5N1 viruses in a mammalian infection model. In this review, we discuss the dominance of the haemagglutinin cleavage site motif as a pathogenicity determinant, the host‐pathogen molecular interactions driving cleavage activation, reverse genetics manipulations and identification of residues key to haemagglutinin cleavage site functionality and the mechanisms of cell and tissue damage during H5N1 infection. We specifically focus on the disease in chickens, as it is in this species that high pathogenicity frequently evolves and from which transmission to the human population occurs. With >75% of emerging infectious diseases being of zoonotic origin, it is necessary to understand pathogenesis in the primary host to explain spillover events into the human population. © 2015 The Authors. Reviews in Medical Virology published by John Wiley & Sons Ltd. PMID:26467906

  4. Identification of E-cadherin signature motifs functioning as cleavage sites for Helicobacter pylori HtrA

    Schmidt, Thomas P.; Perna, Anna M.; Fugmann, Tim; Böhm, Manja; Jan Hiss; Haller, Sarah; Götz, Camilla; Tegtmeyer, Nicole; Hoy, Benjamin; Rau, Tilman T.; Neri, Dario; Backert, Steffen; Schneider, Gisbert; Wessler, Silja

    2016-03-01

    The cell adhesion protein and tumour suppressor E-cadherin exhibits important functions in the prevention of gastric cancer. As a class-I carcinogen, Helicobacter pylori (H. pylori) has developed a unique strategy to interfere with E-cadherin functions. In previous studies, we have demonstrated that H. pylori secretes the protease high temperature requirement A (HtrA) which cleaves off the E-cadherin ectodomain (NTF) on epithelial cells. This opens cell-to-cell junctions, allowing bacterial transmigration across the polarised epithelium. Here, we investigated the molecular mechanism of the HtrA-E-cadherin interaction and identified E-cadherin cleavage sites for HtrA. Mass-spectrometry-based proteomics and Edman degradation revealed three signature motifs containing the [VITA]-[VITA]-x-x-D-[DN] sequence pattern, which were preferentially cleaved by HtrA. Based on these sites, we developed a substrate-derived peptide inhibitor that selectively bound and inhibited HtrA, thereby blocking transmigration of H. pylori. The discovery of HtrA-targeted signature sites might further explain why we detected a stable 90 kDa NTF fragment during H. pylori infection, but also additional E-cadherin fragments ranging from 105 kDa to 48 kDa in in vitro cleavage experiments. In conclusion, HtrA targets E-cadherin signature sites that are accessible in in vitro reactions, but might be partially masked on epithelial cells through functional homophilic E-cadherin interactions.

  5. Positioning the 5'-flap junction in the active site controls the rate of flap endonuclease-1-catalyzed DNA cleavage

    Song, Bo

    2018-02-09

    Flap endonucleases catalyze cleavage of single-stranded DNA flaps formed during replication, repair and recombination, and are therefore essential for genome processing and stability. Recent crystal structures of DNA-bound human flap endonuclease (hFEN1) offer new insights into how conformational changes in the DNA and hFEN1 may facilitate the reaction mechanism. For example, previous biochemical studies of DNA conformation performed under non-catalytic conditions with Ca2+ have suggested that base unpairing at the 5\\'-flap:template junction is an important step in the reaction, but the new structural data suggest otherwise. To clarify the role of DNA changes in the kinetic mechanism, we measured a series of transient steps - from substrate binding to product release - during the hFEN1-catalyzed reaction in the presence of Mg2+. We found that while hFEN1 binds and bends DNA at a fast, diffusion-limited rate, much slower Mg2+-dependent conformational changes in DNA around the active site are subsequently necessary and rate-limiting for 5\\'-flap cleavage. These changes are reported overall by fluorescence of 2-aminopurine at the 5\\'-flap:template junction, indicating that local DNA distortion (e.g., disruption of base stacking observed in structures), associated with positioning the 5\\'-flap scissile phosphodiester bond in the hFEN1 active site, controls catalysis. hFEN1 residues with distinct roles in the catalytic mechanism, including those binding metal ions (Asp-34, Asp-181), steering the 5\\'-flap through the active site and binding the scissile phosphate (Lys-93, Arg-100), and stacking against the base 5\\' to the scissile phosphate (Tyr-40), all contribute to these rate-limiting conformational changes, ensuring efficient and specific cleavage of 5\\'-flaps.

  6. Positioning the 5'-flap junction in the active site controls the rate of flap endonuclease-1-catalyzed DNA cleavage

    Song, Bo; Hamdan, Samir; Hingorani, Manju M

    2018-01-01

    Flap endonucleases catalyze cleavage of single-stranded DNA flaps formed during replication, repair and recombination, and are therefore essential for genome processing and stability. Recent crystal structures of DNA-bound human flap endonuclease (hFEN1) offer new insights into how conformational changes in the DNA and hFEN1 may facilitate the reaction mechanism. For example, previous biochemical studies of DNA conformation performed under non-catalytic conditions with Ca2+ have suggested that base unpairing at the 5'-flap:template junction is an important step in the reaction, but the new structural data suggest otherwise. To clarify the role of DNA changes in the kinetic mechanism, we measured a series of transient steps - from substrate binding to product release - during the hFEN1-catalyzed reaction in the presence of Mg2+. We found that while hFEN1 binds and bends DNA at a fast, diffusion-limited rate, much slower Mg2+-dependent conformational changes in DNA around the active site are subsequently necessary and rate-limiting for 5'-flap cleavage. These changes are reported overall by fluorescence of 2-aminopurine at the 5'-flap:template junction, indicating that local DNA distortion (e.g., disruption of base stacking observed in structures), associated with positioning the 5'-flap scissile phosphodiester bond in the hFEN1 active site, controls catalysis. hFEN1 residues with distinct roles in the catalytic mechanism, including those binding metal ions (Asp-34, Asp-181), steering the 5'-flap through the active site and binding the scissile phosphate (Lys-93, Arg-100), and stacking against the base 5' to the scissile phosphate (Tyr-40), all contribute to these rate-limiting conformational changes, ensuring efficient and specific cleavage of 5'-flaps.

  7. The prototype HIV-1 maturation inhibitor, bevirimat, binds to the CA-SP1 cleavage site in immature Gag particles

    Nguyen Albert T

    2011-12-01

    Full Text Available Abstract Background Bevirimat, the prototype Human Immunodeficiency Virus type 1 (HIV-1 maturation inhibitor, is highly potent in cell culture and efficacious in HIV-1 infected patients. In contrast to inhibitors that target the active site of the viral protease, bevirimat specifically inhibits a single cleavage event, the final processing step for the Gag precursor where p25 (CA-SP1 is cleaved to p24 (CA and SP1. Results In this study, photoaffinity analogs of bevirimat and mass spectrometry were employed to map the binding site of bevirimat to Gag within immature virus-like particles. Bevirimat analogs were found to crosslink to sequences overlapping, or proximal to, the CA-SP1 cleavage site, consistent with previous biochemical data on the effect of bevirimat on Gag processing and with genetic data from resistance mutations, in a region predicted by NMR and mutational studies to have α-helical character. Unexpectedly, a second region of interaction was found within the Major Homology Region (MHR. Extensive prior genetic evidence suggests that the MHR is critical for virus assembly. Conclusions This is the first demonstration of a direct interaction between the maturation inhibitor, bevirimat, and its target, Gag. Information gained from this study sheds light on the mechanisms by which the virus develops resistance to this class of drug and may aid in the design of next-generation maturation inhibitors.

  8. Structure-function analysis of Staphylococcus aureus amidase reveals the determinants of peptidoglycan recognition and cleavage.

    Büttner, Felix Michael; Zoll, Sebastian; Nega, Mulugeta; Götz, Friedrich; Stehle, Thilo

    2014-04-18

    The bifunctional major autolysin AtlA of Staphylococcus aureus cleaves the bacterium's peptidoglycan network (PGN) at two distinct sites during cell division. Deletion of the enzyme results in large cell clusters with disordered division patterns, indicating that AtlA could be a promising target for the development of new antibiotics. One of the two functions of AtlA is performed by the N-acetylmuramyl-l-alanine amidase AmiA, which cleaves the bond between the carbohydrate and the peptide moieties of PGN. To establish the structural requirements of PGN recognition and the enzymatic mechanism of cleavage, we solved the crystal structure of the catalytic domain of AmiA (AmiA-cat) in complex with a peptidoglycan-derived ligand at 1.55 Å resolution. The peptide stem is clearly visible in the structure, forming extensive contacts with protein residues by docking into an elongated groove. Less well defined electron density and the analysis of surface features indicate likely positions of the carbohydrate backbone and the pentaglycine bridge. Substrate specificity analysis supports the importance of the pentaglycine bridge for fitting into the binding cleft of AmiA-cat. PGN of S. aureus with l-lysine tethered with d-alanine via a pentaglycine bridge is completely hydrolyzed, whereas PGN of Bacillus subtilis with meso-diaminopimelic acid directly tethered with d-alanine is not hydrolyzed. An active site mutant, H370A, of AmiA-cat was completely inactive, providing further support for the proposed catalytic mechanism of AmiA. The structure reported here is not only the first of any bacterial amidase in which both the PGN component and the water molecule that carries out the nucleophilic attack on the carbonyl carbon of the scissile bond are present; it is also the first peptidoglycan amidase complex structure of an important human pathogen.

  9. Structure-Function Analysis of Staphylococcus aureus Amidase Reveals the Determinants of Peptidoglycan Recognition and Cleavage*

    Büttner, Felix Michael; Zoll, Sebastian; Nega, Mulugeta; Götz, Friedrich; Stehle, Thilo

    2014-01-01

    The bifunctional major autolysin AtlA of Staphylococcus aureus cleaves the bacterium's peptidoglycan network (PGN) at two distinct sites during cell division. Deletion of the enzyme results in large cell clusters with disordered division patterns, indicating that AtlA could be a promising target for the development of new antibiotics. One of the two functions of AtlA is performed by the N-acetylmuramyl-l-alanine amidase AmiA, which cleaves the bond between the carbohydrate and the peptide moieties of PGN. To establish the structural requirements of PGN recognition and the enzymatic mechanism of cleavage, we solved the crystal structure of the catalytic domain of AmiA (AmiA-cat) in complex with a peptidoglycan-derived ligand at 1.55 Å resolution. The peptide stem is clearly visible in the structure, forming extensive contacts with protein residues by docking into an elongated groove. Less well defined electron density and the analysis of surface features indicate likely positions of the carbohydrate backbone and the pentaglycine bridge. Substrate specificity analysis supports the importance of the pentaglycine bridge for fitting into the binding cleft of AmiA-cat. PGN of S. aureus with l-lysine tethered with d-alanine via a pentaglycine bridge is completely hydrolyzed, whereas PGN of Bacillus subtilis with meso-diaminopimelic acid directly tethered with d-alanine is not hydrolyzed. An active site mutant, H370A, of AmiA-cat was completely inactive, providing further support for the proposed catalytic mechanism of AmiA. The structure reported here is not only the first of any bacterial amidase in which both the PGN component and the water molecule that carries out the nucleophilic attack on the carbonyl carbon of the scissile bond are present; it is also the first peptidoglycan amidase complex structure of an important human pathogen. PMID:24599952

  10. SVM-based prediction of propeptide cleavage sites in spider toxins identifies toxin innovation in an Australian tarantula.

    Emily S W Wong

    Full Text Available Spider neurotoxins are commonly used as pharmacological tools and are a popular source of novel compounds with therapeutic and agrochemical potential. Since venom peptides are inherently toxic, the host spider must employ strategies to avoid adverse effects prior to venom use. It is partly for this reason that most spider toxins encode a protective proregion that upon enzymatic cleavage is excised from the mature peptide. In order to identify the mature toxin sequence directly from toxin transcripts, without resorting to protein sequencing, the propeptide cleavage site in the toxin precursor must be predicted bioinformatically. We evaluated different machine learning strategies (support vector machines, hidden Markov model and decision tree and developed an algorithm (SpiderP for prediction of propeptide cleavage sites in spider toxins. Our strategy uses a support vector machine (SVM framework that combines both local and global sequence information. Our method is superior or comparable to current tools for prediction of propeptide sequences in spider toxins. Evaluation of the SVM method on an independent test set of known toxin sequences yielded 96% sensitivity and 100% specificity. Furthermore, we sequenced five novel peptides (not used to train the final predictor from the venom of the Australian tarantula Selenotypus plumipes to test the accuracy of the predictor and found 80% sensitivity and 99.6% 8-mer specificity. Finally, we used the predictor together with homology information to predict and characterize seven groups of novel toxins from the deeply sequenced venom gland transcriptome of S. plumipes, which revealed structural complexity and innovations in the evolution of the toxins. The precursor prediction tool (SpiderP is freely available on ArachnoServer (http://www.arachnoserver.org/spiderP.html, a web portal to a comprehensive relational database of spider toxins. All training data, test data, and scripts used are available from

  11. The Conserved ATM Kinase RAG2-S365 Phosphorylation Site Limits Cleavage Events in Individual Cells Independent of Any Repair Defect

    Susannah L. Hewitt

    2017-10-01

    Full Text Available Many DNA lesions associated with lymphoid malignancies are linked to off-target cleavage by the RAG1/2 recombinase. However, off-target cleavage has mostly been analyzed in the context of DNA repair defects, confounding any mechanistic understanding of cleavage deregulation. We identified a conserved SQ phosphorylation site on RAG2 365 to 366 that is involved in feedback control of RAG cleavage. Mutation of serine 365 to a non-phosphorylatable alanine permits bi-allelic and bi-locus RAG-mediated breaks in the same cell, leading to reciprocal translocations. This phenomenon is analogous to the phenotype we described for ATM kinase inactivation. Here, we establish deregulated cleavage itself as a driver of chromosomal instability without the associated repair defect. Intriguingly, a RAG2-S365E phosphomimetic rescues the deregulated cleavage of ATM inactivation, reducing the incidence of reciprocal translocations. These data support a model in which feedback control of cleavage and maintenance of genome stability involves ATM-mediated phosphorylation of RAG2.

  12. Functional role of proteolytic cleavage at arginine-275 of human tissue plasminogen activator as assessed by site-directed mutagenesis

    Tate, K.M.; Higgins, D.L.; Holmes, W.E.; Winkler, M.E.; Heyneker, H.L.; Vehar, G.A.

    1987-01-01

    Activation of the zymogen form of a serine protease is associated with a conformational change that follows proteolysis at a specific site. Tissue-type plasminogen activator (t-PA) is homologous to mammalian serine proteases and contains an apparent activation cleavage site at arginine-275. To clarify the functional consequences of cleavage at arginine-275 of t-PA, site-specific mutagenesis was performed to convert arginine-275 to a glutamic acid. The mutant enzyme (designated Arg-275 → Glu t-PA) could be converted to the two-chain form by Staphylococcus aureus V8 protease but not by plasmin. The one-chain form was 8 times less active against the tripeptide substrate H-D-isoleucyl-L-prolyl-L-arginine-rho-nitroanilide (S-2288), and the ability of the enzyme to activate plasminogen in the absence of fibrinogen was reduced 20-50 times compared to the two-chain form. In contrast, one-chain Arg-275 → Glu t-PA has equal activity to the two-chain form when assayed in the presence of physiological levels of fibrinogen and plasminogen. Fibrin bound significantly more of the one-chain form of t-PA than the two-chain form for both the wild-type and mutated enzymes. One- and two-chain forms of the wild-type and mutated plasminogen activators slowly formed complexes with plasma protease inhibitors, although the one-chain forms showed decreased complex formation with → 2 -macroglobulin. The one-chain form of t-PA therefore is fully functional under physiologic conditions and has a increased fibrin binding compared to the two-chain form

  13. A Molecular Sensor To Characterize Arenavirus Envelope Glycoprotein Cleavage by Subtilisin Kexin Isozyme 1/Site 1 Protease

    Oppliger, Joel; da Palma, Joel Ramos; Burri, Dominique J.; Khatib, Abdel-Majid; Spiropoulou, Christina F.

    2015-01-01

    ABSTRACT Arenaviruses are emerging viruses including several causative agents of severe hemorrhagic fevers in humans. The advent of next-generation sequencing technology has greatly accelerated the discovery of novel arenavirus species. However, for many of these viruses, only genetic information is available, and their zoonotic disease potential remains unknown. During the arenavirus life cycle, processing of the viral envelope glycoprotein precursor (GPC) by the cellular subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P) is crucial for productive infection. The ability of newly emerging arenaviruses to hijack human SKI-1/S1P appears, therefore, to be a requirement for efficient zoonotic transmission and human disease potential. Here we implement a newly developed cell-based molecular sensor for SKI-1/S1P to characterize the processing of arenavirus GPC-derived target sequences by human SKI-1/S1P in a quantitative manner. We show that only nine amino acids flanking the putative cleavage site are necessary and sufficient to accurately recapitulate the efficiency and subcellular location of arenavirus GPC processing. In a proof of concept, our sensor correctly predicts efficient processing of the GPC of the newly emergent pathogenic Lujo virus by human SKI-1/S1P and defines the exact cleavage site. Lastly, we employed our sensor to show efficient GPC processing of a panel of pathogenic and nonpathogenic New World arenaviruses, suggesting that GPC cleavage represents no barrier for zoonotic transmission of these pathogens. Our SKI-1/S1P sensor thus represents a rapid and robust test system for assessment of the processing of putative cleavage sites derived from the GPCs of newly discovered arenavirus by the SKI-1/S1P of humans or any other species, based solely on sequence information. IMPORTANCE Arenaviruses are important emerging human pathogens that can cause severe hemorrhagic fevers with high mortality in humans. A crucial step in productive arenavirus

  14. Two tandem RNase III cleavage sites determine betT mRNA stability in response to osmotic stress in Escherichia coli.

    Minji Sim

    Full Text Available While identifying genes regulated by ribonuclease III (RNase III in Escherichia coli, we observed that steady-state levels of betT mRNA, which encodes a transporter mediating the influx of choline, are dependent on cellular concentrations of RNase III. In the present study, we also observed that steady-state levels of betT mRNA are dependent on RNase III activity upon exposure to osmotic stress, indicating the presence of cis-acting elements controlled by RNase III in betT mRNA. Primer extension analyses of betT mRNA revealed two tandem RNase III cleavage sites in its stem-loop region, which were biochemically confirmed via in vitro cleavage assays. Analyses of cleavage sites suggested the stochastic selection of cleavage sites by RNase III, and mutational analyses indicated that RNase III cleavage at either site individually is insufficient for efficient betT mRNA degradation. In addition, both the half-life and abundance of betT mRNA were significantly increased in association with decreased RNase III activity under hyper-osmotic stress conditions. Our findings demonstrate that betT mRNA stability is controlled by RNase III at the post-transcriptional level under conditions of osmotic stress.

  15. Identification of Cleavage Sites Recognized by the 3C-Like Cysteine Protease within the Two Polyproteins of Strawberry Mottle Virus

    Hélène Sanfaçon

    2017-04-01

    Full Text Available Strawberry mottle virus (SMoV, family Secoviridae, order Picornavirales is one of several viruses found in association with strawberry decline disease in Eastern Canada. The SMoV genome consists of two positive-sense single-stranded RNAs, each encoding one large polyprotein. The RNA1 polyprotein (P1 includes the domains for a putative helicase, a VPg, a 3C-like cysteine protease and an RNA-dependent RNA polymerase at its C-terminus, and one or two protein domains at its N-terminus. The RNA2 polyprotein (P2 is predicted to contain the domains for a movement protein (MP and one or several coat proteins at its N-terminus, and one or more additional domains for proteins of unknown function at its C-terminus. The RNA1-encoded 3C-like protease is presumed to cleave the two polyproteins in cis (P1 and in trans (P2. Using in vitro processing assays, we systematically scanned the two polyproteins for cleavage sites recognized by this protease. We identified five cis-cleavage sites in P1, with cleavage between the putative helicase and VPg domains being the most efficient. The presence of six protein domains in the SMoV P1, including two upstream of the putative helicase domain, is a feature shared with nepoviruses but not with comoviruses. Results from trans-cleavage assays indicate that the RNA1-encoded 3C-like protease recognized a single cleavage site, which was between the predicted MP and coat protein domains in the P2 polyprotein. The cleavage site consensus sequence for the SMoV 3C-like protease is AxE (E or Q/(G or S.

  16. Defining a similarity threshold for a functional proteinsequence pattern: The signal peptide cleavage site

    Nielsen, Henrik; Engelbrecht, Jacob; von Heijne, Gunnar

    1996-01-01

    When preparing data sets of amino acid or nucleotide sequences it is necessary to exclude redundant or homologous sequences in order to avoid overestimating the predictive performance of an algorithm. For some time methods for doing this have been available in the area of protein structure...... prediction. We have developed a similar procedure based on pair-wise alignments for sequences with functional sites. We show how a correlation coefficient between sequence similarity and functional homology can be used to compare the efficiency of different similarity measures and choose a nonarbitrary...

  17. A consistency-based feature selection method allied with linear SVMs for HIV-1 protease cleavage site prediction.

    Orkun Oztürk

    Full Text Available BACKGROUND: Predicting type-1 Human Immunodeficiency Virus (HIV-1 protease cleavage site in protein molecules and determining its specificity is an important task which has attracted considerable attention in the research community. Achievements in this area are expected to result in effective drug design (especially for HIV-1 protease inhibitors against this life-threatening virus. However, some drawbacks (like the shortage of the available training data and the high dimensionality of the feature space turn this task into a difficult classification problem. Thus, various machine learning techniques, and specifically several classification methods have been proposed in order to increase the accuracy of the classification model. In addition, for several classification problems, which are characterized by having few samples and many features, selecting the most relevant features is a major factor for increasing classification accuracy. RESULTS: We propose for HIV-1 data a consistency-based feature selection approach in conjunction with recursive feature elimination of support vector machines (SVMs. We used various classifiers for evaluating the results obtained from the feature selection process. We further demonstrated the effectiveness of our proposed method by comparing it with a state-of-the-art feature selection method applied on HIV-1 data, and we evaluated the reported results based on attributes which have been selected from different combinations. CONCLUSION: Applying feature selection on training data before realizing the classification task seems to be a reasonable data-mining process when working with types of data similar to HIV-1. On HIV-1 data, some feature selection or extraction operations in conjunction with different classifiers have been tested and noteworthy outcomes have been reported. These facts motivate for the work presented in this paper. SOFTWARE AVAILABILITY: The software is available at http

  18. The Role of G-Protein-Coupled Receptor Proteolysis Site Cleavage of Polycystin-1 in Renal Physiology and Polycystic Kidney Disease

    Marie Trudel

    2016-01-01

    Full Text Available Polycystin-1 (PC1 plays an essential role in renal tubular morphogenesis, and PC1 dysfunction causes human autosomal dominant polycystic kidney disease. A fundamental characteristic of PC1 is post-translational modification via cleavage at the juxtamembrane GPCR proteolysis site (GPS motif that is part of the larger GAIN domain. Given the considerable biochemical complexity of PC1 molecules generated in vivo by this process, GPS cleavage has several profound implications on the intracellular trafficking and localization in association with their particular function. The critical nature of GPS cleavage is further emphasized by the increasing numbers of PKD1 mutations that significantly affect this cleavage process. The GAIN domain with the GPS motif therefore represents the key structural element with fundamental importance for PC1 and might be polycystic kidney disease’s (PKD Achilles’ heel in a large spectrum of PKD1 missense mutations. We highlight the central roles of PC1 cleavage for the regulation of its biogenesis, intracellular trafficking and function, as well as its significance in polycystic kidney disease.

  19. A catalytic metal ion interacts with the cleavage site G•U wobble in the HDV ribozyme†

    Chen, Jui-Hui; Gong, Bo; Bevilacqua, Philip C.; Carey, Paul R.; Golden, Barbara L.

    2009-01-01

    The HDV ribozyme self-cleaves by a chemical mechanism involving general acid-base catalysis to generate a 2′,3′-cyclic phosphate and a 5′-hydroxyl termini. Biochemical studies from several laboratories have implicated C75 as the general acid and hydrated magnesium as the general base. We have previously shown that C75 has a pKa shifted > 2 pH units toward neutrality [Gong, B., Chen, J. H., Chase, E., Chadalavada, D. M., Yajima, R., Golden, B. L., Bevilacqua, P. C., and Carey, P. R. (2007) J. Am. Chem. Soc. 129, 13335–13342.], while in crystal structures, it is well-positioned for proton transfer. However no crystallographic evidence for a hydrated magnesium poised to serve as a general base in the reaction has been observed in high-resolution crystal structures of various reaction states and mutants. Herein, we use solution kinetic experiments and parallel Raman crystallographic studies to examine the effects of pH on rate and Mg2+-binding properties of wild-type and 7-deazaguanosine mutants of the HDV ribozyme. These data suggest that a previously-unobserved hydrated magnesium ion interacts with the N7 of the cleavage site G•U wobble base pair. Integrating this metal ion binding site with the available crystal structures provides a new three-dimensional model for the active site of the ribozyme that accommodates all available biochemical data and appears competent for catalysis. The position of this metal is consistent with a role of a magnesium-bound hydroxide as a general base as dictated by biochemical data. PMID:19178151

  20. Engineering D-Amino Acid Containing Collagen Like Peptide at the Cleavage Site of Clostridium histolyticum Collagenase for Its Inhibition.

    Punitha Velmurugan

    Full Text Available Collagenase is an important enzyme which plays an important role in degradation of collagen in wound healing, cancer metastasis and even in embryonic development. However, the mechanism of this degradation has not yet been completely understood. In the field of biomedical and protein engineering, the design and development of new peptide based materials is of main concern. In the present work an attempt has been made to study the effect of DAla in collagen like peptide (imino-poor region of type I collagen on the structure and stability of peptide against enzyme hydrolysis. Effect of replacement of DAla in the collagen like peptide has been studied using circular dichroic spectroscopy (CD. Our findings suggest that, DAla substitution leads to conformational changes in the secondary structure and favours the formation of polyproline II conformation than its L-counterpart in the imino-poor region of collagen like peptides. Change in the chirality of alanine at the cleavage site of collagenase in the imino-poor region inhibits collagenolytic activity. This may find application in design of peptides and peptidomimics for enzyme-substrate interaction, specifically with reference to collagen and other extra cellular matrix proteins.

  1. Mutations near the cleavage site of enterocin NKR-5-3B prepeptide reveal new insights into its biosynthesis.

    Perez, Rodney H; Sugino, Haruki; Ishibashi, Naoki; Zendo, Takeshi; Wilaipun, Pongtep; Leelawatcharamas, Vichien; Nakayama, Jiro; Sonomoto, Kenji

    2017-04-01

    Enterocin NKR-5-3B (Ent53B) is a 64-residue novel circular bacteriocin synthesized from an 87-residue prepeptide. Albeit through a still unknown mechanism, the EnkB1234 biosynthetic enzyme complex processes the prepeptide to yield its mature active, circular form. To gain insights into the key region/residue that plays a role in Ent53 maturation, several mutations near the cleavage site on the precursor peptide were generated. The interaction of the precursor peptide and EnkB1234 appeared to be hydrophobic in nature. At the Leu1 position, only mutations with helix structure-promoting hydrophobic residues (Ala, Ile, Val or Phe) were able to yield the mature Ent53B derivative. In this study, we also highlight the possible conformation-stabilizing role of the Ent53B leader peptide on the precursor peptide for its interaction with its biosynthetic enzyme complex. Any truncations of the leader peptide moiety interfered in the processing of the prepeptide. However, when propeptides of other circular bacteriocins (circularin A, leucocyclicin Q or lactocyclicin Q) were cloned at the C-terminus of the leader peptide, EnkB1234 could not process them to yield a mature bacteriocin. Taken together, these findings offer new perspectives in our understanding of the possible molecular mechanism of the biosynthesis of this circular bacteriocin. These new perspectives will help advance our current understanding to eventually elucidate circular bacteriocin biosynthesis. Understanding the biosynthetic mechanism of circular bacteriocins will materialize their application potential.

  2. Crystal structure of Bombyx mori arylphorins reveals a 3:3 heterohexamer with multiple papain cleavage sites

    Hou, Yong; Li, Jianwei; Li, Yi; Dong, Zhaoming; Xia, Qingyou; Yuan, Y Adam

    2014-01-01

    In holometabolous insects, the accumulation and utilization of storage proteins (SPs), including arylphorins and methionine-rich proteins, are critical for the insect metamorphosis. SPs function as amino acids reserves, which are synthesized in fat body, secreted into the larval hemolymph and taken up by fat body shortly before pupation. However, the detailed molecular mechanisms of digestion and utilization of SPs during development are largely unknown. Here, we report the crystal structure of Bombyx mori arylphorins at 2.8 Å, which displays a heterohexameric structural arrangement formed by trimerization of dimers comprising two structural similar arylphorins. Our limited proteolysis assay and microarray data strongly suggest that papain-like proteases are the major players for B. mori arylphorins digestion in vitro and in vivo. Consistent with the biochemical data, dozens of papain cleavage sites are mapped on the surface of the heterohexameric structure of B. mori arylphorins. Hence, our results provide the insightful information to understand the metamorphosis of holometabolous insects at molecular level. PMID:24639361

  3. Inhibition of Lassa virus glycoprotein cleavage and multicycle replication by site 1 protease-adapted alpha(1-antitrypsin variants.

    Anna Maisa

    2009-06-01

    Full Text Available Proteolytic processing of the Lassa virus envelope glycoprotein precursor GP-C by the host proprotein convertase site 1 protease (S1P is a prerequisite for the incorporation of the subunits GP-1 and GP-2 into viral particles and, hence, essential for infectivity and virus spread. Therefore, we tested in this study the concept of using S1P as a target to block efficient virus replication.We demonstrate that stable cell lines inducibly expressing S1P-adapted alpha(1-antitrypsin variants inhibit the proteolytic maturation of GP-C. Introduction of the S1P recognition motifs RRIL and RRLL into the reactive center loop of alpha(1-antitrypsin resulted in abrogation of GP-C processing by endogenous S1P to a similar level observed in S1P-deficient cells. Moreover, S1P-specific alpha(1-antitrypsins significantly inhibited replication and spread of a replication-competent recombinant vesicular stomatitis virus expressing the Lassa virus glycoprotein GP as well as authentic Lassa virus. Inhibition of viral replication correlated with the ability of the different alpha(1-antitrypsin variants to inhibit the processing of the Lassa virus glycoprotein precursor.Our data suggest that glycoprotein cleavage by S1P is a promising target for the development of novel anti-arenaviral strategies.

  4. Albumin Redhill (-1 Arg, 320 Ala → Thr): A glycoprotein variant of human serum albumin whose precursor has an aberrant signal peptidase cleavage site

    Brennan, S.O.; Myles, T.; Peach, R.J.; George, P.M.; Donaldson, D.

    1990-01-01

    Albumin Redhill is an electrophoretically slow genetic variant of human serum albumin that does not bind 63 Ni 2+ and has a molecular mass 2.5 kDa higher than normal albumin. Its inability to bind Ni 2+ was explained by the finding of an additional residue of Arg at position -1. This did not explain the molecular basis of the genetic variation or the increase in apparent molecular mass. Fractionation of tryptic digests on concanavalin A-Sepharose followed by peptide mapping of the bound and unbound fractions and sequence analysis of the glycopeptides identified a mutation of 320 Ala → Thr. This introduces as Asn-Tyr-Thr oligosaccharide attachment sequence centered on Asn-318 and explains the increase in molecular mass. This, however, did not satisfactorily explain the presence of the additional Arg residue at position -1. DNA sequencing of polymerase chain reaction-amplified genomic DNA encoding the prepro sequence of albumin indicated an additional mutation of -2 Arg → Cys. The authors propose that the new Phe-Cys-Arg sequence in the propeptide is an aberrant signal peptidase cleavage site and that the signal peptidase cleaves the propeptide of albumin Redhill in the lumen of the endoplasmic reticulum before it reaches the Golgi vesicles, the site of the diarginyl-specific proalbumin convertase

  5. Novel factor VIII variants with a modified furin cleavage site improve the efficacy of gene therapy for hemophilia A.

    Nguyen, G N; George, L A; Siner, J I; Davidson, R J; Zander, C B; Zheng, X L; Arruda, V R; Camire, R M; Sabatino, D E

    2017-01-01

    Essentials Factor (F) VIII is an inefficiently expressed protein. Furin deletion FVIII variants were purified and characterized using in vitro and in vivo assays. These minimally modified novel FVIII variants have enhanced function. These variants provide a strategy for increasing FVIII expression in hemophilia A gene therapy. Background The major challenge for developing gene-based therapies for hemophilia A is that human factor VIII (hFVIII) has intrinsic properties that result in inefficient biosynthesis. During intracellular processing, hFVIII is predominantly cleaved at a paired basic amino acid cleaving enzyme (PACE) or furin cleavage site to yield a heterodimer that is the major form of secreted protein. Previous studies with B-domain-deleted (BDD) canine FVIII and hFVIII-R1645H, both differing from hFVIII by a single amino acid at this site, suggested that these proteins are secreted mainly in a single polypeptide chain (SC) form and exhibit enhanced function. Objective We hypothesized that deletion(s) of the furin site modulates FVIII biology and may enhance its function. Methods A series of recombinant hFVIII-furin deletion variants were introduced into hFVIII-BDD [Δ1645, 1645-46(Δ2), 1645-47(Δ3), 1645-48(Δ4), or Δ1648] and characterized. Results In vitro, recombinant purified Δ3 and Δ4 were primarily SC and, interestingly, had 2-fold higher procoagulant activity compared with FVIII-BDD. In vivo, the variants also have improved hemostatic function. After adeno-associated viral (AAV) vector delivery, the expression of these variants is 2-4-fold higher than hFVIII-BDD. Protein challenges of each variant in mice tolerant to hFVIII-BDD showed no anti-FVIII immune response. Conclusions These data suggest that the furin deletion hFVIII variants are superior to hFVIII-BDD without increased immunogenicity. In the setting of gene-based therapeutics, these novel variants provide a unique strategy to increase FVIII expression, thus lowering the vector dose, a

  6. 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.

    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.

  7. Analysis by the reductive-cleavage method of linkage positions in a polysaccharide containing 4-linked D-glucopyranosyluronic residues.

    Vodonik, S A; Gray, G R

    1988-04-01

    The fate of 4-linked D-glucopyranosyluronic residues under reductive-cleavage conditions was investigated by using the Klebsiella aerogenes type 54 strain A3 capsular polysaccharide. Treatment of the fully methylated polysaccharide with triethylsilane and trimethylsilyl trifluoromethanesulfonate in dichloromethane, followed by in situ acetylation, yielded 1,5-anhydro-2,3,4,6-tetra-O-methyl-D-glucitol, 3,4-di-O-acetyl-1,5-anhydro-2,6-di-O-methyl-D-glucitol, and 3-O-acetyl-1,5-anhydro-2,4-di-O-methyl-L-fucitol, as expected, but the expected product of reductive cleavage of the 4-linked D-glucopyranosyluronic residue, namely, methyl 3-O-acetyl-2,6-anhydro-4,5-di-O-methyl-L-gulonate, was not observed. Instead, methyl 2-O-acetyl-3,6-anhydro-4,5-di-O-methyl-L-gulonate (6) was identified as the sole product of reductive cleavage of the 4-linked D-glucopyranosyluronic residue. That compound 6 arose as a result of rearrangement during reductive cleavage rather than by reductive cleavage of a 5-linked D-glucofuranosyluronic residue, was established by reductive cleavage of the fully methylated polysaccharide following reduction of its ester groups with either lithium aluminum hydride or lithium aluminum deuteride. The products of the latter reductive cleavage were the same as before, except for the absence of 6 and the presence of 4,6-di-O-acetyl-1,5-anhydro-2,3-di-O-methyl-D-glucitol, or its 6,6-dideuterio isomer. Although the reductive-cleavage technique is suitable for the direct analysis of polysaccharides containing 4-linked D-glucopyranosyluronic residues, it does not establish whether the uronic residue is a 4-linked pyranoside or a 5-linked furanoside. The expected product is, however, derived from the 4-linked D-glucopyranosyluronic residue after sequential methylation, reduction of its ester group and reductive cleavage.

  8. Functional analysis of coordinated cleavage in V(D)J recombination.

    Kim, D R; Oettinger, M A

    1998-08-01

    V(D)J recombination in vivo requires a pair of signals with distinct spacer elements of 12 and 23 bp that separate conserved heptamer and nonamer motifs. Cleavage in vitro by the RAG1 and RAG2 proteins can occur at individual signals when the reaction buffer contains Mn2+, but cleavage is restricted to substrates containing two signals when Mg2+ is the divalent cation. By using a novel V(D)J cleavage substrate, we show that while the RAG proteins alone establish a moderate preference for a 12/23 pair versus a 12/12 pair, a much stricter dependence of cleavage on the 12/23 signal pair is produced by the inclusion of HMG1 and competitor double-stranded DNA. The competitor DNA serves to inhibit the cleavage of substrates carrying a 12/12 or 23/23 pair, as well as the cutting at individual signals in 12/23 substrates. We show that a 23/33 pair is more efficiently recombined than a 12/33 pair, suggesting that the 12/23 rule can be generalized to a requirement for spacers that differ from each other by a single helical turn. Furthermore, we suggest that a fixed spatial orientation of signals is required for cleavage. In general, the same signal variants that can be cleaved singly can function under conditions in which a signal pair is required. However, a chemically modified substrate with one noncleavable signal enables us to show that formation of a functional cleavage complex is mechanistically separable from the cleavage reaction itself and that although cleavage requires a pair of signals, cutting does not have to occur simultaneously at both. The implications of these results are discussed with respect to the mechanism of V(D)J recombination and the generation of chromosomal translocations.

  9. Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations

    van Aelst, Kara; Saikrishnan, Kayarat; Szczelkun, Mark D.

    2015-01-01

    The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1–2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand breaks are introduced at random wherever two translocating enzymes form a so-called collision complex following long-range communication between a pair of target sites in inverted (head-to-head) repeat. Paradoxically, structural models for collision suggest that the nuclease domains are too far apart (>30 bp) to dimerise and produce a double-strand DNA break using just two strand-cleavage events. Here, we examined the organisation of different collision complexes and how these lead to nuclease activation. We mapped DNA cleavage when a translocating enzyme collides with a static enzyme bound to its site. By following communication between sites in both head-to-head and head-to-tail orientations, we could show that motor activity leads to activation of the nuclease domains via distant interactions of the helicase or MTase-TRD. Direct nuclease dimerization is not required. To help explain the observed cleavage patterns, we also used exonuclease footprinting to demonstrate that individual Type ISP domains can swing off the DNA. This study lends further support to a model where DNA breaks are generated by multiple random nicks due to mobility of a collision complex with an overall DNA-binding footprint of ∼30 bp. PMID:26507855

  10. Electrochemical bond cleavage in pesticide ioxynil. Kinetic analysis by voltammetry and impedance spectroscopy

    Sokolová, R.; Giannarelli, S.; Fanelli, N.; Pospíšil, Lubomír

    2017-01-01

    Roč. 49, SI C (2017), s. 134-138 ISSN 0324-1130 Institutional support: RVO:61388963 Keywords : electrochemical impedance spectroscopy * rate constant * self-protonation * faradaic phase angle * halogen cleavage * EC processes fitting Subject RIV: CG - Electrochemistry OBOR OECD: Electrochemistry (dry cells, batteries, fuel cells, corrosion metals, electrolysis) Impact factor: 0.238, year: 2016

  11. Regional energy facility siting analysis

    Eberhart, R.C.; Eagles, T.W.

    1976-01-01

    Results of the energy facility siting analysis portion of a regional pilot study performed for the anticipated National Energy Siting and Facility Report are presented. The question of cell analysis versus site-specific analysis is explored, including an evaluation of the difference in depth between the two approaches. A discussion of the possible accomplishments of regional analysis is presented. It is concluded that regional sitting analysis could be of use in a national siting study, if its inherent limits are recognized

  12. Fracture mechanics and physics approach to cleavage analysis in bcc monocrystals

    Ivanova, V.S.; Plastinin, V.M.

    1980-01-01

    On monocrystals of molybdenum obtained by electron--beam zone melting studied are the bonds between micro-and macroparameters of fracture controlling the limit state. Monocrystals of three orientations have been studied, namely >001 110 111<. Confirmed is an important role of plastic deformation in the (110) family planes at cleavage forming in the (100) family planes. A correlation connection is established between threshold value of the stress intensity coefficient and activation energy of plastic deformation

  13. Fluorescence quenching of graphene oxide combined with the site-specific cleavage of restriction endonuclease for deoxyribonucleic acid demethylase activity assay

    Ji, Lijuan; Qian, Yingdan; Wu, Ping; Zhang, Hui; Cai, Chenxin, E-mail: cxcai@njnu.edu.cn

    2015-04-15

    Highlights: • An approach for sensitive and selective DNA demethylase activity assay is reported. • This assay is based on the fluorescence quenching of GO and site-specific cleavage of endonuclease. • It can determine as low as 0.05 ng mL{sup −1} of MBD2 with a linear range of 0.2–300 ng mL{sup −1}. • It has an ability to recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. • It can avoid false signals, requiring no bisulfite conversion, PCR amplification, radioisotope-labeling. - Abstract: We report on the development of a sensitive and selective deoxyribonucleic acid (DNA) demethylase (using MBD2 as an example) activity assay by coupling the fluorescence quenching of graphene oxide (GO) with the site-specific cleavage of HpaII endonuclease to improve the selectivity. This approach was developed by designing a single-stranded probe (P1) that carries a binding region to facilitate the interaction with GO, which induces fluorescence quenching of the labeled fluorophore (FAM, 6-carboxyfluorescein), and a sensing region, which contains a hemi-methylated site of 5′-CmCGG-3′, to specifically recognize the target (T1, a 32-mer DNA from the promoter region of p53 gene) and hybridize with it to form a P1/T1 duplex. After demethylation with MBD2, the duplex can be specifically cleaved using HpaII, which releases the labeled FAM from the GO surface and results in the recovery of fluorescence. However, this cleavage is blocked by the hemi-methylation of this site. Thus, the magnitude of the recovered fluorescence signal is related to the MBD2 activity, which establishes the basis of the DNA demethylase activity assay. This assay can determine as low as ∼(0.05 ± 0.01) ng mL{sup −1} (at a signal/noise of 3) of MBD2 with a linear range of 0.2–300 ng mL{sup −1} and recognize MBD2 from other possibly coexisting proteins and cancer cell extracts. The advantage of this assay is its ability to avoid false signals and no

  14. Detection of siRNA Mediated Target mRNA Cleavage Activities in Human Cells by a Novel Stem-Loop Array RT-PCR Analysis

    2016-09-07

    sequences of the target mRNA, and a double stranded stem at the 5′ end that forms a stem -loop to function as a forceps to stabilize the secondary...E-mjournal homepage: www.elsevier.com/locate/bbrepDetection of siRNA-mediated target mRNA cleavage activities in human cells by a novel stem -loop...challenges for the accurate and efficient detection and verification of cleavage sites on target mRNAs. Here we used a sensitive stem -loop array reverse

  15. Beneficial effect of antibodies against β- secretase cleavage site of APP on Alzheimer's-like pathology in triple-transgenic mice.

    Inna Rabinovich-Nikitin

    Full Text Available The toxicity of amyloid β and tau, the two hallmark proteins in Alzheimer's disease (AD, has been extensively studied individually. Recently new data suggest their possible interactions and synergistic effects in the disease. In this study, we investigate the ability of antibodies against the β secretase cleavage site on APP, named BBS1, to affect tau pathology, besides their well established effect on intracellular Aβ and amyloid load. For this purpose we treated the triple transgenic mice model of AD (3x Tg-AD with mAb BBS1 intracerebroventricularly, using mini osmotic pumps for one month. The experimental data demonstrated reduction in total and phosphorylated tau levels, explained by significant reduction in GSK3β which phosphorylates tau on sites recognized by antibodies against PHF1 and AT-8. The treatment increased the cognitive capabilities and reduced the brain inflammation levels which accompany AD pathology. The data showing that tau pathology was significantly reduced by BBS1 antibodies suggest a close interaction between tau and Aβ in the development of AD, and may serve as an efficient novel immunotherapy against both hallmarks of this disease.

  16. Group I-like ribozymes with a novel core organization perform obligate sequential hydrolytic cleavages at two processing sites

    Einvik, C; Nielsen, Henrik; Westhof, E

    1998-01-01

    A new category of self-splicing group I introns with conserved structural organization and function is found among the eukaryotic microorganisms Didymium and Naegleria. These complex rDNA introns contain two distinct ribozymes with different functions: a regular group I splicing...... available GIR1 sequences and propose a common RNA secondary structure resembling that of group I splicing-ribozymes, but with some important differences. The GIR1s lack most peripheral sequence components, as well as a P1 segment, and, at approximately 160-190 nt, they are the smallest functional group I...... ribozymes known from nature. All GIR1s were found to contain a novel 6-bp pseudoknot (P15) within their catalytic core region. Experimental support of the proposed structure was obtained from the Didymium GIR1 by RNA structure probing and site-directed mutagenesis. Three-dimensional modeling indicates...

  17. Vertebrate Embryonic Cleavage Pattern Determination.

    Hasley, Andrew; Chavez, Shawn; Danilchik, Michael; Wühr, Martin; Pelegri, Francisco

    2017-01-01

    The pattern of the earliest cell divisions in a vertebrate embryo lays the groundwork for later developmental events such as gastrulation, organogenesis, and overall body plan establishment. Understanding these early cleavage patterns and the mechanisms that create them is thus crucial for the study of vertebrate development. This chapter describes the early cleavage stages for species representing ray-finned fish, amphibians, birds, reptiles, mammals, and proto-vertebrate ascidians and summarizes current understanding of the mechanisms that govern these patterns. The nearly universal influence of cell shape on orientation and positioning of spindles and cleavage furrows and the mechanisms that mediate this influence are discussed. We discuss in particular models of aster and spindle centering and orientation in large embryonic blastomeres that rely on asymmetric internal pulling forces generated by the cleavage furrow for the previous cell cycle. Also explored are mechanisms that integrate cell division given the limited supply of cellular building blocks in the egg and several-fold changes of cell size during early development, as well as cytoskeletal specializations specific to early blastomeres including processes leading to blastomere cohesion. Finally, we discuss evolutionary conclusions beginning to emerge from the contemporary analysis of the phylogenetic distributions of cleavage patterns. In sum, this chapter seeks to summarize our current understanding of vertebrate early embryonic cleavage patterns and their control and evolution.

  18. Systematic Design of Trypsin Cleavage Site Mutated Exendin4-Cysteine 1, an Orally Bioavailable Glucagon-Like Peptide-1 Receptor Agonist

    Wenbo Sai

    2017-03-01

    Full Text Available Exendin-4 is a strong therapeutic candidate for the treatment of metabolic syndrome. Related receptor agonist drugs have been on the market since 2005. However, technical limitations and the pain caused by subcutaneous injection have severely limited patient compliance. The goal of the study is to investigate a biologically active exendin-4 analog could be administered orally. Using intraperitoneal glucose tolerance tests, we discovered that exendin4-cysteine administered by oral gavage had a distinct hypoglycemic effect in C57BL/6J mice. Using Rosetta Design and Amber, we designed and screened a series of exendin4-cysteine analogs to identify those that retained biological activity while resisting trypsin digestion. Trypsin Cleavage Site Mutated Exendin4-cysteine 1 (TSME-1, an analog whose bioactivity was similar to exendin-4 and was almost completely resistant to trypsin, was screened out. In addition, TSME-1 significantly normalized the blood glucose levels and the availability of TSME-1 was significantly higher than that of exendin-4 and exendin4-cysteine. Collectively orally administered TSME-1, a trypsin-resistant exendin-4 analog obtained by the system, is a strong candidate for future treatments of type 2 diabetes.

  19. Mutation in the protease cleavage site of GDF9 increases ovulation rate and litter size in heterozygous ewes and causes infertility in homozygous ewes.

    Souza, C J H; McNeilly, A S; Benavides, M V; Melo, E O; Moraes, J C F

    2014-10-01

    Litter size (LS) in sheep is determined mainly by ovulation rate (OR). Several polymorphisms have been identified in the growth differentiation factor 9 (GDF9) gene that result in an increase in OR and prolificacy of sheep. Screening the databank of the Brazilian Sheep Breeders Association for triplet delivery, we identified flocks of prolific Ile de France ewes. After resequencing of GDF9, a point mutation (c.943C>T) was identified, resulting in a non-conservative amino acid change (p.Arg315Cys) in the cleavage site of the propeptide. This new allele was called Vacaria (FecG(v) ). A flock of half-sib ewes was evaluated for OR in the first three breeding seasons, and Vacaria heterozygotes had higher OR (P develop up to small antral stages, although with abnormal oocyte morphology and altered arrangement of granulosa cells. After the collapse of the oocyte in most follicles, the remaining cells formed clusters that persisted in the ovary. This SNP is useful to improve selection for dam prolificacy and also as a model to investigate GDF9 post-translation processing and the fate of the follicular cells that remain after the oocyte demise. © 2014 Stichting International Foundation for Animal Genetics.

  20. Analysis of the in vitro cleavage products of the tomato black ring virus RNA-1-encoded 250K polyprotein.

    Demangeat, Gerard; Greif, Charles; Hemmer, O; Fritsch, C

    1990-01-01

    Tomato black ring virus RNA-1 was translated in a rabbit reticulocyte lysate. The primary translation product of Mr 250K, which corresponds to its whole coding capacity, was synthesized within 45 min and, during further incubation in the translation medium, was proteolytically processed. Essentially, four cleavage products (P190, P120, P60 and P50) were detected and located within P250 by pulse-chase and immunoprecipitation experiments. P190 is an intermediate cleavage product which is furthe...

  1. Mutation in cpsf6/CFIm68 (Cleavage and Polyadenylation Specificity Factor Subunit 6 causes short 3'UTRs and disturbs gene expression in developing embryos, as revealed by an analysis of primordial germ cell migration using the medaka mutant naruto.

    Takao Sasado

    Full Text Available Our previous studies analyzing medaka mutants defective in primordial germ cell (PGC migration identified cxcr4b and cxcr7, which are both receptors of the chemokine sdf1/cxcl12, as key regulators of PGC migration. Among PGC migration mutants, naruto (nar is unique in that the mutant phenotype includes gross morphological abnormalities of embryos, suggesting that the mutation affects a broader range of processes. A fine genetic linkage mapping and genome sequencing showed the nar gene encodes Cleavage and Polyadenylation Specificity Factor subunit 6 (CPSF6/CFIm68. CPSF6 is a component of the Cleavage Factor Im complex (CFIm which plays a key role in pre-mRNA 3'-cleavage and polyadenylation. 3'RACE of sdf1a/b and cxcr7 transcripts in the mutant embryos indicated shorter 3'UTRs with poly A additions occurring at more upstream positions than wild-type embryos, suggesting CPSF6 functions to prevent premature 3'UTR cleavage. In addition, expression of the coding region sequences of sdf1a/b in nar mutants was more anteriorly extended in somites than wild-type embryos, accounting for the abnormally extended distribution of PGCs in nar mutants. An expected consequence of shortening 3'UTR is the escape from the degradation mechanism mediated by microRNAs interacting with distal 3'UTR sequence. The abnormal expression pattern of sdf1a coding sequence may be at least partially accounted for by this mechanism. Given the pleiotropic effects of nar mutation, further analysis using the nar mutant will reveal processes in which CPSF6 plays essential regulatory roles in poly A site selection and involvement of 3'UTRs in posttranscriptional gene regulation in various genes in vivo.

  2. Mutation in cpsf6/CFIm68 (Cleavage and Polyadenylation Specificity Factor Subunit 6) causes short 3'UTRs and disturbs gene expression in developing embryos, as revealed by an analysis of primordial germ cell migration using the medaka mutant naruto.

    Sasado, Takao; Kondoh, Hisato; Furutani-Seiki, Makoto; Naruse, Kiyoshi

    2017-01-01

    Our previous studies analyzing medaka mutants defective in primordial germ cell (PGC) migration identified cxcr4b and cxcr7, which are both receptors of the chemokine sdf1/cxcl12, as key regulators of PGC migration. Among PGC migration mutants, naruto (nar) is unique in that the mutant phenotype includes gross morphological abnormalities of embryos, suggesting that the mutation affects a broader range of processes. A fine genetic linkage mapping and genome sequencing showed the nar gene encodes Cleavage and Polyadenylation Specificity Factor subunit 6 (CPSF6/CFIm68). CPSF6 is a component of the Cleavage Factor Im complex (CFIm) which plays a key role in pre-mRNA 3'-cleavage and polyadenylation. 3'RACE of sdf1a/b and cxcr7 transcripts in the mutant embryos indicated shorter 3'UTRs with poly A additions occurring at more upstream positions than wild-type embryos, suggesting CPSF6 functions to prevent premature 3'UTR cleavage. In addition, expression of the coding region sequences of sdf1a/b in nar mutants was more anteriorly extended in somites than wild-type embryos, accounting for the abnormally extended distribution of PGCs in nar mutants. An expected consequence of shortening 3'UTR is the escape from the degradation mechanism mediated by microRNAs interacting with distal 3'UTR sequence. The abnormal expression pattern of sdf1a coding sequence may be at least partially accounted for by this mechanism. Given the pleiotropic effects of nar mutation, further analysis using the nar mutant will reveal processes in which CPSF6 plays essential regulatory roles in poly A site selection and involvement of 3'UTRs in posttranscriptional gene regulation in various genes in vivo.

  3. In vitro maturation of Drosophila melanogaster Spätzle protein with refolded Easter reveals a novel cleavage site within the prodomain.

    Ursel, Christian; Fandrich, Uwe; Hoffmann, Anita; Sieg, Torsten; Ihling, Christian; Stubbs, Milton T

    2013-08-01

    Dorsoventral patterning during Drosophila melanogaster embryogenesis is mediated by a well-defined gradient of the mature NGF-like ligand Spätzle. Easter, the ultimate protease of a ventrally-restricted serine protease cascade, plays a key role in the regulation of the morphogenic gradient, catalyzing the activation cleavage of proSpätzle. As a result of alternative splicing, proSpätzle exists in multiple isoforms, almost all of which differ only in their prodomain. Although this domain is unstructured in isolation, it has a stabilizing influence on the mature cystine knot domain and is involved in the binding to the Toll receptor. Here, we report the expression and refolding of Easter, and show that the renatured enzyme performs the activation cleavage of two Spätzle isoforms. We determine the affinity of the prodomain for the cystine knot domain, and show that Easter performs a previously unknown secondary cleavage in each prodomain.

  4. Flanking signal and mature peptide residues influence signal peptide cleavage

    Ranganathan Shoba

    2008-12-01

    Full Text Available Abstract Background Signal peptides (SPs mediate the targeting of secretory precursor proteins to the correct subcellular compartments in prokaryotes and eukaryotes. Identifying these transient peptides is crucial to the medical, food and beverage and biotechnology industries yet our understanding of these peptides remains limited. This paper examines the most common type of signal peptides cleavable by the endoprotease signal peptidase I (SPase I, and the residues flanking the cleavage sites of three groups of signal peptide sequences, namely (i eukaryotes (Euk (ii Gram-positive (Gram+ bacteria, and (iii Gram-negative (Gram- bacteria. Results In this study, 2352 secretory peptide sequences from a variety of organisms with amino-terminal SPs are extracted from the manually curated SPdb database for analysis based on physicochemical properties such as pI, aliphatic index, GRAVY score, hydrophobicity, net charge and position-specific residue preferences. Our findings show that the three groups share several similarities in general, but they display distinctive features upon examination in terms of their amino acid compositions and frequencies, and various physico-chemical properties. Thus, analysis or prediction of their sequences should be separated and treated as distinct groups. Conclusion We conclude that the peptide segment recognized by SPase I extends to the start of the mature protein to a limited extent, upon our survey of the amino acid residues surrounding the cleavage processing site. These flanking residues possibly influence the cleavage processing and contribute to non-canonical cleavage sites. Our findings are applicable in defining more accurate prediction tools for recognition and identification of cleavage site of SPs.

  5. Analysis of the in vitro cleavage products of the tomato black ring virus RNA-1-encoded 250K polyprotein.

    Demangeat, G; Greif, C; Hemmer, O; Fritsch, C

    1990-08-01

    Tomato black ring virus RNA-1 was translated in a rabbit reticulocyte lysate. The primary translation product of Mr 250K, which corresponds to its whole coding capacity, was synthesized within 45 min and, during further incubation in the translation medium, was proteolytically processed. Essentially, four cleavage products (P190, P120, P60 and P50) were detected and located within P250 by pulse-chase and immunoprecipitation experiments. P190 is an intermediate cleavage product which is further cleaved to form P60 and P120. P120, which contains the region that has been assigned to the virus protease and the virus polymerase, was not further cleaved in vitro.

  6. Capillary gas chromatographic analysis of mycolic acid cleavage products, cellular fatty acids, and alcohols of Mycobacterium xenopi.

    Luquin, M; Lopez, F; Ausina, V

    1989-01-01

    The fatty acids, alcohols, and mycolic acids of 26 strains of Mycobacterium xenopi were studied by capillary gas chromatography and thin-layer chromatography. All strains contained alpha-, keto-, and omega-carboxymycolates. The primary mycolic acid cleavage product was hexacosanoic acid. The fatty acid patterns and, especially, the presence of 2-docosanol are characteristic markers of M. xenopi.

  7. An evaluation of analysis methodologies for predicting cleavage arrest of a deep crack in an RPV subjected to PTS loading conditions

    Keeney-Walker, J.; Bass, B.R.

    1992-01-01

    Several calculational procedures are compared for predicting cleavage arrest of a deep crack in the wall of a prototypical reactor pressure vessel (RPV) subjected to pressurized-thermal-shock (PTS) types of loading conditions. Three procedures examined in this study utilized the following models: (1) a static finite-element model (full bending); (2) a radially constrained static model; and (3) a thermoelastic dynamic finite-element model. A PTS transient loading condition was selected that produced a deep arrest of an axially-oriented initially shallow crack according to calculational results obtained from the static (full-bending) model. Results from the two static models were compared with those generated from the detailed thermoelastic dynamic finite-element analysis. The dynamic analyses modeled cleavage-crack propagation using node-release technique and an application-mode methodology based on dynamic fracture toughness curves generated from measured data. Comparisons presented here indicate that the degree to which dynamic solutions can be approximated by static models is highly dependent on several factors, including the material dynamic fracture curves and the propensity for cleavage reinitiation of the arrested crack under PTS loading conditions. Additional work is required to develop and validate a satisfactory dynamic fracture toughness model applicable to postcleavage arrest conditions in an RPV

  8. The large terminase DNA packaging motor grips DNA with its ATPase domain for cleavage by the flexible nuclease domain

    Hilbert, Brendan J.; Hayes, Janelle A.; Stone, Nicholas P.; Xu, Rui-Gang

    2017-01-01

    Abstract Many viruses use a powerful terminase motor to pump their genome inside an empty procapsid shell during virus maturation. The large terminase (TerL) protein contains both enzymatic activities necessary for packaging in such viruses: the adenosine triphosphatase (ATPase) that powers DNA translocation and an endonuclease that cleaves the concatemeric genome at both initiation and completion of genome packaging. However, how TerL binds DNA during translocation and cleavage remains mysterious. Here we investigate DNA binding and cleavage using TerL from the thermophilic phage P74-26. We report the structure of the P74-26 TerL nuclease domain, which allows us to model DNA binding in the nuclease active site. We screened a large panel of TerL variants for defects in binding and DNA cleavage, revealing that the ATPase domain is the primary site for DNA binding, and is required for nuclease activity. The nuclease domain is dispensable for DNA binding but residues lining the active site guide DNA for cleavage. Kinetic analysis of DNA cleavage suggests flexible tethering of the nuclease domains during DNA cleavage. We propose that interactions with the procapsid during DNA translocation conformationally restrict the nuclease domain, inhibiting cleavage; TerL release from the capsid upon completion of packaging unlocks the nuclease domains to cleave DNA. PMID:28082398

  9. N-Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

    Niu, Canfang; Luo, Huiying; Shi, Pengjun; Huang, Huoqing; Wang, Yaru; Yang, Peilong

    2015-01-01

    N-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases from Yersinia kristensenii (YkAPPA) and Yersinia rohdei (YrAPPA), each having an N-glycosylation motif, and one pepsin-sensitive HAP phytase from Yersinia enterocolitica (YeAPPA) that lacked an N-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering the N-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed in Pichia pastoris for biochemical characterization. Compared with those of the N-glycosylation site deletion mutants and N-deglycosylated enzymes, all N-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of the N-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of the N-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced in Escherichia coli but had no effect on the pepsin resistance of N-glycosylated enzymes produced in P. pastoris. Thus, N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation of N-glycosylation, for improvement of phytase properties for use in animal feed. PMID:26637601

  10. Calicivirus 3C-like proteinase inhibits cellular translation by cleavage of poly(A)-binding protein.

    Kuyumcu-Martinez, Muge; Belliot, Gaël; Sosnovtsev, Stanislav V; Chang, Kyeong-Ok; Green, Kim Y; Lloyd, Richard E

    2004-08-01

    Caliciviruses are single-stranded RNA viruses that cause a wide range of diseases in both humans and animals, but little is known about the regulation of cellular translation during infection. We used two distinct calicivirus strains, MD145-12 (genus Norovirus) and feline calicivirus (FCV) (genus Vesivirus), to investigate potential strategies used by the caliciviruses to inhibit cellular translation. Recombinant 3C-like proteinases (r3CL(pro)) from norovirus and FCV were found to cleave poly(A)-binding protein (PABP) in the absence of other viral proteins. The norovirus r3CL(pro) PABP cleavage products were indistinguishable from those generated by poliovirus (PV) 3C(pro) cleavage, while the FCV r3CL(pro) products differed due to cleavage at an alternate cleavage site 24 amino acids downstream of one of the PV 3C(pro) cleavage sites. All cleavages by calicivirus or PV proteases separated the C-terminal domain of PABP that binds translation factors eIF4B and eRF3 from the N-terminal RNA-binding domain of PABP. The effect of PABP cleavage by the norovirus r3CL(pro) was analyzed in HeLa cell translation extracts, and the presence of r3CL(pro) inhibited translation of both endogenous and exogenous mRNAs. Translation inhibition was poly(A) dependent, and replenishment of the extracts with PABP restored translation. Analysis of FCV-infected feline kidney cells showed that the levels of de novo cellular protein synthesis decreased over time as virus-specific proteins accumulated, and cleavage of PABP occurred in virus-infected cells. Our data indicate that the calicivirus 3CL(pro), like PV 3C(pro), mediates the cleavage of PABP as part of its strategy to inhibit cellular translation. PABP cleavage may be a common mechanism among certain virus families to manipulate cellular translation.

  11. Analysis of ILRS Site Ties

    Husson, V. S.; Long, J. L.; Pearlman, M.

    2001-12-01

    By the end of 2000, 94% of ILRS stations had completed station and site information forms (i.e. site logs). These forms contain six types of information. These six categories include site identifiers, contact information, approximate coordinates, system configuration history, system ranging capabilities, and local survey ties. The ILRS Central Bureau, in conjunction with the ILRS Networks and Engineering Working Group, has developed procedures to quality control site log contents. Part of this verification entails data integrity checks of local site ties and is the primary focus of this paper. Local survey ties are critical to the combination of space geodetic network coordinate solutions (i.e. GPS, SLR, VLBI, DORIS) of the International Terrestrial Reference Frame (ITRF). Approximately 90% of active SLR sites are collocated with at least one other space geodetic technique. The process used to verify these SLR ties, at collocated sites, is identical to the approach used in ITRF2000. Local vectors (X, Y, Z) from each ILRS site log are differenced from its corresponding ITRF2000 position vectors (i.e. no transformations). These X, Y, and Z deltas are converted into North, East, and Up. Any deltas, in any component, larger than 5 millimeter is flagged for investigation. In the absence of ITRF2000 SLR positions, CSR positions were used. To further enhance this comparison and to fill gaps in information, local ties contained in site logs from the other space geodetic services (i.e. IGS, IVS, IDS) were used in addition to ITRF2000 ties. Case studies of two collocated sites (McDonald/Ft. Davis and Hartebeeshtoek) will be explored in-depth. Recommendations on how local site surveys should be conducted and how this information should be managed will also be presented.

  12. Multilevel magnetic resonance imaging analysis of multifidus-longissimus cleavage planes in the lumbar spine and potential clinical applications to Wiltse's paraspinal approach.

    Palmer, Daniel Kyle; Allen, Jonathan L; Williams, Paul A; Voss, Ashley Elizabeth; Jadhav, Vikram; Wu, David S; Cheng, Wayne K

    2011-07-15

    Retrospective magnetic resonance imaging (MRI)-based study. Our goal was to develop Wiltse's paraspinal surgical approach by determining the precise anatomic locations of the intermuscular cleavage planes formed by the multifidus and longissimus muscles. The primary objective was to measure the distances between the midline and the intermuscular planes, bilaterally, on MRI scans at each of the five disc levels between L1 and S1. Secondary objectives included identifying the existence of any correlations between patient demographics and the measured outcomes. In 1968, Wiltse described an approach to the spine using the natural cleavage plane of the multifidus and longissimus muscles as an entry to the posterior spinal elements. The small direct incisions lessened bleeding, tissue violation, and muscle retraction, which popularized Wiltse's approach among surgeons. A detailed description of the locations of the intermuscular cleavage planes at each lumbar disc level, however, is not available. MRI scans of 200 patients taken during routine care (2007-2009) were retrospectively reviewed to gather measurements of the distances from the intermuscular cleavage planes to the midline, bilaterally, at each disc level from L1 to S1. Age, sex, and BMI (body mass index) were obtained to determine correlations. Mean measurements significantly differed between all disc levels. At L5-S1, the mean distance was 37.8 mm; at L4-L5, 28.4 mm; at L3-L4, 16.2 mm; at L2-L3, 10.4 mm; and at L1-L2, 7.9 mm. The mean female distances were significantly greater than males (2 mm) on both sides of L5-S1 only. No correlation was discovered between BMI, age, height (N = 50), or weight (N = 50) with respect to measured distances. In the absence of any significant clinical correlation between patient demographics and the entry site in Wiltse's approach, the spine surgeon may use distances described in this paper to apply to a broad base of spine patients regardless of BMI, sex, or age.

  13. Crystallization and preliminary X-ray diffraction analysis of two N-terminal fragments of the DNA-cleavage domain of topoisomerase IV from Staphylococcus aureus

    Carr, Stephen B., E-mail: bmbsbc@bmb.leeds.ac.uk [Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT (United Kingdom); Makris, George [Omega Mediation Hellas Ltd, Clinical and Pharma Consulting, 11525 N. Psychiko, Athens (Greece); Phillips, Simon E. V.; Thomas, Christopher D. [Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT (United Kingdom)

    2006-11-01

    The crystallization and data collection of topoisomerase IV from S. aureus is described. Phasing by molecular replacement proved difficult owing to the presence of translational NCS and strategies used to overcome this are discussed. DNA topoisomerase IV removes undesirable topological features from DNA molecules in order to help maintain chromosome stability. Two constructs of 56 and 59 kDa spanning the DNA-cleavage domain of the A subunit of topoisomerase IV from Staphylococcus aureus (termed GrlA56 and GrlA59) have been crystallized. Crystals were grown at 291 K using the sitting-drop vapour-diffusion technique with PEG 3350 as a precipitant. Preliminary X-ray analysis revealed that GrlA56 crystals belong to space group P2{sub 1}, diffract to a resolution of 2.9 Å and possess unit-cell parameters a = 83.6, b = 171.5, c = 87.8 Å, β = 90.1°, while crystals of GrlA59 belong to space group P2{sub 1}2{sub 1}2, with unit-cell parameters a = 41.5, b = 171.89, c = 87.9 Å. These crystals diffract to a resolution of 2.8 Å. This is the first report of the crystallization and preliminary X-ray analysis of the DNA-cleavage domain of a topoisomerase IV from a Gram-positive organism.

  14. Crystallization and preliminary X-ray diffraction analysis of two N-terminal fragments of the DNA-cleavage domain of topoisomerase IV from Staphylococcus aureus

    Carr, Stephen B.; Makris, George; Phillips, Simon E. V.; Thomas, Christopher D.

    2006-01-01

    The crystallization and data collection of topoisomerase IV from S. aureus is described. Phasing by molecular replacement proved difficult owing to the presence of translational NCS and strategies used to overcome this are discussed. DNA topoisomerase IV removes undesirable topological features from DNA molecules in order to help maintain chromosome stability. Two constructs of 56 and 59 kDa spanning the DNA-cleavage domain of the A subunit of topoisomerase IV from Staphylococcus aureus (termed GrlA56 and GrlA59) have been crystallized. Crystals were grown at 291 K using the sitting-drop vapour-diffusion technique with PEG 3350 as a precipitant. Preliminary X-ray analysis revealed that GrlA56 crystals belong to space group P2 1 , diffract to a resolution of 2.9 Å and possess unit-cell parameters a = 83.6, b = 171.5, c = 87.8 Å, β = 90.1°, while crystals of GrlA59 belong to space group P2 1 2 1 2, with unit-cell parameters a = 41.5, b = 171.89, c = 87.9 Å. These crystals diffract to a resolution of 2.8 Å. This is the first report of the crystallization and preliminary X-ray analysis of the DNA-cleavage domain of a topoisomerase IV from a Gram-positive organism

  15. Capturing the Alternative Cleavage and Polyadenylation Sites of 14 NAC Genes in Populus Using a Combination of 3′-RACE and High-Throughput Sequencing

    Haoran Wang

    2018-03-01

    Full Text Available Detection of complex splice sites (SSs and polyadenylation sites (PASs of eukaryotic genes is essential for the elucidation of gene regulatory mechanisms. Transcriptome-wide studies using high-throughput sequencing (HTS have revealed prevalent alternative splicing (AS and alternative polyadenylation (APA in plants. However, small-scale and high-depth HTS aimed at detecting genes or gene families are very few and limited. We explored a convenient and flexible method for profiling SSs and PASs, which combines rapid amplification of 3′-cDNA ends (3′-RACE and HTS. Fourteen NAC (NAM, ATAF1/2, CUC2 transcription factor genes of Populus trichocarpa were analyzed by 3′-RACE-seq. Based on experimental reproducibility, boundary sequence analysis and reverse transcription PCR (RT-PCR verification, only canonical SSs were considered to be authentic. Based on stringent criteria, candidate PASs without any internal priming features were chosen as authentic PASs and assumed to be PAS-rich markers. Thirty-four novel canonical SSs, six intronic/internal exons and thirty 3′-UTR PAS-rich markers were revealed by 3′-RACE-seq. Using 3′-RACE and real-time PCR, we confirmed that three APA transcripts ending in/around PAS-rich markers were differentially regulated in response to plant hormones. Our results indicate that 3′-RACE-seq is a robust and cost-effective method to discover SSs and label active regions subjected to APA for genes or gene families. The method is suitable for small-scale AS and APA research in the initial stage.

  16. H9N2 influenza virus acquires intravenous pathogenicity on the introduction of a pair of di-basic amino acid residues at the cleavage site of the hemagglutinin and consecutive passages in chickens

    Sakoda Yoshihiro

    2011-02-01

    Full Text Available Abstract Background Outbreaks of avian influenza (AI caused by infection with low pathogenic H9N2 viruses have occurred in poultry, resulting in serious economic losses in Asia and the Middle East. It has been difficult to eradicate the H9N2 virus because of its low pathogenicity, frequently causing in apparent infection. It is important for the control of AI to assess whether the H9N2 virus acquires pathogenicity as H5 and H7 viruses. In the present study, we investigated whether a non-pathogenic H9N2 virus, A/chicken/Yokohama/aq-55/2001 (Y55 (H9N2, acquires pathogenicity in chickens when a pair of di-basic amino acid residues is introduced at the cleavage site of its HA molecule. Results rgY55sub (H9N2, which had four basic amino acid residues at the HA cleavage site, replicated in MDCK cells in the absence of trypsin after six consecutive passages in the air sacs of chicks, and acquired intravenous pathogenicity to chicken after four additional passages. More than 75% of chickens inoculated intravenously with the passaged virus, rgY55sub-P10 (H9N2, died, indicating that it is pathogenic comparable to that of highly pathogenic avian influenza viruses (HPAIVs defined by World Organization for Animal Health (OIE. The chickens inoculated with the virus via the intranasal route, however, survived without showing any clinical signs. On the other hand, an avirulent H5N1 strain, A/duck/Hokkaido/Vac-1/2004 (Vac1 (H5N1, acquired intranasal pathogenicity after a pair of di-basic amino acid residues was introduced into the cleavage site of the HA, followed by two passages by air sac inoculation in chicks. Conclusion The present results demonstrate that an H9N2 virus has the potential to acquire intravenous pathogenicity in chickens although the morbidity via the nasal route of infection is lower than that of H5N1 HPAIV.

  17. Analysis of embryo morphokinetics, multinucleation and cleavage anomalies using continuous time-lapse monitoring in blastocyst transfer cycles.

    Desai, Nina; Ploskonka, Stephanie; Goodman, Linnea R; Austin, Cynthia; Goldberg, Jeffrey; Falcone, Tommaso

    2014-06-20

    Time-lapse imaging combined with embryo morphokinetics may offer a non-invasive means for improving embryo selection. Data from clinics worldwide are necessary to compare and ultimately develop embryo classifications models using kinetic data. The primary objective of this study was to determine if there were kinetic differences between embryos with limited potential and those more often associated with in vitro blastocyst formation and/or implantation. We also wanted to compare putative kinetic markers for embryo selection as proposed by other laboratories to what we were observing in our own laboratory setting. Kinetic data and cycle outcomes were retrospectively analyzed in patients age 39 and younger with 7 or more zygotes cultured in the Embryoscope. Timing of specific events from the point of insemination were determined using time-lapse (TL) imaging. The following kinetic markers were assessed: time to syngamy (tPNf), t2, time to two cells (c), 3c (t3), 4c ( t4), 5c (t5), 8c (t8), morula (tMor), start of blastulation (tSB); tBL, blastocyst (tBL); expanded blastocyst (tEBL). Durations of the second (cc2) and third (cc3) cell cycles, the t5-t2 interval as well as time to complete synchronous divisions s1, s2 and s3 were calculated. Incidence and impact on development of nuclear and cleavage anomalies were also assessed. A total of 648 embryos transferred on day 5 were analyzed. The clinical pregnancy and implantation rate were 72% and 50%, respectively. Morphokinetic data showed that tPNf, t2,t4, t8, s1, s2,s3 and cc2 were significantly different in embryos forming blastocysts (ET or frozen) versus those with limited potential either failing to blastulate or else forming poor quality blastocysts ,ultimately discarded. Comparison of embryo kinetics in cycles with all embryos implanting (KID+) versus no implantation (KID-) suggested that markers of embryo competence to implant may be different from ability to form a blastocyst. The incidence of multinucleation

  18. Mapping DNA cleavage by the Type ISP restriction-modification enzymes following long-range communication between DNA sites in different orientations

    van Aelst, Kara; Saikrishnan, Kayarat; Szczelkun, Mark D

    2015-01-01

    The prokaryotic Type ISP restriction-modification enzymes are single-chain proteins comprising an Mrr-family nuclease, a superfamily 2 helicase-like ATPase, a coupler domain, a methyltransferase, and a DNA-recognition domain. Upon recognising an unmodified DNA target site, the helicase-like domain hydrolyzes ATP to cause site release (remodeling activity) and to then drive downstream translocation consuming 1-2 ATP per base pair (motor activity). On an invading foreign DNA, double-strand brea...

  19. The dimer interfaces of protease and extra-protease domains influence the activation of protease and the specificity of GagPol cleavage.

    Pettit, Steven C; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H

    2003-01-01

    Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation.

  20. Green Infrastructure Siting and Cost Effectiveness Analysis

    Allegheny County / City of Pittsburgh / Western PA Regional Data Center — Parcel scale green infrastructure siting and cost effectiveness analysis. You can find more details at the project's website.

  1. Comparative analysis of safety related site characteristics

    Andersson, Johan

    2010-12-01

    This document presents a comparative analysis of site characteristics related to long-term safety for the two candidate sites for a final repository for spent nuclear fuel in Forsmark (municipality of Oesthammar) and in Laxemar (municipality of Oskarshamn) from the point of view of site selection. The analyses are based on the updated site descriptions of Forsmark /SKB 2008a/ and Laxemar /SKB 2009a/, together with associated updated repository layouts and designs /SKB 2008b and SKB 2009b/. The basis for the comparison is thus two equally and thoroughly assessed sites. However, the analyses presented here are focussed on differences between the sites rather than evaluating them in absolute terms. The document serves as a basis for the site selection, from the perspective of long-term safety, in SKB's application for a final repository. A full evaluation of safety is made for a repository at the selected site in the safety assessment SR-Site /SKB 2011/, referred to as SR-Site main report in the following

  2. Comparative analysis of safety related site characteristics

    Andersson, Johan (ed.)

    2010-12-15

    This document presents a comparative analysis of site characteristics related to long-term safety for the two candidate sites for a final repository for spent nuclear fuel in Forsmark (municipality of Oesthammar) and in Laxemar (municipality of Oskarshamn) from the point of view of site selection. The analyses are based on the updated site descriptions of Forsmark /SKB 2008a/ and Laxemar /SKB 2009a/, together with associated updated repository layouts and designs /SKB 2008b and SKB 2009b/. The basis for the comparison is thus two equally and thoroughly assessed sites. However, the analyses presented here are focussed on differences between the sites rather than evaluating them in absolute terms. The document serves as a basis for the site selection, from the perspective of long-term safety, in SKB's application for a final repository. A full evaluation of safety is made for a repository at the selected site in the safety assessment SR-Site /SKB 2011/, referred to as SR-Site main report in the following

  3. Analysis of positional isotope exchange in ATP by cleavage of the βP-OγP bond. Demonstration of negligible positional isotope exchange by myosin

    Dale, M.P.; Hackney, D.D.

    1987-01-01

    A method for analysis of positional isotope exchange (PIX) during ATP ↔ HOH oxygen exchange is presented that uses a two-step degradation of ATP resulting in cleavage of the βP-OγP bond. This cleavage yields P/sub i/ derived from the γ-phosphoryl of ATP that contains all four of the γ oxygens. Both PIX between the β, γ-bridge and β-nonbridge positions and washout of the γ-nonbridge oxygens can be simultaneously followed by using ATP labeled with 17 O at the β-nonbridge positions and 18 O at the β,γ-bridge and γ-nonbridge positions. Application of this method to ATP ↔ HOH exchange during single turnovers of myosin indicates that the bulk of the ATP undergoes rapid washout of γ-nonbridge oxygens in the virtual absence of PIX. At 25 0 C with subfragment 1 the scrambling rate is at the limit of detectability of approximately 0.001 s -1 , which is 50-fold slower than the steady-state rate. This corresponds to a probability of scrambling for the β-oxygens of bound ADP of 1 in 10,000 for each cycle of reversible hydrolysis of bound ATP. A fraction of the ATP, however, does not undergo rapid washout. With myosin and stoichiometric ATP at 0 0 C, this fraction correspond to 10% of the ATP remaining at 36 s, or 2% of the initial ATP, and an equivalent level of ATP is found that does not bind irreversibly to myosin in a cold chase experiment. A significant level of apparent PIX is observed with subfragment 1 in the fraction that resists washout, and this apparent PIX is shown to be due to contaminant adenylate kinase activity. This apparent PIX due to adenylate kinase provides a possible explanation for the PIX observed by Geeves et al. with subfragment 1

  4. A flow cytometry-based method for a high-throughput analysis of drug-stabilized topoisomerase II cleavage complexes in human cells.

    de Campos-Nebel, Marcelo; Palmitelli, Micaela; González-Cid, Marcela

    2016-09-01

    Topoisomerase II (Top2) is an important target for anticancer therapy. A variety of drugs that poison Top2, including several epipodophyllotoxins, anthracyclines, and anthracenediones, are widely used in the clinic for both hematologic and solid tumors. The poisoning of Top2 involves the formation of a reaction intermediate Top2-DNA, termed Top2 cleavage complex (Top2cc), which is persistent in the presence of the drug and involves a 5' end of DNA covalently bound to a tyrosine from the enzyme through a phosphodiester group. Drug-induced Top2cc leads to Top2 linked-DNA breaks which are the major responsible for their cytotoxicity. While biochemical detection is very laborious, quantification of drug-induced Top2cc by immunofluorescence-based microscopy techniques is time consuming and requires extensive image segmentation for the analysis of a small population of cells. Here, we developed a flow cytometry-based method for the analysis of drug-induced Top2cc. This method allows a rapid analysis of a high number of cells in their cell cycle phase context. Moreover, it can be applied to almost any human cell type, including clinical samples. The methodology is useful for a high-throughput analysis of drugs that poison Top2, allowing not just the discrimination of the Top2 isoform that is targeted but also to track its removal. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry.

  5. LANDSAFE: LANDING SITE RISK ANALYSIS SOFTWARE FRAMEWORK

    R. Schmidt

    2012-08-01

    Full Text Available The European Space Agency (ESA is planning a Lunar Lander mission in the 2018 timeframe that will demonstrate precise soft landing at the polar regions of the Moon. To ensure a safe and successful landing a careful risk analysis has to be carried out. This is comprised of identifying favorable target areas and evaluating the surface conditions in these areas. Features like craters, boulders, steep slopes, rough surfaces and shadow areas have to be identified in order to assess the risk associated to a landing site in terms of a successful touchdown and subsequent surface operation of the lander. In addition, global illumination conditions at the landing site have to be simulated and analyzed. The Landing Site Risk Analysis software framework (LandSAfe is a system for the analysis, selection and certification of safe landing sites on the lunar surface. LandSAfe generates several data products including high resolution digital terrain models (DTMs, hazard maps, illumination maps, temperature maps and surface reflectance maps which assist the user in evaluating potential landing site candidates. This paper presents the LandSAfe system and describes the methods and products of the different modules. For one candidate landing site on the rim of Shackleton crater at the south pole of the Moon a high resolution DTM is showcased.

  6. Genome-wide analysis of carotenoid cleavage oxygenase genes and their responses to various phytohormones and abiotic stresses in apple (Malus domestica).

    Chen, Hongfei; Zuo, Xiya; Shao, Hongxia; Fan, Sheng; Ma, Juanjuan; Zhang, Dong; Zhao, Caiping; Yan, Xiangyan; Liu, Xiaojie; Han, Mingyu

    2018-02-01

    Carotenoid cleavage oxygenases (CCOs) are able to cleave carotenoids to produce apocarotenoids and their derivatives, which are important for plant growth and development. In this study, 21 apple CCO genes were identified and divided into six groups based on their phylogenetic relationships. We further characterized the apple CCO genes in terms of chromosomal distribution, structure and the presence of cis-elements in the promoter. We also predicted the cellular localization of the encoded proteins. An analysis of the synteny within the apple genome revealed that tandem, segmental, and whole-genome duplication events likely contributed to the expansion of the apple carotenoid oxygenase gene family. An additional integrated synteny analysis identified orthologous carotenoid oxygenase genes between apple and Arabidopsis thaliana, which served as references for the functional analysis of the apple CCO genes. The net photosynthetic rate, transpiration rate, and stomatal conductance of leaves decreased, while leaf stomatal density increased under drought and saline conditions. Tissue-specific gene expression analyses revealed diverse spatiotemporal expression patterns. Finally, hormone and abiotic stress treatments indicated that many apple CCO genes are responsive to various phytohormones as well as drought and salinity stresses. The genome-wide identification of apple CCO genes and the analyses of their expression patterns described herein may provide a solid foundation for future studies examining the regulation and functions of this gene family. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  7. Analysis of site-specific dispersion conditions

    Paesler-Sauer, J.

    1989-03-01

    This report presents an analysis of atmospheric dispersion conditions in the environs of nuclear power stations in the Federal Republic of Germany. The analysis is based on meteorological data measured on the power station sites (KFUe = nuclear reactor remote control records) and by neighbouring stations operated by the German Weather Service. The data are series of hourly mean values of wind and temperature gradient or stability class over the period of one or more years. The aim of the data analysis is to find types of dispersion conditions characterized by the flow field and stratification, and to assess the feasibility of calculating these quantities in the case of an emergency. Influences of terrain structures in the environs of the site are considered. The annual frequencies of types of dispersion situations are assessed, the capability to recognize the dispersion situation from meteorological data measured on the site and the applicability of dispersion models are discussed. (orig.) [de

  8. Summary Analysis: Hanford Site Composite Analysis Update

    Nichols, W. E. [CH2M HILL Plateau Remediation Company, Richland, WA (United States); Lehman, L. L. [CH2M HILL Plateau Remediation Company, Richland, WA (United States)

    2017-06-05

    The Hanford Site’s currently maintained Composite Analysis, originally completed in 1998, requires an update. A previous update effort was undertaken by the U.S. Department of Energy (DOE) in 2001-2005, but was ended before completion to allow the Tank Closure & Waste Management Environmental Impact Statement (TC&WM EIS) (DOE/EIS-0391) to be prepared without potential for conflicting sitewide models. This EIS was issued in 2012, and the deferral was ended with guidance in memorandum “Modeling to Support Regulatory Decision Making at Hanford” (Williams, 2012) provided with the aim of ensuring subsequent modeling is consistent with the EIS.

  9. Photoaffinity labeling of human serum vitamin D binding protein and chemical cleavage of the labeled protein: Identification of an 11.5-kDa peptide containing the putative 25-hydroxyvitamin D3 binding site

    Ray, R.; Holick, M.F.; Bouillon, R.; Baelen, H.V.

    1991-01-01

    In this paper, the authors describe photoaffinity labeling and related studies of human serum vitamin D binding protein (hDBP) with 25-hydroxyvitamin D 3 3β-3'-[N-(4-azido-2-nitrophenyl)amino]propyl ether (25-ANE) and its radiolabeled counterpart, i.e., 25-hydroxyvitamin D 3 3β-3'-[N-(4-azido-2-nitro-[3,5- 3 H]phenyl)amino]propyl ether ( 3 H-25-ANE). They have carried out studies to demonstrate that (1) 25-ANE competes with 25-OH-D 3 for the binding site of the latter in hDBP and (2) 3 H-25-ANE is capable of covalently labeling the hDBP molecule when exposed ot UV light. Treatment of a sample of purified hDBP, labeled with 3 H-25-ANE, with BNPS-skatole produced two Coomassie Blue stained peptide fragments, and the majority of the radioactivity was assoicated with the smaller of the two peptide fragments (16.5 kDa). On the other hand, cleavage of the labeled protein with cyanogen bromide produced a peptide (11.5 kDa) containing most of the covalently attached radioactivity. Considering the primary amino acid structure of hDBP, this peptide fragment (11.5 kDa) represents the N-terminus through residue 108 of the intact protein. Thus, the results tentatively identify this segment of the protein containing the binding pocket for 25-OH-D 3

  10. Detection of nucleic acid sequences by invader-directed cleavage

    Brow, Mary Ann D.; Hall, Jeff Steven Grotelueschen; Lyamichev, Victor; Olive, David Michael; Prudent, James Robert

    1999-01-01

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The 5' nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based by charge.

  11. EPOR-Based Purification and Analysis of Erythropoietin Mimetic Peptides from Human Urine by Cys-Specific Cleavage and LC/MS/MS

    Vogel, Matthias; Thomas, Andreas; Schänzer, Wilhelm; Thevis, Mario

    2015-09-01

    The development of a new class of erythropoietin mimetic agents (EMA) for treating anemic conditions has been initiated with the discovery of oligopeptides capable of dimerizing the erythropoietin (EPO) receptor and thus stimulating erythropoiesis. The most promising amino acid sequences have been mounted on various different polymeric structures or carrier molecules to obtain highly active EPO-like drugs exhibiting beneficial and desirable pharmacokinetic profiles. Concomitant with creating new therapeutic options, erythropoietin mimetic peptide (EMP)-based drug candidates represent means to artificially enhance endurance performance and necessitate coverage by sports drug testing methods. Therefore, the aim of the present study was to develop a strategy for the comprehensive detection of EMPs in doping controls, which can be used complementary to existing protocols. Three model EMPs were used to provide proof-of-concept data. Following EPO receptor-facilitated purification of target analytes from human urine, the common presence of the cysteine-flanked core structure of EMPs was exploited to generate diagnostic peptides with the aid of a nonenzymatic cleavage procedure. Sensitive detection was accomplished by targeted-SIM/data-dependent MS2 analysis. Method characterization was conducted for the EMP-based drug peginesatide concerning specificity, linearity, precision, recovery, stability, ion suppression/enhancement, and limit of detection (LOD, 0.25 ng/mL). Additionally, first data for the identification of the erythropoietin mimetic peptides EMP1 and BB68 were generated, demonstrating the multi-analyte testing capability of the presented approach.

  12. Putney Basketville Site Biomass CHP Analysis

    Hunsberger, Randolph [National Renewable Energy Lab. (NREL), Golden, CO (United States); Mosey, Gail [National Renewable Energy Lab. (NREL), Golden, CO (United States)

    2013-10-01

    The U.S. Environmental Protection Agency (EPA) Office of Solid Waste and Emergency Response Center for Program Analysis developed the RE-Powering America's Land initiative to reuse contaminated sites for renewable energy generation when aligned with the community's vision for the site. The Putney, Vermont, Basketville site, formerly the location of a basket-making facility and a paper mill andwoolen mill, was selected for a feasibility study under the program. Biomass was chosen as the renewable energy resource based on abundant woody-biomass resources available in the area. Biomass combined heat and power (CHP) was selected as the technology due to nearby loads, including Putney Paper and Landmark College.

  13. Intermolecular cleavage by UmuD-like mutagenesis proteins

    McDonald, John P.; Frank, Ekaterina G.; Levine, Arthur S.; Woodgate, Roger

    1998-01-01

    The activity of a number of proteins is regulated by self-processing reactions. Elegant examples are the cleavage of the prokaryotic LexA and λCI transcriptional repressors and the UmuD-like mutagenesis proteins. Various studies support the hypothesis that LexA and λCI cleavage reactions are predominantly intramolecular in nature. The recently described crystal structure of the Escherichia coli UmuD′ protein (the posttranslational cleavage product of the UmuD protein) suggests, however, that the region of the protein corresponding to the cleavage site is at least 50 Å away from the catalytic active site. We considered the possibility, therefore, that the UmuD-like proteins might undergo self-processing that, in contrast to LexA and λCI, occurs via an intermolecular rather than intramolecular reaction. To test this hypothesis, we introduced into E. coli compatible plasmids with mutations at either the cleavage or the catalytic site of three UmuD-like proteins. Cleavage of these proteins only occurs in the presence of both plasmids, indicating that the reaction is indeed intermolecular in nature. Furthermore, this intermolecular reaction is completely dependent upon the multifunctional RecA protein and leads to the restoration of cellular mutagenesis in nonmutable E. coli strains. Intermolecular cleavage of a biotinylated UmuD active site mutant was also observed in vitro in the presence of the wild-type UmuD′ protein, indicating that in addition to the intact UmuD protein, the normal cleavage product (UmuD′) can also act as a classical enzyme. PMID:9465040

  14. Preliminary safety analysis of the Gorleben site

    Bracke, G.; Fischer-Appelt, K.

    2014-01-01

    The safety requirements governing the final disposal of heat-generating radioactive waste in Germany were implemented by the Federal Ministry of Environment, Natural Conservation and Nuclear Safety (BMU) in 2010. The Ministry considers as a fundamental objective the protection of man and the environment against the hazards of radioactive waste. Unreasonable burdens and obligation for future generations shall be avoided. The main safety principles are concentration and inclusion of radioactive and other pollutants in a containment-providing rock zone. Any release of radioactive nuclides may increase the risk for men and the environment only negligibly compared to natural radiation exposure. No intervention or maintenance work shall be necessary in the post-closure phase. Retrieval/recovery of the waste shall be possible up to 500 years after closure. The Gorleben salt dome has been discussed since the 1970's as a possible repository site for heat-generating radioactive waste in Germany. The objective of the project preliminary safety analysis of the Gorleben site (VSG) was to assess if repository concepts at the Gorleben site or other sites with a comparable geology could comply with these requirements based on currently available knowledge (Fischer-Appelt, 2013; Bracke, 2013). In addition to this it was assessed if methodological approaches can be used for a future site selection procedure and which technological and conceptual considerations can be transferred to other geological situations. The objective included the compilation and review of the available exploration data of the Gorleben site and on disposal in salt rock, the development of repository designs, and the identification of the needs for future R and D work and further site investigations. (authors)

  15. Prediction of proprotein convertase cleavage sites

    Duckert, Peter; Brunak, Søren; Blom, Nikolaj

    2004-01-01

    by members of the subtilisin/kexin-like proprotein convertase (PC) family. In mammals, seven members have been identified, with furin being the one first discovered and best characterized. Recently, the involvement of furin in diseases ranging from Alzheimer's disease and cancer to anthrax and Ebola fever...

  16. Repository surface design site layout analysis

    Montalvo, H.R.

    1998-01-01

    The purpose of this analysis is to establish the arrangement of the Yucca Mountain Repository surface facilities and features near the North Portal. The analysis updates and expands the North Portal area site layout concept presented in the ACD, including changes to reflect the resizing of the Waste Handling Building (WHB), Waste Treatment Building (WTB), Carrier Preparation Building (CPB), and site parking areas; the addition of the Carrier Washdown Buildings (CWBs); the elimination of the Cask Maintenance Facility (CMF); and the development of a concept for site grading and flood control. The analysis also establishes the layout of the surface features (e.g., roads and utilities) that connect all the repository surface areas (North Portal Operations Area, South Portal Development Operations Area, Emplacement Shaft Surface Operations Area, and Development Shaft Surface Operations Area) and locates an area for a potential lag storage facility. Details of South Portal and shaft layouts will be covered in separate design analyses. The objective of this analysis is to provide a suitable level of design for the Viability Assessment (VA). The analysis was revised to incorporate additional material developed since the issuance of Revision 01. This material includes safeguards and security input, utility system input (size and location of fire water tanks and pump houses, potable water and sanitary sewage rates, size of wastewater evaporation pond, size and location of the utility building, size of the bulk fuel storage tank, and size and location of other exterior process equipment), main electrical substation information, redundancy of water supply and storage for the fire support system, and additional information on the storm water retention pond

  17. Staggering in the cleavage pattern of E. coli ABC-excinuclease

    Myles, G.M.; Van Houten, B.; Sancar, A.

    1986-01-01

    E. coli ABC excinuclease is a complex of three proteins encoded by the uvrA, uvrB, and uvrC genes. The enzyme repairs DNA mono and diadducts by the single strand cleavage of DNA eight phosphodiester bond 5' and four or five phosphodiester bonds 3' to a DNA lesion and facilitates the removal of the resulting twelve or thirteen nucleotide fragment. In this study, the authors have investigated the excision pattern for ultraviolet (UV) induced diadducts, i.e. cyclobutane pyrimidine dimers and pyrimidine-pyrimidone (6-4) photoproducts. Terminally (5' or 3') labeled DNA was irradiated with 254nm UV and treated with ABC excinuclease before and after photoreactivation of cyclobutane dimers by E. coli DNA photolyase. In this way, the authors were able to differentiate between the cleavage pattern of pyrimidine dimers and of (6-4) photoproducts. Their results show that certain TT cyclobutane dimers and rare TT (6-4) photoproducts are excised by cleavage seven and, less frequently, six phosphodiester bonds to the 5' side of the DNA lesion in addition to the primary cutting site at the eight 5' phosphodiester bond. The 3' cleavage sites are maintained at the fourth and fifth phosphodiester bonds for the these UV induced lesions. These data indicate that the cleavage pattern of the ABC excinuclease may be dependent upon both the type of DNA lesion as well as it surrounding nucleotide sequence. In addition, the authors analysis shows that (6-4) photoproducts are much better substrates for ABC excinuclease than are pyrimidine dimers

  18. Sequence specific inhibition of DNA restriction enzyme cleavage by PNA

    Nielsen, P.E.; Egholm, M.; Berg, R.H.

    1993-01-01

    Plasmids containing double-stranded 10-mer PNA (peptide nucleic acid chimera) targets proximally flanked by two restriction enzyme sites were challenged with the complementary PNA or PNAs having one or two mismatches, and the effect on the restriction enzyme cleavage of the flanking sites was ass...

  19. Mission analysis for cross-site transfer

    Riesenweber, S.D.; Fritz, R.L.; Shipley, L.E.

    1995-11-01

    The Mission Analysis Report describes the requirements and constraints associated with the Transfer Waste Function as necessary to support the Manage Tank Waste, Retrieve Waste, and Process Tank Waste Functions described in WHC-SD-WM-FRD-020, Tank Waste Remediation System (TWRS) Functions and Requirements Document and DOE/RL-92-60, Revision 1, TWRS Functions and Requirements Document, March 1994. It further assesses the ability of the ''initial state'' (or current cross-site transfer system) to meet the requirements and constraints

  20. Decontamination analysis of a radiologically contaminated site

    Tawil, J.J.; Strenge, D.L.

    1984-01-01

    This paper presents a post-exercise analysis of decontamination options for the NUWAX-83 exercise site. Held in May 1983, the purpose of NUWAX-83 was to evaluate the ability of federal, state and local officials to respond to an accident involving nuclear weapons. A computer program, called DECON, was developed by Pacific Northwest Laboratory and used to conduct the decontamination analysis in November 1983. DECON was designed to assist personnel engaged in the planning of decontamination activities. The many features of DECON that are demonstrated in this paper contribute to its potential usefulness as a planning tool for site restoration. Strategies that are analyzed with DECON include: 1) using a Quick-Vac option, under which exterior surfaces are vacuumed before it rains; 2) protecting surfaces against precipitation; 3) prohibiting specific operations on selected surfaces; 4) requiring that specific methods be used on selected surfaces; 5) evaluating the trade-off between cleanup standards and decontamination costs; and 6) varying clean-up standards according to expected human exposure to the surface

  1. Decontamination analysis of a radiologically contaminated site

    Tawil, J.J.; Strenge, D.L.

    1984-02-01

    This paper presents an analysis of decontamination options at the NUWAZX-83 exercise site. Held in May 1983, the purpose of the exercise was to evaluate the ability of federal, state and local officials to respond to a radiological accident involving nuclear weapons. A computer program developed by Pacific Northwest Laboratory was used to conduct the decontamination analysis. The program, called DECON, was designed to assist personnel engaged in the planning of decontamination activities. The many features of DECON that are demonstrated in this paper contribute to its potential usefulness as a planning tool for site restoration. Strategies that are analyzed with DECON include: (1) using a Quick-Vac option, under which exterior surfaces are vacuumed before it rains; (2) protecting surfaces against precipitation; (3) prohibiting specific operations on selected surfaces; (4) requiring that specific methods be used on selected surfaces; (5) evaluating the trade-off between cleanup standards and decontamination costs; and (6) varying clean-up standards according to expected human exposure to the surface

  2. Prediction of proteasome cleavage motifs by neural networks

    Kesimir, C.; Nussbaum, A.K.; Schild, H.

    2002-01-01

    physiological conditions. Our algorithm has been trained not only on in vitro data, but also on MHC Class I ligand data, which reflect a combination of immunoproteasome and constitutive proteasome specificity. This feature, together with the use of neural networks, a non-linear classification technique, make...... the prediction of MHC Class I ligand boundaries more accurate: 65% of the cleavage sites and 85% of the non-cleavage sites are correctly determined. Moreover, we show that the neural networks trained on the constitutive proteasome data learns a specificity that differs from that of the networks trained on MHC...

  3. Maximizing Selective Cleavages at Aspartic Acid and Proline Residues for the Identification of Intact Proteins

    Foreman, David J.; Dziekonski, Eric T.; McLuckey, Scott A.

    2018-04-01

    A new approach for the identification of intact proteins has been developed that relies on the generation of relatively few abundant products from specific cleavage sites. This strategy is intended to complement standard approaches that seek to generate many fragments relatively non-selectively. Specifically, this strategy seeks to maximize selective cleavage at aspartic acid and proline residues via collisional activation of precursor ions formed via electrospray ionization (ESI) under denaturing conditions. A statistical analysis of the SWISS-PROT database was used to predict the number of arginine residues for a given intact protein mass and predict a m/z range where the protein carries a similar charge to the number of arginine residues thereby enhancing cleavage at aspartic acid residues by limiting proton mobility. Cleavage at aspartic acid residues is predicted to be most favorable in the m/z range of 1500-2500, a range higher than that normally generated by ESI at low pH. Gas-phase proton transfer ion/ion reactions are therefore used for precursor ion concentration from relatively high charge states followed by ion isolation and subsequent generation of precursor ions within the optimal m/z range via a second proton transfer reaction step. It is shown that the majority of product ion abundance is concentrated into cleavages C-terminal to aspartic acid residues and N-terminal to proline residues for ions generated by this process. Implementation of a scoring system that weights both ion fragment type and ion fragment area demonstrated identification of standard proteins, ranging in mass from 8.5 to 29.0 kDa. [Figure not available: see fulltext.

  4. Analysis of digitalis genin receptor site in Na,K-ATPase

    Ahmed, K.; McParland, R.; Becker, R.; From, A.; Fullerton, D.S.

    1987-01-01

    Na,K-ATPase is believed to be the receptor for digitalis glycosides, with binding site located in the α-subunit. To identify this binding site, the enzyme was covalently labeled with a photoactive probe localized in C17 side group of the cardenolide ([ 3 H]24-azidodigitoxoside). 3 H-labeled α-subunit was purified, and subjected to trypsin digestion. Fractions containing 3 H-labeled material were pooled. Amino acid sequence analysis of this material suggested the presence of two peptides (residues 68-146; residues 263-342). Additional studies have employed purification of the 3 H-labeled material by chromatography on Sepharose-6B, and CNBr cleavage followed by chromatography on hydroxylapatite. Amino acid sequence analysis of the purified 3 H-labeled peptide thus isolated indicated sequence containing amino acid residues 263-342. These data suggest that this is the peptide containing the digitalis genin binding site, and rule out such a role for the other peptide (amino acids 68 - 146). Preliminary data also hint that binding of the 3 H-probe occurs at the leu residue in the sequence glu tyr thr try leu glu .. present in the peptide containing residues 263 - 342

  5. Analysis of digitalis genin receptor site in Na,K-ATPase

    Ahmed, K.; McParland, R.; Becker, R.; From, A.; Fullerton, D.S.

    1987-05-01

    Na,K-ATPase is believed to be the receptor for digitalis glycosides, with binding site located in the ..cap alpha..-subunit. To identify this binding site, the enzyme was covalently labeled with a photoactive probe localized in C17 side group of the cardenolide ((/sup 3/H)24-azidodigitoxoside). /sup 3/H-labeled ..cap alpha..-subunit was purified, and subjected to trypsin digestion. Fractions containing /sup 3/H-labeled material were pooled. Amino acid sequence analysis of this material suggested the presence of two peptides (residues 68-146; residues 263-342). Additional studies have employed purification of the /sup 3/H-labeled material by chromatography on Sepharose-6B, and CNBr cleavage followed by chromatography on hydroxylapatite. Amino acid sequence analysis of the purified /sup 3/H-labeled peptide thus isolated indicated sequence containing amino acid residues 263-342. These data suggest that this is the peptide containing the digitalis genin binding site, and rule out such a role for the other peptide (amino acids 68 - 146). Preliminary data also hint that binding of the /sup 3/H-probe occurs at the leu residue in the sequence glu tyr thr try leu glu .. present in the peptide containing residues 263 - 342.

  6. Site suitability analysis and route optimization for solid waste ...

    Solid waste management system is a tedious task that is facing both developing and developed countries. Site Suitability analysis and route optimization for solid waste disposal can make waste management cheap and can be used for sustainable development. However, if the disposal site(s) is/are not sited and handle ...

  7. Description of the Northwest hazardous waste site data base and preliminary analysis of site characteristics

    Woodruff, D.L.; Hartz, K.E.; Triplett, M.B.

    1988-08-01

    The Northwest Hazardous Waste RD and D Center (the Center) conducts research, development, and demonstration (RD and D) activities for hazardous and radioactive mixed-waste technologies applicable to remediating sites in the states of Idaho, Montana, Oregon, and Washington. To properly set priorities for these RD and D activities and to target development efforts it is necessary to understand the nature of the sites requiring remediation. A data base of hazardous waste site characteristics has been constructed to facilitate this analysis. The data base used data from EPA's Region X Comprehensive Environmental Response, Compensation, and Liability Information System (CERCLIS) and from Preliminary Assessment/Site Investigation (PA/SI) forms for sites in Montana. The Center's data base focuses on two sets of sites--those on the National Priorities List (NPL) and other sites that are denoted as ''active'' CERCLIS sites. Active CERCLIS sites are those sites that are undergoing active investigation and analysis. The data base contains information for each site covering site identification and location, type of industry associated with the site, waste categories present (e.g., heavy metals, pesticides, etc.), methods of disposal (e.g., tanks, drums, land, etc.), waste forms (e.g., liquid, solid, etc.), and hazard targets (e.g., surface water, groundwater, etc.). As part of this analysis, the Northwest region was divided into three geographic subregions to identify differences in disposal site characteristics within the Northwest. 2 refs., 18 figs., 5 tabs

  8. Cleavage of desmin by cysteine proteases: Calpains and cathepsin B

    Baron, Caroline; Jacobsen, S.; Purslow, P.P.

    2004-01-01

    The intermediate filament protein, desmin, was purified from pork longissimus dorsi and incubated with either P-calpain, m-calpain or cathepsin B. Proteolysis of desmin was followed using SDS-PAGE and Western blotting. After incubation of desmin with the proteases, cleavage sites on the desmin mo...

  9. Unexpected tolerance of alpha-cleavage of the prion protein to sequence variations.

    José B Oliveira-Martins

    Full Text Available The cellular form of the prion protein, PrP(C, undergoes extensive proteolysis at the alpha site (109K [see text]H110. Expression of non-cleavable PrP(C mutants in transgenic mice correlates with neurotoxicity, suggesting that alpha-cleavage is important for PrP(C physiology. To gain insights into the mechanisms of alpha-cleavage, we generated a library of PrP(C mutants with mutations in the region neighbouring the alpha-cleavage site. The prevalence of C1, the carboxy adduct of alpha-cleavage, was determined for each mutant. In cell lines of disparate origin, C1 prevalence was unaffected by variations in charge and hydrophobicity of the region neighbouring the alpha-cleavage site, and by substitutions of the residues in the palindrome that flanks this site. Instead, alpha-cleavage was size-dependently impaired by deletions within the domain 106-119. Almost no cleavage was observed upon full deletion of this domain. These results suggest that alpha-cleavage is executed by an alpha-PrPase whose activity, despite surprisingly limited sequence specificity, is dependent on the size of the central region of PrP(C.

  10. ANALYSIS OF CHP POTENTIAL AT FEDERAL SITES

    HADLEY, S.W.

    2002-03-11

    This document was prepared at the request of the U.S. Department of Energy's (DOE's) Federal Energy Management Program (FEMP) under its Technical Guidance and Assistance and Project Financing Programs. The purpose was to provide an estimate of the national potential for combined heat and power (also known as CHP; cogeneration; or cooling, heating, and power) applications at federal facilities and the associated costs and benefits including energy and emission savings. The report provides a broad overview for the U.S. Department of Energy (DOE) and other agencies on when and where CHP systems are most likely to serve the government's best interest. FEMP's mission is to reduce the cost to and environmental impact of the federal government by advancing energy efficiency and water conservation, promoting the use of renewable energy, and improving utility management decisions at federal sites. FEMP programs are driven by its customers: federal agency sites. FEMP monitors energy efficiency and renewable energy technology developments and mounts ''technology-specific'' programs to make technologies that are in strong demand by agencies more accessible. FEMP's role is often one of helping the federal government ''lead by example'' through the use of advanced energy efficiency/renewable energy (EERE) technologies in its own buildings and facilities. CHP was highlighted in the Bush Administration's National Energy Policy Report as a commercially available technology offering extraordinary benefits in terms of energy efficiencies and emission reductions. FEMP's criteria for emphasizing a technology are that it must be commercially available; be proven but underutilized; have a strong constituency and momentum; offer large energy savings and other benefits of interest to federal sites and FEMP mission; be in demand; and carry sufficient federal market potential. As discussed in the report, CHP meets all

  11. Abyssal fiction: common shares, colonial cleavages

    Alexandre Montaury

    2016-12-01

    Full Text Available The paper aims to develop a reflection on the interaction between the legacies of colonialism and traditional symbolic and cultural practices in African Portuguese-speaking spaces. From a preliminary analysis of fictional texts of wide circulation in Brazil, aims to examine the cleavages, or “abyssal lines” that constitute experiences printed in the daily life of the former Portuguese colony of Cape Verde, Mozambique and Angola.---DOI: http://dx.doi.org/10.21881/abriluff.2016n17a378

  12. Short RNA guides cleavage by eukaryotic RNase III.

    Bruno Lamontagne

    Full Text Available In eukaryotes, short RNAs guide a variety of enzymatic activities that range from RNA editing to translation repression. It is hypothesized that pre-existing proteins evolved to bind and use guide RNA during evolution. However, the capacity of modern proteins to adopt new RNA guides has never been demonstrated. Here we show that Rnt1p, the yeast orthologue of the bacterial dsRNA-specific RNase III, can bind short RNA transcripts and use them as guides for sequence-specific cleavage. Target cleavage occurred at a constant distance from the Rnt1p binding site, leaving the guide RNA intact for subsequent cleavage. Our results indicate that RNase III may trigger sequence-specific RNA degradation independent of the RNAi machinery, and they open the road for a new generation of precise RNA silencing tools that do not trigger a dsRNA-mediated immune response.

  13. The Detection and Analysis of Chromosome Fragile Sites

    Bjerregaard, Victoria A; Özer, Özgün; Hickson, Ian D

    2018-01-01

    A fragile site is a chromosomal locus that is prone to form a gap or constriction visible within a condensed metaphase chromosome, particularly following exposure of cells to DNA replication stress. Based on their frequency, fragile sites are classified as either common (CFSs; present in all...... for detection and analysis of chromosome fragile sites....

  14. Genome-wide analysis of poly(A) site selection in Schizosaccharomyces pombe

    Schlackow, M.

    2013-10-23

    Polyadenylation of pre-mRNAs, a critical step in eukaryotic gene expression, is mediated by cis elements collectively called the polyadenylation signal. Genome-wide analysis of such polyadenylation signals was missing in fission yeast, even though it is an important model organism. We demonstrate that the canonical AATAAA motif is the most frequent and functional polyadenylation signal in Schizosaccharomyces pombe. Using analysis of RNA-Seq data sets from cells grown under various physiological conditions, we identify 3\\' UTRs for nearly 90% of the yeast genes. Heterogeneity of cleavage sites is common, as is alternative polyadenylation within and between conditions. We validated the computationally identified sequence elements likely to promote polyadenylation by functional assays, including qRT-PCR and 3\\'RACE analysis. The biological importance of the AATAAA motif is underlined by functional analysis of the genes containing it. Furthermore, it has been shown that convergent genes require trans elements, like cohesin for efficient transcription termination. Here we show that convergent genes lacking cohesin (on chromosome 2) are generally associated with longer overlapping mRNA transcripts. Our bioinformatic and experimental genome-wide results are summarized and can be accessed and customized in a user-friendly database Pomb(A).

  15. Genome-wide analysis of poly(A) site selection in Schizosaccharomyces pombe

    Schlackow, M.; Marguerat, S.; Proudfoot, N. J.; Bahler, J.; Erban, R.; Gullerova, M.

    2013-01-01

    Polyadenylation of pre-mRNAs, a critical step in eukaryotic gene expression, is mediated by cis elements collectively called the polyadenylation signal. Genome-wide analysis of such polyadenylation signals was missing in fission yeast, even though it is an important model organism. We demonstrate that the canonical AATAAA motif is the most frequent and functional polyadenylation signal in Schizosaccharomyces pombe. Using analysis of RNA-Seq data sets from cells grown under various physiological conditions, we identify 3' UTRs for nearly 90% of the yeast genes. Heterogeneity of cleavage sites is common, as is alternative polyadenylation within and between conditions. We validated the computationally identified sequence elements likely to promote polyadenylation by functional assays, including qRT-PCR and 3'RACE analysis. The biological importance of the AATAAA motif is underlined by functional analysis of the genes containing it. Furthermore, it has been shown that convergent genes require trans elements, like cohesin for efficient transcription termination. Here we show that convergent genes lacking cohesin (on chromosome 2) are generally associated with longer overlapping mRNA transcripts. Our bioinformatic and experimental genome-wide results are summarized and can be accessed and customized in a user-friendly database Pomb(A).

  16. Structure of the Cpf1 endonuclease R-loop complex after target DNA cleavage

    Stella, Stefano; Alcón, Pablo; Montoya, Guillermo

    2017-01-01

    involved in DNA unwinding to form a CRISPR RNA (crRNA)-DNA hybrid and a displaced DNA strand. The protospacer adjacent motif (PAM) is recognized by the PAM-interacting domain. The loop-lysine helix-loop motif in this domain contains three conserved lysine residues that are inserted in a dentate manner...... and the crRNA-DNA hybrid, avoiding DNA re-annealing. Mutations in key residues reveal a mechanism linking the PAM and DNA nuclease sites. Analysis of the Cpf1 structures proposes a singular working model of RNA-guided DNA cleavage, suggesting new avenues for redesign of Cpf1....

  17. Post-transcription cleavage generates the 3' end of F17R transcripts in vaccinia virus

    D'Costa, Susan M.; Antczak, James B.; Pickup, David J.; Condit, Richard C.

    2004-01-01

    Most vaccinia virus intermediate and late mRNAs possess 3' ends that are extremely heterogeneous in sequence. However, late mRNAs encoding the cowpox A-type inclusion protein (ATI), the second largest subunit of the RNA polymerase, and the late telomeric transcripts possess homogeneous 3' ends. In the case of the ATI mRNA, it has been shown that the homogeneous 3' end is generated by a post-transcriptional endoribonucleolytic cleavage event. We have determined that the F17R gene also produces homogeneous transcripts generated by a post-transcriptional cleavage event. Mapping of in vivo mRNA shows that the major 3' end of the F17R transcript maps 1262 nt downstream of the F17R translational start site. In vitro transcripts spanning the in vivo 3' end are cleaved in an in vitro reaction using extracts from virus infected cells, and the site of cleavage is the same both in vivo and in vitro. Cleavage is not observed using extract from cells infected in the presence of hydroxyurea; therefore, the cleavage factor is either virus-coded or virus-induced during the post-replicative phase of virus replication. The cis-acting sequence responsible for cleavage is orientation specific and the factor responsible for cleavage activity has biochemical properties similar to the factor required for cleavage of ATI transcripts. Partially purified cleavage factor generates cleavage products of expected size when either the ATI or F17R substrates are used in vitro, strongly suggesting that cleavage of both transcripts is mediated by the same factor

  18. Specificity of N-terminal methionyl peptidase: analysis by site-directed mutagenesis

    Kasper, T.J.; Boissel, J.P.; Bunn, H.F.

    1987-01-01

    The start site of eukaryotic translation is normally an AUG codon. The corresponding N-terminal methionine is most often removed when the nascent chain reaches about 30 residues. Data from a survey of 1764 eukaryotic protein sequences suggest that the residue adjacent to the initiator Met determines Met cleavage. In order to investigate the mechanism of this reaction, the authors have prepared oligonucleotide-directed mutants of human β-globin from gapped heteroduplexes of a T3/T7 plasmid containing a globin cDNA clone. To date, the authors have produced mutants encoding for 15 of 19 possible amino acid replacements at position 1 in the β-globin chain. These mutants have been confirmed by dideoxy sequencing, transcribed in vitro, and translated in a rabbit reticulocyte lysate in the presence of 35 S-methionine. Labeled translation products were then isolated by cation exchange HPLC, and tryptic peptides were analyzed by RP-HPLC. Thus far, this structural analysis has shown that for β-1 Val, Ala, and Ser, the initiator Met is cleaved, whereas for β-1 Lys, Met, Glu, Trp, Asn, Tyr, and Glu, initiator Met is retained. For β-1 Leu initiator Met is cleaved with a frequency of about 50%. These results are consistent with the data obtained from the previous survey. The expression of site-directed mutants in a cell-free system can also be used to investigate other N-terminal processing events, such as acetylation and myristylation

  19. Altered cleavage patterns in human tripronuclear embryos and their association to fertilization method

    Joergensen, Mette Warming; Agerholm, Inge; Hindkjaer, Johnny

    2014-01-01

    PURPOSE: To analyze the cleavage patterns in dipronuclear (2PN) and tripronuclear (3PN) embryos in relation to fertilization method. METHOD: Time-lapse analysis. RESULTS: Compared to 2PN, more 3PN IVF embryos displayed early cleavage into 3 cells (p ... stage (p embryos, the 2nd and 3rd cleavage cycles were completed within the expected time frame. However, timing of the cell divisions within the cleavage cycles differed between the two groups. In contrast......, the completion of the 1st, 2nd, and 3rd cleavage cycle was delayed, but with a similar division pattern for 3PN ICSI compared with the 2PN ICSI embryos. 3PN, more often than 2PN ICSI embryos, displayed early cleavage into 3 cells (p = 0.03) and arrested development from the compaction stage and onwards (p = 0...

  20. Rings and sector : intrasite spatial analysis of stone age sites

    Stapert, Durk

    1992-01-01

    This thesis deals with intrasite spatial analysis: the analysis of spatial patterns on site level. My main concern has been to develop a simple method for analysing Stone Age sites of a special type: those characterised by the presence of a hearth closely associated in space with an artefact

  1. Site Specific Ground Response Analysis for Quantifying Site Amplification at A Regolith Site

    Bambang Setiawan

    2017-08-01

    Full Text Available DOI: 10.17014/ijog.4.3.159-167A numerical model has demonstrated that it can simulate reasonably well earthquake motions at the ground level during a seismic event. The most widely used model is an equivalent linear approach. The equivalent linear model was used to compute the free-field response of Adelaide regolith during the 1997 Burra earthquake. The aim of this study is to quantify the amplification at the investigated site. The model computed the ground response of horizontally layered soil deposits subjected to transient and vertically propagating shear waves through a one-dimensional-soil column. Each soil layer was assumed to be homogeneous, visco-elastic, and infinite in the horizontal extent. The results of this study were compared to other studies and forward computation of the geotechnical dynamic parameters of the investigated site. The amplification triggered by the 1997 Burra seismic event was deduced. This study reveals the amplification factor up to 3.6 at the studied site.

  2. Structural and functional basis for RNA cleavage by Ire1

    Stroud Robert M

    2011-07-01

    Full Text Available Abstract Background The unfolded protein response (UPR controls the protein folding capacity of the endoplasmic reticulum (ER. Central to this signaling pathway is the ER-resident bifunctional transmembrane kinase/endoribonuclease Ire1. The endoribonuclease (RNase domain of Ire1 initiates a non-conventional mRNA splicing reaction, leading to the production of a transcription factor that controls UPR target genes. The mRNA splicing reaction is an obligatory step of Ire1 signaling, yet its mechanism has remained poorly understood due to the absence of substrate-bound crystal structures of Ire1, the lack of structural similarity between Ire1 and other RNases, and a scarcity of quantitative enzymological data. Here, we experimentally define the active site of Ire1 RNase and quantitatively evaluate the contribution of the key active site residues to catalysis. Results This analysis and two new crystal structures suggest that Ire1 RNase uses histidine H1061 and tyrosine Y1043 as the general acid-general base pair contributing ≥ 7.6 kcal/mol and 1.4 kcal/mol to transition state stabilization, respectively, and asparagine N1057 and arginine R1056 for coordination of the scissile phosphate. Investigation of the stem-loop recognition revealed that additionally to the stem-loops derived from the classic Ire1 substrates HAC1 and Xbp1 mRNA, Ire1 can site-specifically and rapidly cleave anticodon stem-loop (ASL of unmodified tRNAPhe, extending known substrate specificity of Ire1 RNase. Conclusions Our data define the catalytic center of Ire1 RNase and suggest a mechanism of RNA cleavage: each RNase monomer apparently contains a separate catalytic apparatus for RNA cleavage, whereas two RNase subunits contribute to RNA stem-loop docking. Conservation of the key residues among Ire1 homologues suggests that the mechanism elucidated here for yeast Ire1 applies to Ire1 in metazoan cells, and to the only known Ire1 homologue RNase L.

  3. Perturbation with intrabodies reveals that calpain cleavage is required for degradation of huntingtin exon 1.

    Amber L Southwell

    2011-01-01

    Full Text Available Proteolytic processing of mutant huntingtin (mHtt, the protein that causes Huntington's disease (HD, is critical for mHtt toxicity and disease progression. mHtt contains several caspase and calpain cleavage sites that generate N-terminal fragments that are more toxic than full-length mHtt. Further processing is then required for the degradation of these fragments, which in turn, reduces toxicity. This unknown, secondary degradative process represents a promising therapeutic target for HD.We have used intrabodies, intracellularly expressed antibody fragments, to gain insight into the mechanism of mutant huntingtin exon 1 (mHDx-1 clearance. Happ1, an intrabody recognizing the proline-rich region of mHDx-1, reduces the level of soluble mHDx-1 by increasing clearance. While proteasome and macroautophagy inhibitors reduce turnover of mHDx-1, Happ1 is still able to reduce mHDx-1 under these conditions, indicating Happ1-accelerated mHDx-1 clearance does not rely on these processes. In contrast, a calpain inhibitor or an inhibitor of lysosomal pH block Happ1-mediated acceleration of mHDx-1 clearance. These results suggest that mHDx-1 is cleaved by calpain, likely followed by lysosomal degradation and this process regulates the turnover rate of mHDx-1. Sequence analysis identifies amino acid (AA 15 as a potential calpain cleavage site. Calpain cleavage of recombinant mHDx-1 in vitro yields fragments of sizes corresponding to this prediction. Moreover, when the site is blocked by binding of another intrabody, V(L12.3, turnover of soluble mHDx-1 in living cells is blocked.These results indicate that calpain-mediated removal of the 15 N-terminal AAs is required for the degradation of mHDx-1, a finding that may have therapeutic implications.

  4. Analysis of laparoscopic port site complications: A descriptive study.

    Karthik, Somu; Augustine, Alfred Joseph; Shibumon, Mundunadackal Madhavan; Pai, Manohar Varadaraya

    2013-04-01

    The rate of port site complications following conventional laparoscopic surgery is about 21 per 100,000 cases. It has shown a proportional rise with increase in the size of the port site incision and trocar. Although rare, complications that occur at the port site include infection, bleeding, and port site hernia. To determine the morbidity associated with ports at the site of their insertion in laparoscopic surgery and to identify risk factors for complications. Prospective descriptive study. In the present descriptive study, a total of 570 patients who underwent laparoscopic surgeries for various ailments between August 2009 and July 2011 at our institute were observed for port site complications prospectively and the complications were reviewed. Descriptive statistical analysis was carried out in the present study. The statistical software, namely, SPSS 15.0 was used for the analysis of the data. Of the 570 patients undergoing laparoscopic surgery, 17 (3%) had developed complications specifically related to the port site during a minimum follow-up of three months; port site infection (PSI) was the most frequent (n = 10, 1.8%), followed by port site bleeding (n = 4, 0.7%), omentum-related complications (n = 2; 0.35%), and port site metastasis (n = 1, 0.175%). Laparoscopic surgeries are associated with minimal port site complications. Complications are related to the increased number of ports. Umbilical port involvement is the commonest. Most complications are manageable with minimal morbidity, and can be further minimized with meticulous surgical technique during entry and exit.

  5. Site-specific analysis of the cobbly soils at the Grand Junction processing site

    1992-06-01

    This report describes a recent site-specific analysis to evaluate the necessity of a recommendation to install a slurry trench around the Grand Junction processing site. The following analysis addresses the cobbly nature of the site's radiologically contaminated foundation soil, reassesses the excavation depths based on bulk radionuclide concentrations, and presents data-based arguments that support the elimination of the initially proposed slurry trench. The slurry trench around the processing site was proposed by the Remedial Action Contractor (RAC) to minimize the amount of water encountered during excavation. The initial depths of excavation developed during conceptual design, which indicated the need for a slurry wall, were reexamined as part of this analysis. This reanalysis, based on bulk concentrations of a cobbly subsoil, supports decreasing the original excavation depth, limiting the dewatering quantities to those which can be dissipated by normal construction activities. This eliminates the need for a slurry trench andseparate water treatment prior to permitted discharge

  6. Cell-surface acceleration of urokinase-catalyzed receptor cleavage

    Høyer-Hansen, G; Ploug, M; Behrendt, N

    1997-01-01

    by a prior incubation of the cells with uPA inactivated by diisopropyl fluorophosphate, demonstrating a requirement for specific receptor binding of the active uPA to obtain the high-efficiency cleavage of cell-bound uPAR. Furthermore, amino-terminal sequence analysis revealed that uPAR(2+3), purified from U...

  7. LANDSAFE: LANDING SITE RISK ANALYSIS SOFTWARE FRAMEWORK

    Schmidt, Ralph; Bostelmann, Jonas; Cornet, Yves; Heipke, Christian; Philippe, Christian; Poncelet, Nadia; de Rosa, Diego; Vandeloise, Yannick

    2012-01-01

    The European Space Agency (ESA) is planning a Lunar Lander mission in the 2018 timeframe that will demonstrate precise soft landing at the polar regions of the Moon. To ensure a safe and successful landing a careful risk analysis has to be carried out. This is comprised of identifying favorable target areas and evaluating the surface conditions in these areas. Features like craters, boulders, steep slopes, rough surfaces and shadow areas have to be identified in order to assess the risk assoc...

  8. Development of a site analysis tool for distributed wind projects

    Shaw, Shawn [The Cadmus Group, Inc., Waltham MA (United States)

    2012-02-28

    The Cadmus Group, Inc., in collaboration with the National Renewable Energy Laboratory (NREL) and Encraft, was awarded a grant from the Department of Energy (DOE) to develop a site analysis tool for distributed wind technologies. As the principal investigator for this project, Mr. Shawn Shaw was responsible for overall project management, direction, and technical approach. The product resulting from this project is the Distributed Wind Site Analysis Tool (DSAT), a software tool for analyzing proposed sites for distributed wind technology (DWT) systems. This user-friendly tool supports the long-term growth and stability of the DWT market by providing reliable, realistic estimates of site and system energy output and feasibility. DSAT-which is accessible online and requires no purchase or download of software-is available in two account types; Standard: This free account allows the user to analyze a limited number of sites and to produce a system performance report for each; and Professional: For a small annual fee users can analyze an unlimited number of sites, produce system performance reports, and generate other customizable reports containing key information such as visual influence and wind resources. The tool’s interactive maps allow users to create site models that incorporate the obstructions and terrain types present. Users can generate site reports immediately after entering the requisite site information. Ideally, this tool also educates users regarding good site selection and effective evaluation practices.

  9. Glutamic Acid Selective Chemical Cleavage of Peptide Bonds.

    Nalbone, Joseph M; Lahankar, Neelam; Buissereth, Lyssa; Raj, Monika

    2016-03-04

    Site-specific hydrolysis of peptide bonds at glutamic acid under neutral aqueous conditions is reported. The method relies on the activation of the backbone amide chain at glutamic acid by the formation of a pyroglutamyl (pGlu) imide moiety. This activation increases the susceptibility of a peptide bond toward hydrolysis. The method is highly specific and demonstrates broad substrate scope including cleavage of various bioactive peptides with unnatural amino acid residues, which are unsuitable substrates for enzymatic hydrolysis.

  10. Robust detection of discordant sites in regional frequency analysis

    Neykov, N.M.; Neytchev, P.N.; Van Gelder, P.H.A.J.M.; Todorov, V.K.

    2007-01-01

    The discordancy measure in terms of the sample L?moment ratios (L?CV, L?skewness, L?kurtosis) of the at?site data is widely recommended in the screening process of atypical sites in the regional frequency analysis (RFA). The sample mean and the covariance matrix of the L?moments ratios, on which the

  11. DECISION ANALYSIS OF INCINERATION COSTS IN SUPERFUND SITE REMEDIATION

    This study examines the decision-making process of the remedial design (RD) phase of on-site incineration projects conducted at Superfund sites. Decisions made during RD affect the cost and schedule of remedial action (RA). Decision analysis techniques are used to determine the...

  12. [Modular risk analysis for assessing multiple waste sites]: Proceedings

    Whelan, G.

    1994-01-01

    This document contains proceedings from the Integrated Planning Workshop from Strategic Planning to Baselining and Other Objectives. Topics discussed include: stakeholder involvement; regulations; future site use planning; site integration and baseline methods; risk analysis in decision making; land uses; and economics in decision making. Individual records have been processed separately for the database

  13. Alternatives Analysis For Selecting ET 3 Site

    Collard, L.; Hamm, L.

    2012-01-01

    Engineered trenches (ETs) are considered to be a cost-effective method for disposing Low Level Waste (LLW). Based on waste forecasts from waste generators, the last engineered trench in operation (ET No.2) is anticipated to close in FY14, requiring development of a new ET. Solid Waste requested that SRNL develop an assessment report that reviews four disposal options for this new ET (ET No.3) and determine which option would provide the 'best' Performance Assessment (PA) disposal limits for LLW (Appendix A). Those four options (see option footprint locations in Figure 1-1) are: (1) Disposal at grade on TRU Pads 7-13 where soil would be mounded over waste packages; (2) Excavation at a slightly modified SLIT No.13 location - near the Used Equipment Storage Area; (3) Excavation at a modified SLIT No.12 location - near the 643-26E Naval Reactor Component Disposal Area; and (4) Excavation east of TRU Pad No.26 that replaces northeast portions of four slit trench (ST) disposal units in the eastern set of STs. The assessment consisted of both quantitative and qualitative analyses. The quantitative analysis captured key aspects that were readily quantifiable and had predictable impacts on limits and doses. A simplified modeling strategy stemming from current Special Analysis (SA) practices was employed. Both inventory capacity for a specific nuclide (a quasi-inventory limit) and overall performance for specified inventory mixtures (doses resulting from historical inventories) were considered. The qualitative analysis evaluated other key aspects based on engineering judgment in the form of pros and cons.

  14. High-resolution characterization of sequence signatures due to non-random cleavage of cell-free DNA.

    Chandrananda, Dineika; Thorne, Natalie P; Bahlo, Melanie

    2015-06-17

    High-throughput sequencing of cell-free DNA fragments found in human plasma has been used to non-invasively detect fetal aneuploidy, monitor organ transplants and investigate tumor DNA. However, many biological properties of this extracellular genetic material remain unknown. Research that further characterizes circulating DNA could substantially increase its diagnostic value by allowing the application of more sophisticated bioinformatics tools that lead to an improved signal to noise ratio in the sequencing data. In this study, we investigate various features of cell-free DNA in plasma using deep-sequencing data from two pregnant women (>70X, >50X) and compare them with matched cellular DNA. We utilize a descriptive approach to examine how the biological cleavage of cell-free DNA affects different sequence signatures such as fragment lengths, sequence motifs at fragment ends and the distribution of cleavage sites along the genome. We show that the size distributions of these cell-free DNA molecules are dependent on their autosomal and mitochondrial origin as well as the genomic location within chromosomes. DNA mapping to particular microsatellites and alpha repeat elements display unique size signatures. We show how cell-free fragments occur in clusters along the genome, localizing to nucleosomal arrays and are preferentially cleaved at linker regions by correlating the mapping locations of these fragments with ENCODE annotation of chromatin organization. Our work further demonstrates that cell-free autosomal DNA cleavage is sequence dependent. The region spanning up to 10 positions on either side of the DNA cleavage site show a consistent pattern of preference for specific nucleotides. This sequence motif is present in cleavage sites localized to nucleosomal cores and linker regions but is absent in nucleosome-free mitochondrial DNA. These background signals in cell-free DNA sequencing data stem from the non-random biological cleavage of these fragments. This

  15. Cleavage and creep fracture of rock salt

    Chan, K.S.; Munson, D.E.; Bodner, S.R.

    1996-01-01

    The dominant failure mechanism in rock salt at ambient temperature is either cleavage or creep fracture. Since the transition of creep fracture to cleavage in a compressive stress field is not well understood, failure of rock salt by cleavage and creep fracture is analyzed in this paper to elucidate the effect of stress state on the competition between these two fracture mechanisms. For cleavage fracture, a shear crack is assumed to cause the formation and growth of a symmetric pair of wing cracks in a predominantly compressive stress field. The conditions for wing-crack instability are derived and presented as the cleavage fracture boundary in the fracture mechanism map. Using an existing creep fracture model, stress conditions for the onset of creep fracture and isochronous failure curves of specified times-to-rupture are calculated and incorporated into the fracture mechanism map. The regimes of dominance by cleavage and creep fracture are established and compared with experimental data. The result indicates that unstable propagation of cleavage cracks occurs only in the presence of tensile stress. The onset of creep fracture is promoted by a tensile stress, but can be totally suppressed by a high confining pressure. Transition of creep fracture to cleavage occurs when critical conditions of stress difference and tensile stress for crack instability are exceeded

  16. Hanford Site Composite Analysis Technical Approach Description: Radionuclide Inventory and Waste Site Selection Process.

    Nichols, Will E.; Mehta, Sunil

    2017-09-13

    The updated Hanford Site Composite Analysis will provide an all-pathways dose projection to a hypothetical future member of the public from all planned low-level radioactive waste disposal facilities and potential contributions from all other projected end-state sources of radioactive material left at Hanford following site closure. Its primary purpose is to support the decision-making process of the U.S. Department of Energy (DOE) under DOE O 435.1-1, Radioactive Waste Management (DOE, 2001), related to managing low-level waste disposal facilities at the Hanford Site.

  17. Microstructure and cleavage in lath martensitic steels

    Morris, John W Jr; Kinney, Chris; Pytlewski, Ken; Adachi, Y

    2013-01-01

    In this paper we discuss the microstructure of lath martensitic steels and the mechanisms by which it controls cleavage fracture. The specific experimental example is a 9Ni (9 wt% Ni) steel annealed to have a large prior austenite grain size, then examined and tested in the as-quenched condition to produce a relatively coarse lath martensite. The microstructure is shown to approximate the recently identified ‘classic’ lath martensite structure: prior austenite grains are divided into packets, packets are subdivided into blocks, and blocks contain interleaved laths whose variants are the two Kurjumov–Sachs relations that share the same Bain axis of the transformation. When the steel is fractured in brittle cleavage, the laths in the block share {100} cleavage planes and cleave as a unit. However, cleavage cracks deflect or blunt at the boundaries between blocks with different Bain axes. It follows that, as predicted, the block size governs the effective grain size for cleavage. (paper)

  18. Downstream element determines RNase Y cleavage of the saePQRS operon in Staphylococcus aureus.

    Marincola, Gabriella; Wolz, Christiane

    2017-06-02

    In gram-positive bacteria, RNase J1, RNase J2 and RNase Y are thought to be major contributors to mRNA degradation and maturation. In Staphylococcus aureus, RNase Y activity is restricted to regulating the mRNA decay of only certain transcripts. Here the saePQRS operon was used as a model to analyze RNase Y specificity in living cells. A RNase Y cleavage site is located in an intergenic region between saeP and saeQ. This cleavage resulted in rapid degradation of the upstream fragment and stabilization of the downstream fragment. Thereby, the expression ratio of the different components of the operon was shifted towards saeRS, emphasizing the regulatory role of RNase Y activity. To assess cleavage specificity different regions surrounding the sae CS were cloned upstream of truncated gfp, and processing was analyzed in vivo using probes up- and downstream of CS. RNase Y cleavage was not determined by the cleavage site sequence. Instead a 24-bp double-stranded recognition structure was identified that was required to initiate cleavage 6 nt upstream. The results indicate that RNase Y activity is determined by secondary structure recognition determinants, which guide cleavage from a distance. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Analysis of laparoscopic port site complications: A descriptive study

    Somu Karthik

    2013-01-01

    Full Text Available Context: The rate of port site complications following conventional laparoscopic surgery is about 21 per 100,000 cases. It has shown a proportional rise with increase in the size of the port site incision and trocar. Although rare, complications that occur at the port site include infection, bleeding, and port site hernia. Aims: To determine the morbidity associated with ports at the site of their insertion in laparoscopic surgery and to identify risk factors for complications. Settings and Design: Prospective descriptive study. Materials and Methods: In the present descriptive study, a total of 570 patients who underwent laparoscopic surgeries for various ailments between August 2009 and July 2011 at our institute were observed for port site complications prospectively and the complications were reviewed. Statistical Analysis Used: Descriptive statistical analysis was carried out in the present study. The statistical software, namely, SPSS 15.0 was used for the analysis of the data. Results: Of the 570 patients undergoing laparoscopic surgery, 17 (3% had developed complications specifically related to the port site during a minimum follow-up of three months; port site infection (PSI was the most frequent (n = 10, 1.8%, followed by port site bleeding (n = 4, 0.7%, omentum-related complications (n = 2; 0.35%, and port site metastasis (n = 1, 0.175%. Conclusions: Laparoscopic surgeries are associated with minimal port site complications. Complications are related to the increased number of ports. Umbilical port involvement is the commonest. Most complications are manageable with minimal morbidity, and can be further minimized with meticulous surgical technique during entry and exit.

  20. Analysis of laparoscopic port site complications: A descriptive study

    Karthik, Somu; Augustine, Alfred Joseph; Shibumon, Mundunadackal Madhavan; Pai, Manohar Varadaraya

    2013-01-01

    CONTEXT: The rate of port site complications following conventional laparoscopic surgery is about 21 per 100,000 cases. It has shown a proportional rise with increase in the size of the port site incision and trocar. Although rare, complications that occur at the port site include infection, bleeding, and port site hernia. AIMS: To determine the morbidity associated with ports at the site of their insertion in laparoscopic surgery and to identify risk factors for complications. SETTINGS AND DESIGN: Prospective descriptive study. MATERIALS AND METHODS: In the present descriptive study, a total of 570 patients who underwent laparoscopic surgeries for various ailments between August 2009 and July 2011 at our institute were observed for port site complications prospectively and the complications were reviewed. STATISTICAL ANALYSIS USED: Descriptive statistical analysis was carried out in the present study. The statistical software, namely, SPSS 15.0 was used for the analysis of the data. RESULTS: Of the 570 patients undergoing laparoscopic surgery, 17 (3%) had developed complications specifically related to the port site during a minimum follow-up of three months; port site infection (PSI) was the most frequent (n = 10, 1.8%), followed by port site bleeding (n = 4, 0.7%), omentum-related complications (n = 2; 0.35%), and port site metastasis (n = 1, 0.175%). CONCLUSIONS: Laparoscopic surgeries are associated with minimal port site complications. Complications are related to the increased number of ports. Umbilical port involvement is the commonest. Most complications are manageable with minimal morbidity, and can be further minimized with meticulous surgical technique during entry and exit. PMID:23741110

  1. Site Response Analysis Using DeepSoil: Case Study of Bangka Site, Indonesia

    Iswanto, Eko Rudi; Yee, Eric [KEPCO International Nuclear Graduate School, Ulsan (Korea, Republic of)

    2015-10-15

    Indonesia government declared through Act No. 17 year 2007 on the National Long-Term Development Plant Year 2005-2025 and Presidential Decree No. 5 year 2006 on the National Energy Policy (Indonesia 2007; Indonesia 2006), that nuclear energy is stated as a part of the national energy system. In order to undertake the above national policy, National Nuclear Energy Agency of Indonesia, as the promotor for the utilization of nuclear energy will conduct site study, which is a part of infrastructure preparation for NPP construction. Thorough preparation and steps are needed to operate an NPP and it takes between 10 to 15 years from the preliminary study (site selection, financial study, etc.) up to project implementation (manufacturing, construction, commissioning). During project implementation, it is necessary to prepare various documents relevant for permit application such as Safety Evaluation Report for site permit, Preliminary Safety Analysis Report and Environment Impact Assessment Report for construction permit. Considering the continuously increasing electricity energy demand, it is necessary to prepare for alternative NPP sites. The safety requirements of NPP's are stringent; amongst the various requirements is the ability to safely shut down in the wake of a possible earthquake. Ground response analysis of a potential site therefore needs to be carried out, parameter that affect the resistance of an NPP to earthquakes such as peak strain profiles is analysed. The objective of this paper is to analyse the ground response of the selected site for a NPP, using The Mw 7.9 in Sikuai Island, West Sumatra on September 12, 2007 as present input motion. This analysis will be carried out using a ground response analysis program, DeepSoil. In addition to this, an attempt was made to define the site specific input motion characteristics of the selected site for use in DeepSoil (DeepSoil 5.0). A site investigation at the WB site was performed primarily on the PS

  2. Two Divalent Metal Ions and Conformational Changes Play Roles in the Hammerhead Ribozyme Cleavage Reaction

    Mir, Aamir; Chen, Ji; Robinson, Kyle; Lendy, Emma; Goodman, Jaclyn; Neau, David; Golden, Barbara L.

    2016-01-01

    The hammerhead ribozyme is a self-cleaving RNA broadly dispersed across all kingdoms of life. Although it was the first of the small, nucleolytic ribozymes discovered, the mechanism by which it catalyzes its reaction remains elusive. The nucleobase of G12 is well positioned to be a general base, but it is unclear if or how this guanine base becomes activated for proton transfer. Metal ions have been implicated in the chemical mechanism, but no interactions between divalent metal ions and the cleavage site have been observed crystallographically. To better understand how this ribozyme functions, we have solved crystal structures of wild-type and G12A mutant ribozymes. We observe a pH-dependent conformational change centered around G12, consistent with this nucleotide becoming deprotonated. Crystallographic and kinetic analysis of the G12A mutant reveals a Zn2+ specificity switch suggesting a direct interaction between a divalent metal ion and the purine at position 12. The metal ion specificity switch and the pH–rate profile of the G12A mutant suggest that the minor imino tautomer of A12 serves as the general base in the mutant ribozyme. We propose a model in which the hammerhead ribozyme rearranges prior to the cleavage reaction, positioning two divalent metal ions in the process. The first metal ion, positioned near G12, becomes directly coordinated to the O6 keto oxygen, to lower the pKa of the general base and organize the active site. The second metal ion, positioned near G10.1, bridges the N7 of G10.1 and the scissile phosphate and may participate directly in the cleavage reaction. PMID:26398724

  3. Cleavage and Cell Adhesion Properties of Human Epithelial Cell Adhesion Molecule (HEPCAM)*

    Tsaktanis, Thanos; Kremling, Heidi; Pavšič, Miha; von Stackelberg, Ricarda; Mack, Brigitte; Fukumori, Akio; Steiner, Harald; Vielmuth, Franziska; Spindler, Volker; Huang, Zhe; Jakubowski, Jasmine; Stoecklein, Nikolas H.; Luxenburger, Elke; Lauber, Kirsten; Lenarčič, Brigita; Gires, Olivier

    2015-01-01

    Human epithelial cell adhesion molecule (HEPCAM) is a tumor-associated antigen frequently expressed in carcinomas, which promotes proliferation after regulated intramembrane proteolysis. Here, we describe extracellular shedding of HEPCAM at two α-sites through a disintegrin and metalloprotease (ADAM) and at one β-site through BACE1. Transmembrane cleavage by γ-secretase occurs at three γ-sites to generate extracellular Aβ-like fragments and at two ϵ-sites to release human EPCAM intracellular domain HEPICD, which is efficiently degraded by the proteasome. Mapping of cleavage sites onto three-dimensional structures of HEPEX cis-dimer predicted conditional availability of α- and β-sites. Endocytosis of HEPCAM warrants acidification in cytoplasmic vesicles to dissociate protein cis-dimers required for cleavage by BACE1 at low pH values. Intramembrane cleavage sites are accessible and not part of the structurally important transmembrane helix dimer crossing region. Surprisingly, neither chemical inhibition of cleavage nor cellular knock-out of HEPCAM using CRISPR-Cas9 technology impacted the adhesion of carcinoma cell lines. Hence, a direct function of HEPCAM as an adhesion molecule in carcinoma cells is not supported and appears to be questionable. PMID:26292218

  4. OLEM Center for Program Analysis Site Analysis Data

    U.S. Environmental Protection Agency — This asset includes environmental justice-related analyses of population located within a mile of Superfund and RCRA Corrective Action sites. It characterizes...

  5. Characterization of SNARE Cleavage Products Generated by Formulated Botulinum Neurotoxin Type-A Drug Products

    Jack Xie

    2010-08-01

    Full Text Available The study evaluated substrate cleavage product(s generated by three botulinum neurotoxin serotype A (BoNT/A medicinal drug products utilizing a novel and highly specific, light-chain activity, high-performance liquid chromatography (LCA-HPLC method. Samples were reacted with a commercially available BoNT/A fluorescent substrate derived from the SNAP-25 sequence. Reaction products were separated by reversed-phase HPLC. The method detected an atypical cleavage pattern by one of the formulated drug products. IncobotulinumtoxinA produced two cleavage fragments rather than the single fragment typically generated by BoNT/A. Identification confirmed the secondary cleavage at a position corresponding to SNAP-25 Arg198–Ala199 (normal BoNT/A cleavage is Gln197–Arg198. Arg198–Ala199 is also the cleavage site for trypsin and serotype C toxin. Normal cleavage was observed for all other BoNT/A drug product samples, as well as 900-kD and 150-kD bulk toxin BoNT/A. The reason for this unexpected secondary cleavage pattern by one formulated BoNT/A drug product is unknown. Possible explanations include a contaminating protease and/or damage to the 150-kD type-A toxin causing nonspecific substrate recognition and subsequent cleavage uncharacteristic of type-A toxin. The BoNT/A drug products were also analyzed via the LCA-HPLC assay using a commercial BoNT/C fluorescent substrate derived from the syntaxin sequence. Cleavage of the serotype C substrate by incobotulinumtoxinA was also confirmed whilst neither of the other drug products cleaved the syntaxin substrate.

  6. Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes

    Ma, Enbo; Harrington, Lucas B.; O’Connell, Mitchell R.; Zhou, Kaihong; Doudna, Jennifer A.

    2015-01-01

    Summary Double-stranded DNA (dsDNA) cleavage by Cas9 is a hallmark of type II CRISPR-Cas immune systems. Cas9–guide RNA complexes recognize 20-base-pair sequences in DNA and generate a site-specific double-strand break, a robust activity harnessed for genome editing. DNA recognition by all studied Cas9 enzymes requires a protospacer adjacent motif (PAM) next to the target site. We show that Cas9 enzymes from evolutionarily divergent bacteria can recognize and cleave single-stranded DNA (ssDNA) by an RNA-guided, PAM-independent recognition mechanism. Comparative analysis shows that in contrast to the type II-A S. pyogenes Cas9 that is widely used for genome engineering, the smaller type II-C Cas9 proteins have limited dsDNA binding and unwinding activity and promiscuous guide-RNA specificity. These results indicate that inefficiency of type II-C Cas9 enzymes for genome editing results from a limited ability to cleave dsDNA, and suggest that ssDNA cleavage was an ancestral function of the Cas9 enzyme family. PMID:26545076

  7. NRC staff site characterization analysis of the Department of Energy`s Site Characterization Plan, Yucca Mountain Site, Nevada

    NONE

    1989-08-01

    This Site Characterization Analysis (SCA) documents the NRC staff`s concerns resulting from its review of the US Department of Energy`s (DOE`s) Site Characterization Plan (SCP) for the Yucca Mountain site in southern Nevada, which is the candidate site selected for characterization as the nation`s first geologic repository for high-level radioactive waste. DOE`s SCP explains how DOE plans to obtain the information necessary to determine the suitability of the Yucca Mountain site for a repository. NRC`s specific objections related to the SCP, and major comments and recommendations on the various parts of DOE`s program, are presented in SCA Section 2, Director`s Comments and Recommendations. Section 3 contains summaries of the NRC staff`s concerns for each specific program, and Section 4 contains NRC staff point papers which set forth in greater detail particular staff concerns regarding DOE`s program. Appendix A presents NRC staff evaluations of those NRC staff Consultation Draft SCP concerns that NRC considers resolved on the basis of the SCP. This SCA fulfills NRC`s responsibilities with respect to DOE`s SCP as specified by the Nuclear Waste Policy Act (NWPA) and 10 CFR 60.18. 192 refs., 2 tabs.

  8. NRC staff site characterization analysis of the Department of Energy's Site Characterization Plan, Yucca Mountain Site, Nevada

    1989-08-01

    This Site Characterization Analysis (SCA) documents the NRC staff's concerns resulting from its review of the US Department of Energy's (DOE's) Site Characterization Plan (SCP) for the Yucca Mountain site in southern Nevada, which is the candidate site selected for characterization as the nation's first geologic repository for high-level radioactive waste. DOE's SCP explains how DOE plans to obtain the information necessary to determine the suitability of the Yucca Mountain site for a repository. NRC's specific objections related to the SCP, and major comments and recommendations on the various parts of DOE's program, are presented in SCA Section 2, Director's Comments and Recommendations. Section 3 contains summaries of the NRC staff's concerns for each specific program, and Section 4 contains NRC staff point papers which set forth in greater detail particular staff concerns regarding DOE's program. Appendix A presents NRC staff evaluations of those NRC staff Consultation Draft SCP concerns that NRC considers resolved on the basis of the SCP. This SCA fulfills NRC's responsibilities with respect to DOE's SCP as specified by the Nuclear Waste Policy Act (NWPA) and 10 CFR 60.18. 192 refs., 2 tabs

  9. Sequence specificity of DNA cleavage by Micrococcus luteus γ endonuclease

    Hentosh, P.; Henner, W.D.; Reynolds, R.J.

    1985-01-01

    DNA fragments of defined sequence have been used to determine the sites of cleavage by γ-endonuclease activity in extracts prepared from Micrococcus luteus. End-labeled DNA restriction fragments of pBR322 DNA that had been irradiated under nitrogen in the presence of potassium iodide or t-butanol were treated with M. luteus γ endonuclease and analyzed on irradiated DNA preferentially at the positions of cytosines and thymines. DNA cleavage occurred immediately to the 3' side of pyrimidines in irradiated DNA and resulted in fragments that terminate in a 5'-phosphoryl group. These studies indicate that both altered cytosines and thymines may be important DNA lesions requiring repair after exposure to γ radiation

  10. Cleavage of nicotinamide adenine dinucleotide by the ribosome-inactivating protein from Momordica charantia.

    Vinkovic, M; Dunn, G; Wood, G E; Husain, J; Wood, S P; Gill, R

    2015-09-01

    The interaction of momordin, a type 1 ribosome-inactivating protein from Momordica charantia, with NADP(+) and NADPH has been investigated by X-ray diffraction analysis of complexes generated by co-crystallization and crystal soaking. It is known that the proteins of this family readily cleave the adenine-ribose bond of adenosine and related nucleotides in the crystal, leaving the product, adenine, bound to the enzyme active site. Surprisingly, the nicotinamide-ribose bond of oxidized NADP(+) is cleaved, leaving nicotinamide bound in the active site in the same position but in a slightly different orientation to that of the five-membered ring of adenine. No binding or cleavage of NADPH was observed at pH 7.4 in these experiments. These observations are in accord with current views of the enzyme mechanism and may contribute to ongoing searches for effective inhibitors.

  11. Site-specific Probabilistic Analysis of DCGLs Using RESRAD Code

    Kim, Jeongju; Yoon, Suk Bon; Sohn, Wook [KHNP CRI, Daejeon (Korea, Republic of)

    2016-10-15

    In general, DCGLs can be conservative (screening DCGL) if they do not take into account site specific factors. Use of such conservative DCGLs can lead to additional remediation that would not be required if the effort was made to develop site-specific DCGLs. Therefore, the objective of this work is to provide an example on the use of the RESRAD 6.0 probabilistic (site-specific) dose analysis to compare with the screening DCGL. Site release regulations state that a site will be considered acceptable for unrestricted use if the residual radioactivity that is distinguishable from background radiation results in a Total Effective Dose Equivalent (TEDE) to an average member of the critical group of less than the site release criteria, for example 0.25 mSv per year in U.S. Utilities use computer dose modeling codes to establish an acceptable level of contamination, the derived concentration guideline level (DCGL) that will meet this regulatory limit. Since the DCGL value is the principal measure of residual radioactivity, it is critical to understand the technical basis of these dose modeling codes. The objective this work was to provide example on nuclear power plant decommissioning dose analysis in a probabilistic analysis framework. The focus was on the demonstration of regulatory compliance for surface soil contamination using the RESRAD 6.0 code. Both the screening and site-specific probabilistic dose analysis methodologies were examined. Example analyses performed with the screening probabilistic dose analysis confirmed the conservatism of the NRC screening values and indicated the effectiveness of probabilistic dose analysis in reducing the conservatism in DCGL derivation.

  12. Comparative and phylogenetic perspectives of the cleavage process in tailed amphibians.

    Desnitskiy, Alexey G; Litvinchuk, Spartak N

    2015-10-01

    The order Caudata includes about 660 species and displays a variety of important developmental traits such as cleavage pattern and egg size. However, the cleavage process of tailed amphibians has never been analyzed within a phylogenetic framework. We use published data on the embryos of 36 species concerning the character of the third cleavage furrow (latitudinal, longitudinal or variable) and the magnitude of synchronous cleavage period (up to 3-4 synchronous cell divisions in the animal hemisphere or a considerably longer series of synchronous divisions followed by midblastula transition). Several species from basal caudate families Cryptobranchidae (Andrias davidianus and Cryptobranchus alleganiensis) and Hynobiidae (Onychodactylus japonicus) as well as several representatives from derived families Plethodontidae (Desmognathus fuscus and Ensatina eschscholtzii) and Proteidae (Necturus maculosus) are characterized by longitudinal furrows of the third cleavage and the loss of synchrony as early as the 8-cell stage. By contrast, many representatives of derived families Ambystomatidae and Salamandridae have latitudinal furrows of the third cleavage and extensive period of synchronous divisions. Our analysis of these ontogenetic characters mapped onto a phylogenetic tree shows that the cleavage pattern of large, yolky eggs with short series of synchronous divisions is an ancestral trait for the tailed amphibians, while the data on the orientation of third cleavage furrows seem to be ambiguous with respect to phylogeny. Nevertheless, the midblastula transition, which is characteristic of the model species Ambystoma mexicanum (Caudata) and Xenopus laevis (Anura), might have evolved convergently in these two amphibian orders.

  13. Structural and functional characterization of cleavage and inactivation of human serine protease inhibitors by the bacterial SPATE protease EspPα from enterohemorrhagic E. coli.

    André Weiss

    Full Text Available EspPα and EspI are serine protease autotransporters found in enterohemorrhagic Escherichia coli. They both belong to the SPATE autotransporter family and are believed to contribute to pathogenicity via proteolytic cleavage and inactivation of different key host proteins during infection. Here, we describe the specific cleavage and functional inactivation of serine protease inhibitors (serpins by EspPα and compare this activity with the related SPATE EspI. Serpins are structurally related proteins that regulate vital protease cascades, such as blood coagulation and inflammatory host response. For the rapid determination of serpin cleavage sites, we applied direct MALDI-TOF-MS or ESI-FTMS analysis of coincubations of serpins and SPATE proteases and confirmed observed cleavage positions using in-gel-digest of SDS-PAGE-separated degradation products. Activities of both serpin and SPATE protease were assessed in a newly developed photometrical assay using chromogenic peptide substrates. EspPα cleaved the serpins α1-protease inhibitor (α1-PI, α1-antichymotrypsin, angiotensinogen, and α2-antiplasmin. Serpin cleavage led to loss of inhibitory function as demonstrated for α1-PI while EspPα activity was not affected. Notably, EspPα showed pronounced specificity and cleaved procoagulatory serpins such as α2-antiplasmin while the anticoagulatory antithrombin III was not affected. Together with recently published research, this underlines the interference of EspPα with hemostasis or inflammatory responses during infection, while the observed interaction of EspI with serpins is likely to be not physiologically relevant. EspPα-mediated serpin cleavage occurred always in flexible loops, indicating that this structural motif might be required for substrate recognition.

  14. On-site and off-site forensic analysis capabilities for proliferation and terrorism prevention

    Whipple, R.E.; Nunes, P.J.; Reynolds, J.G.; Alcaraz, A.; Hart, B.R.

    2004-01-01

    Full text: We will present current on-site technologies that can be utilized for the screening of explosives, chemical agents, and environmental contaminants. These techniques must have the capability to detect various hazardous materials at very low levels, since they pose a major challenge for first responders. Specifically, the technology must detect concealed explosives or chemical agents on-site rapidly. Lawrence Livermore National Laboratory (LLNL) is currently developing several different high explosive screening and detection technologies for field use. Two technologies that have demonstrated an ability to screen for explosives at low levels are colorimetric spot tests and thin layer chromatography (TLC). Another technology that has demonstrated usefulness for the on-site analysis of unknowns is portable gas chromatography-mass spectrometry (GC-MS) with solid phase micro-extraction (SPME) sampling. Several examples utilizing these technologies and their usefulness will be presented. In addition to developing on-site screening methods, LLNL is an Organization for the Prohibition of Chemical Weapons (OPCW) designated laboratory and is certified to accept CW suspect samples. Currently, LLNL is expanding its ISO-17025 certification to include nuclear forensics and explosives. These off-site forensic analysis capabilities and certified procedures will support the needs for homeland security. We will highlight some of the ISO-17025 requirements to accredit procedures, handle samples, and reports. Work performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under contract W-7405-ENG-48

  15. On-site and off-site forensic analysis capabilities for proliferation and terrorism prevention

    Hart, B.R.; Whipple, R.E.; Nunes, P.J.; Reynolds, J.G.; Alcaraz, A.

    2004-01-01

    We present current on-site technologies that can be utilized for the screening of explosives, chemical agents, and environmental contaminants. These techniques must have the capability to detect various hazardous materials at very low levels, since they pose a major challenge for first responders. Specifically, the technology must detect concealed explosives or chemical agents on-site rapidly. Lawrence Livermore National Laboratory (LLNL) is currently developing several different high explosive screening and detection technologies for field use. Two technologies that have demonstrated an ability to screen for explosives at low levels are colorimetric spot tests and thin layer chromatography (TLC). Another technology that has demonstrated usefulness for the on-site analysis of unknowns is portable gas chromatography-mass spectrometry (GC-MS) with solid phase micro-extraction (SPME) sampling. Several examples utilizing these technologies and their usefulness will be presented. In addition to developing on-site screening methods, LLNL is an Organization for the Prohibition of Chemical Weapons (OPCW) designated laboratory and is certified to accept CW suspect samples. Currently, LLNL is expanding its ISO-17025 certification to include nuclear forensics and explosives. These off-site forensic analysis capabilities and certified procedures will support the needs for homeland security. We will highlight some of the ISO-17025 requirements to accredit procedures, handle samples, and reports. Work performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under contract W-7405-ENG-48. (author)

  16. Methods for the analysis and remediation of contaminated sites

    Mariani, M.; Bemporad, E.; Berardi, S.; Marino, A.; Paglietti, F.

    2008-01-01

    In Italy, in recent years, the number of contaminated sites has multiplied disproportionately. In essence, contamination is caused by accidental spills or intentional discharge of pollutants into the soils or waters from industrial activities, or non-controlled deposits of urban and/or industrial waste, mostly part toxic and harmful. Contaminated sites clearly pose risks to human health and the environment; hence the need to remediate these sites. The remediation of soil and water and the restoration of degraded areas are complex operations requiring specific technical and scientific know-how, including knowledge of the methodologies and tools required to tackle problems arising during the different phases of the remediation process. These include, in particular: - health and environmental risk assessment procedures for the quantification of risks to human health (general population and workers) and the environment from a contaminated site; - remote sensing and the Geographical Information Systems (GIS), which are a fundamentally important IT support for each phase of planning and management of remediation interventions; - criteria for the management of sites contaminated by asbestos, a highly carcinogenic and therefore hazardous substance that was widely used in the past due to its particular mechanical and thermal characteristics; - analysis of the issues relating to waste management in contaminated sites; - relationship between safety procedures for workers and the general population. Identification of the best available techniques for an efficient, integrated management of contaminated sites, which will also take into account the health protection of workers and of the general population living near such sites

  17. Probabilistic approaches for geotechnical site characterization and slope stability analysis

    Cao, Zijun; Li, Dianqing

    2017-01-01

    This is the first book to revisit geotechnical site characterization from a probabilistic point of view and provide rational tools to probabilistically characterize geotechnical properties and underground stratigraphy using limited information obtained from a specific site. This book not only provides new probabilistic approaches for geotechnical site characterization and slope stability analysis, but also tackles the difficulties in practical implementation of these approaches. In addition, this book also develops efficient Monte Carlo simulation approaches for slope stability analysis and implements these approaches in a commonly available spreadsheet environment. These approaches and the software package are readily available to geotechnical practitioners and alleviate them from reliability computational algorithms. The readers will find useful information for a non-specialist to determine project-specific statistics of geotechnical properties and to perform probabilistic analysis of slope stability.

  18. Mesoscale carbon sequestration site screening and CCS infrastructure analysis.

    Keating, Gordon N; Middleton, Richard S; Stauffer, Philip H; Viswanathan, Hari S; Letellier, Bruce C; Pasqualini, Donatella; Pawar, Rajesh J; Wolfsberg, Andrew V

    2011-01-01

    We explore carbon capture and sequestration (CCS) at the meso-scale, a level of study between regional carbon accounting and highly detailed reservoir models for individual sites. We develop an approach to CO(2) sequestration site screening for industries or energy development policies that involves identification of appropriate sequestration basin, analysis of geologic formations, definition of surface sites, design of infrastructure, and analysis of CO(2) transport and storage costs. Our case study involves carbon management for potential oil shale development in the Piceance-Uinta Basin, CO and UT. This study uses new capabilities of the CO(2)-PENS model for site screening, including reservoir capacity, injectivity, and cost calculations for simple reservoirs at multiple sites. We couple this with a model of optimized source-sink-network infrastructure (SimCCS) to design pipeline networks and minimize CCS cost for a given industry or region. The CLEAR(uff) dynamical assessment model calculates the CO(2) source term for various oil production levels. Nine sites in a 13,300 km(2) area have the capacity to store 6.5 GtCO(2), corresponding to shale-oil production of 1.3 Mbbl/day for 50 years (about 1/4 of U.S. crude oil production). Our results highlight the complex, nonlinear relationship between the spatial deployment of CCS infrastructure and the oil-shale production rate.

  19. Comparison Analysis among Large Amount of SNS Sites

    Toriumi, Fujio; Yamamoto, Hitoshi; Suwa, Hirohiko; Okada, Isamu; Izumi, Kiyoshi; Hashimoto, Yasuhiro

    In recent years, application of Social Networking Services (SNS) and Blogs are growing as new communication tools on the Internet. Several large-scale SNS sites are prospering; meanwhile, many sites with relatively small scale are offering services. Such small-scale SNSs realize small-group isolated type of communication while neither mixi nor MySpace can do that. However, the studies on SNS are almost about particular large-scale SNSs and cannot analyze whether their results apply for general features or for special characteristics on the SNSs. From the point of view of comparison analysis on SNS, comparison with just several types of those cannot reach a statistically significant level. We analyze many SNS sites with the aim of classifying them by using some approaches. Our paper classifies 50,000 sites for small-scale SNSs and gives their features from the points of network structure, patterns of communication, and growth rate of SNS. The result of analysis for network structure shows that many SNS sites have small-world attribute with short path lengths and high coefficients of their cluster. Distribution of degrees of the SNS sites is close to power law. This result indicates the small-scale SNS sites raise the percentage of users with many friends than mixi. According to the analysis of their coefficients of assortativity, those SNS sites have negative values of assortativity, and that means users with high degree tend to connect users with small degree. Next, we analyze the patterns of user communication. A friend network of SNS is explicit while users' communication behaviors are defined as an implicit network. What kind of relationships do these networks have? To address this question, we obtain some characteristics of users' communication structure and activation patterns of users on the SNS sites. By using new indexes, friend aggregation rate and friend coverage rate, we show that SNS sites with high value of friend coverage rate activate diary postings

  20. Structural and Biochemical Characterization of a Copper-Binding Mutant of the Organomercurial Lyase MerB: Insight into the Key Role of the Active Site Aspartic Acid in Hg-Carbon Bond Cleavage and Metal Binding Specificity.

    Wahba, Haytham M; Lecoq, Lauriane; Stevenson, Michael; Mansour, Ahmed; Cappadocia, Laurent; Lafrance-Vanasse, Julien; Wilkinson, Kevin J; Sygusch, Jurgen; Wilcox, Dean E; Omichinski, James G

    2016-02-23

    In bacterial resistance to mercury, the organomercurial lyase (MerB) plays a key role in the detoxification pathway through its ability to cleave Hg-carbon bonds. Two cysteines (C96 and C159; Escherichia coli MerB numbering) and an aspartic acid (D99) have been identified as the key catalytic residues, and these three residues are conserved in all but four known MerB variants, where the aspartic acid is replaced with a serine. To understand the role of the active site serine, we characterized the structure and metal binding properties of an E. coli MerB mutant with a serine substituted for D99 (MerB D99S) as well as one of the native MerB variants containing a serine residue in the active site (Bacillus megaterium MerB2). Surprisingly, the MerB D99S protein copurified with a bound metal that was determined to be Cu(II) from UV-vis absorption, inductively coupled plasma mass spectrometry, nuclear magnetic resonance, and electron paramagnetic resonance studies. X-ray structural studies revealed that the Cu(II) is bound to the active site cysteine residues of MerB D99S, but that it is displaced following the addition of either an organomercurial substrate or an ionic mercury product. In contrast, the B. megaterium MerB2 protein does not copurify with copper, but the structure of the B. megaterium MerB2-Hg complex is highly similar to the structure of the MerB D99S-Hg complexes. These results demonstrate that the active site aspartic acid is crucial for both the enzymatic activity and metal binding specificity of MerB proteins and suggest a possible functional relationship between MerB and its only known structural homologue, the copper-binding protein NosL.

  1. On the formation and nature of quasi-cleavage fracture surfaces in hydrogen embrittled steels

    Martin, May L.; Fenske, Jamey A.; Liu, Grace S.; Sofronis, Petros [University of Illinois, Dept. of Materials Science and Engineering, 1304 W. Green St., Urbana, IL 61801 (United States); Robertson, Ian M., E-mail: ianr@illinois.edu [University of Illinois, Dept. of Materials Science and Engineering, 1304 W. Green St., Urbana, IL 61801 (United States)

    2011-02-15

    Quasi-cleavage, a common feature of hydrogen-induced fracture surfaces, is generally taken as being cleavage-like but not along a known cleavage plane. Despite the frequency with which this surface is observed, the relationship to the underlying microstructure remains unknown. Through a combination of topographical reconstruction of secondary electron microscope fractographs and a transmission electron microscopy study of the microstructure from site-specific locations, it will be shown that the features on quasi-cleavage surfaces are ridges that can be correlated with sub-surface intense and highly localized deformation bands. It will be demonstrated that the fracture surface arises from the growth and coalescence of voids that initiate at and extend along slip band intersections. This mechanism and process is fully consistent with hydrogen enhancing and localizing plastic processes.

  2. Structure of the cleavage-activated prefusion form of the parainfluenza virus 5 fusion protein.

    Welch, Brett D; Liu, Yuanyuan; Kors, Christopher A; Leser, George P; Jardetzky, Theodore S; Lamb, Robert A

    2012-10-09

    The paramyxovirus parainfluenza virus 5 (PIV5) enters cells by fusion of the viral envelope with the plasma membrane through the concerted action of the fusion (F) protein and the receptor binding protein hemagglutinin-neuraminidase. The F protein folds initially to form a trimeric metastable prefusion form that is triggered to undergo large-scale irreversible conformational changes to form the trimeric postfusion conformation. It is thought that F refolding couples the energy released with membrane fusion. The F protein is synthesized as a precursor (F0) that must be cleaved by a host protease to form a biologically active molecule, F1,F2. Cleavage of F protein is a prerequisite for fusion and virus infectivity. Cleavage creates a new N terminus on F1 that contains a hydrophobic region, known as the FP, which intercalates target membranes during F protein refolding. The crystal structure of the soluble ectodomain of the uncleaved form of PIV5 F is known; here we report the crystal structure of the cleavage-activated prefusion form of PIV5 F. The structure shows minimal movement of the residues adjacent to the protease cleavage site. Most of the hydrophobic FP residues are buried in the uncleaved F protein, and only F103 at the newly created N terminus becomes more solvent-accessible after cleavage. The conformational freedom of the charged arginine residues that compose the protease recognition site increases on cleavage of F protein.

  3. Decontamination analysis of the NUWAX-83 accident site using DECON

    Tawil, J.J.

    1983-11-01

    This report presents an analysis of the site restoration options for the NUWAX-83 site, at which an exercise was conducted involving a simulated nuclear weapons accident. This analysis was performed using a computer program deveoped by Pacific Northwest Laboratory. The computer program, called DECON, was designed to assist personnel engaged in the planning of decontamination activities. The many features of DECON that are used in this report demonstrate its potential usefulness as a site restoration planning tool. Strategies that are analyzed with DECON include: (1) employing a Quick-Vac option, under which selected surfaces are vacuumed before they can be rained on; (2) protecting surfaces against precipitation; (3) prohibiting specific operations on selected surfaces; (4) requiring specific methods to be used on selected surfaces; (5) evaluating the trade-off between cleanup standards and decontamination costs; and (6) varying of the cleanup standards according to expected exposure to surface

  4. Solid waste dumping site suitability analysis using geographic ...

    Solid waste dumping site suitability analysis using geographic information system (GIS) and remote sensing for Bahir Dar Town, North Western Ethiopia. ... The PDF file you selected should load here if your Web browser has a PDF reader plug-in installed (for example, a recent version of Adobe Acrobat Reader).

  5. Young Adult Capacity Initiative Cross-Site Analysis

    Academy for Educational Development, 2012

    2012-01-01

    This cross-site analysis presents findings about the implementation, impact, and outcomes of the Young Adult Capacity Initiative (YACI), at 13 community-based organizations in New York City. These agencies received technical assistance and small incentive grants from the Fund for the City of New York Youth Development Institute (YDI) to build…

  6. Analysis of a photovoltaic dwelling in remote sites

    Eliot, P

    1982-01-01

    This work is a design analysis of autonomous or quasi-autonomous photovolaic system suited to dwellings on remote sites in France; this study allowed to establish ''curves'' of sizing related to several performance, from which it is possible to estimate costs (under many conditions of house occupancy, of climates). The interest of a back-up generating sit for the permanent resident is studied. Then, an adaptation and an improvement of existing analysis tools are looked.

  7. 3' RNA ligase mediated rapid amplification of cDNA ends for validating viroid induced cleavage at the 3' extremity of the host mRNA.

    Adkar-Purushothama, Charith Raj; Bru, Pierrick; Perreault, Jean-Pierre

    2017-12-01

    5' RNA ligase-mediated rapid amplification of cDNA ends (5' RLM-RACE) is a widely-accepted method for the validation of direct cleavage of a target gene by a microRNA (miRNA) and viroid-derived small RNA (vd-sRNA). However, this method cannot be used if cleavage takes place in the 3' extremity of the target RNA, as this gives insufficient sequence length to design nested PCR primers for 5' RLM RACE. To overcome this hurdle, we have developed 3' RNA ligase-mediated rapid amplification of cDNA ends (3' RLM RACE). In this method, an oligonucleotide adapter having 5' adenylated and 3' blocked is ligated to the 3' end of the cleaved RNA followed by PCR amplification using gene specific primers. In other words, in 3' RLM RACE, 3' end is mapped using 5' fragment instead of small 3' fragment. The method developed here was verified by examining the bioinformatics predicted and parallel analysis of RNA ends (PARE) proved cleavage sites of chloride channel protein CLC-b-like mRNA in Potato spindle tuber viroid infected tomato plants. The 3' RLM RACE developed in this study has the potential to validate the miRNA and vd-sRNA mediated cleavage of mRNAs at its 3' untranslated region (3' UTR). Copyright © 2017 Elsevier B.V. All rights reserved.

  8. The On-Site Analysis of the Cherenkov Telescope Array

    Bulgarelli, Andrea; Zoli, Andrea; Aboudan, Alessio; Rodríguez-Vázquez, Juan José; De Cesare, Giovanni; De Rosa, Adriano; Maier, Gernot; Lyard, Etienne; Bastieri, Denis; Lombardi, Saverio; Tosti, Gino; Bergamaschi, Sonia; Beneventano, Domenico; Lamanna, Giovanni; Jacquemier, Jean; Kosack, Karl; Antonelli, Lucio Angelo; Boisson, Catherine; Borkowski, Jerzy; Buson, Sara; Carosi, Alessandro; Conforti, Vito; Colomé, Pep; Reyes, Raquel de los; Dumm, Jon; Evans, Phil; Fortson, Lucy; Fuessling, Matthias; Gotz, Diego; Graciani, Ricardo; Gianotti, Fulvio; Grandi, Paola; Hinton, Jim; Humensky, Brian; Inoue, Susumu; Knödlseder, Jürgen; Flour, Thierry Le; Lindemann, Rico; Malaguti, Giuseppe; Markoff, Sera; Marisaldi, Martino; Neyroud, Nadine; Nicastro, Luciano; Ohm, Stefan; Osborne, Julian; Oya, Igor; Rodriguez, Jerome; Rosen, Simon; Ribo, Marc; Tacchini, Alessandro; Schüssler, Fabian; Stolarczyk, Thierry; Torresi, Eleonora; Testa, Vincenzo; Wegner, Peter

    2015-01-01

    The Cherenkov Telescope Array (CTA) observatory will be one of the largest ground-based very high-energy gamma-ray observatories. The On-Site Analysis will be the first CTA scientific analysis of data acquired from the array of telescopes, in both northern and southern sites. The On-Site Analysis will have two pipelines: the Level-A pipeline (also known as Real-Time Analysis, RTA) and the level-B one. The RTA performs data quality monitoring and must be able to issue automated alerts on variable and transient astrophysical sources within 30 seconds from the last acquired Cherenkov event that contributes to the alert, with a sensitivity not worse than the one achieved by the final pipeline by more than a factor of 3. The Level-B Analysis has a better sensitivity (not be worse than the final one by a factor of 2) and the results should be available within 10 hours from the acquisition of the data: for this reason this analysis could be performed at the end of an observation or next morning. The latency (in part...

  9. Rainfall frequency analysis for ungauged sites using satellite precipitation products

    Gado, Tamer A.; Hsu, Kuolin; Sorooshian, Soroosh

    2017-11-01

    The occurrence of extreme rainfall events and their impacts on hydrologic systems and society are critical considerations in the design and management of a large number of water resources projects. As precipitation records are often limited or unavailable at many sites, it is essential to develop better methods for regional estimation of extreme rainfall at these partially-gauged or ungauged sites. In this study, an innovative method for regional rainfall frequency analysis for ungauged sites is presented. The new method (hereafter, this is called the RRFA-S) is based on corrected annual maximum series obtained from a satellite precipitation product (e.g., PERSIANN-CDR). The probability matching method (PMM) is used here for bias correction to match the CDF of satellite-based precipitation data with the gauged data. The RRFA-S method was assessed through a comparative study with the traditional index flood method using the available annual maximum series of daily rainfall in two different regions in USA (11 sites in Colorado and 18 sites in California). The leave-one-out cross-validation technique was used to represent the ungauged site condition. Results of this numerical application have found that the quantile estimates obtained from the new approach are more accurate and more robust than those given by the traditional index flood method.

  10. Accurate and rapid modeling of iron-bleomycin-induced DNA damage using tethered duplex oligonucleotides and electrospray ionization ion trap mass spectrometric analysis.

    Harsch, A; Marzilli, L A; Bunt, R C; Stubbe, J; Vouros, P

    2000-05-01

    Bleomycin B(2)(BLM) in the presence of iron [Fe(II)] and O(2)catalyzes single-stranded (ss) and double-stranded (ds) cleavage of DNA. Electrospray ionization ion trap mass spectrometry was used to monitor these cleavage processes. Two duplex oligonucleotides containing an ethylene oxide tether between both strands were used in this investigation, allowing facile monitoring of all ss and ds cleavage events. A sequence for site-specific binding and cleavage by Fe-BLM was incorporated into each analyte. One of these core sequences, GTAC, is a known hot-spot for ds cleavage, while the other sequence, GGCC, is a hot-spot for ss cleavage. Incubation of each oligo-nucleotide under anaerobic conditions with Fe(II)-BLM allowed detection of the non-covalent ternary Fe-BLM/oligonucleotide complex in the gas phase. Cleavage studies were then performed utilizing O(2)-activated Fe(II)-BLM. No work-up or separation steps were required and direct MS and MS/MS analyses of the crude reaction mixtures confirmed sequence-specific Fe-BLM-induced cleavage. Comparison of the cleavage patterns for both oligonucleotides revealed sequence-dependent preferences for ss and ds cleavages in accordance with previously established gel electrophoresis analysis of hairpin oligonucleotides. This novel methodology allowed direct, rapid and accurate determination of cleavage profiles of model duplex oligonucleotides after exposure to activated Fe-BLM.

  11. Revised analysis of in-migrating workers during site characterization

    1987-10-01

    The Deaf Smith Environmental Assessment's analysis of in-migrating workers and community service impacts was predicated on the assumption that a peak of approximately 480 workers would be needed on location to conduct site characterization activities. This analysis assumed that DOE's prime contractor(s) would have a limited staff in the area; the majority of the workers would be on site for the construction of the exploratory shaft and to conduct geologic and environmental studies. Since the time when the Environmental Assessment was prepared, the prime contractors [Battelle-ISSC and the Technical Field Service Contractor (TFSC)] were requested to move their offices to the site area. Therefore, many more administrative and technical workers would be expected to relocate in the Deaf Smith County regions. A change in the expected number of in-migrants could also change the expected nature of community service impacts. It is the purpose of this analysis to evaluate the site characterization workforce and thresholds for local community services. 22 refs., 24 tabs

  12. Programmable RNA recognition and cleavage by CRISPR/Cas9.

    O'Connell, Mitchell R; Oakes, Benjamin L; Sternberg, Samuel H; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A

    2014-12-11

    The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.

  13. Drosha regulates gene expression independently of RNA cleavage function

    Gromak, Natalia; Dienstbier, Martin; Macias, Sara

    2013-01-01

    Drosha is the main RNase III-like enzyme involved in the process of microRNA (miRNA) biogenesis in the nucleus. Using whole-genome ChIP-on-chip analysis, we demonstrate that, in addition to miRNA sequences, Drosha specifically binds promoter-proximal regions of many human genes in a transcription......-dependent manner. This binding is not associated with miRNA production or RNA cleavage. Drosha knockdown in HeLa cells downregulated nascent gene transcription, resulting in a reduction of polyadenylated mRNA produced from these gene regions. Furthermore, we show that this function of Drosha is dependent on its N......-terminal protein-interaction domain, which associates with the RNA-binding protein CBP80 and RNA Polymerase II. Consequently, we uncover a previously unsuspected RNA cleavage-independent function of Drosha in the regulation of human gene expression....

  14. Structural analysis by reductive cleavage with LiAlH4 of an allyl ether choline-phospholipid, archaetidylcholine, from the hyperthermophilic methanoarchaeon Methanopyrus kandleri

    Masateru Nishihara

    2002-01-01

    Full Text Available A choline-containing phospholipid (PL-4 in Methanopyrus kandleri cells was identified as archaetidylcholine, which has been described by Sprott et al. (1997. The PL-4 consisted of a variety of molecular species differing in hydrocarbon composition. Most of the PL-4 was acid-labile because of its allyl ether bond. The identity of PL-4 was confirmed by thin-layer chromatography (TLC followed by positive staining with Dragendorff-reagent and fast-atom bombardment–mass spectrometry. A new method of LiAlH4 hydrogenolysis was developed to cleave allyl ether bonds and recover the corresponding hydrocarbons. We confirmed the validity of the LiAlH4 method in a study of the model compound synthetic unsaturated archaetidic acid (2,3-di-O-geranylgeranyl-sn-glycerol-1-phosphate. Saturated ether bonds were not cleaved by the LiAlH4 method. The hydrocarbons formed following LiAlH4 hydrogenolysis of PL-4 were identified by gas–liquid chromatography and mass spectrometry. Four kinds of hydrocarbons with one to four double bonds were detected: 47% of the hydrocarbons had four double bonds; 11% had three double bonds; 14% had two double bonds; 7% had one double bond; and 6% were saturated species. The molecular species composition of PL-4 was also estimated based on acid lability: 77% of the molecular species had two acid-labile hydrocarbons; 11% had one acid-labile and one acid-stable hydrocarbon; and 11% had two acid-stable hydrocarbons. To our knowledge, this is the first report of a specific chemical degradation method for the structural analysis of allyl ether phospholipid in archaea.

  15. RCRA groundwater data analysis protocol for the Hanford Site, Washington

    Chou, C.J.; Jackson, R.L.

    1992-04-01

    The Resource Conservation and Recovery Act of 1976 (RCRA) groundwater monitoring program currently involves site-specific monitoring of 20 facilities on the Hanford Site in southeastern Washington. The RCRA groundwater monitoring program has collected abundant data on groundwater quality. These data are used to assess the impact of a facility on groundwater quality or whether remediation efforts under RCRA corrective action programs are effective. Both evaluations rely on statistical analysis of groundwater monitoring data. The need for information on groundwater quality by regulators and environmental managers makes statistical analysis of monitoring data an important part of RCRA groundwater monitoring programs. The complexity of groundwater monitoring programs and variabilities (spatial, temporal, and analytical) exhibited in groundwater quality variables indicate the need for a data analysis protocol to guide statistical analysis. A data analysis protocol was developed from the perspective of addressing regulatory requirements, data quality, and management information needs. This data analysis protocol contains four elements: data handling methods; graphical evaluation techniques; statistical tests for trend, central tendency, and excursion analysis; and reporting procedures for presenting results to users

  16. Fast dynamics perturbation analysis for prediction of protein functional sites

    Cohn Judith D

    2008-01-01

    Full Text Available Abstract Background We present a fast version of the dynamics perturbation analysis (DPA algorithm to predict functional sites in protein structures. The original DPA algorithm finds regions in proteins where interactions cause a large change in the protein conformational distribution, as measured using the relative entropy Dx. Such regions are associated with functional sites. Results The Fast DPA algorithm, which accelerates DPA calculations, is motivated by an empirical observation that Dx in a normal-modes model is highly correlated with an entropic term that only depends on the eigenvalues of the normal modes. The eigenvalues are accurately estimated using first-order perturbation theory, resulting in a N-fold reduction in the overall computational requirements of the algorithm, where N is the number of residues in the protein. The performance of the original and Fast DPA algorithms was compared using protein structures from a standard small-molecule docking test set. For nominal implementations of each algorithm, top-ranked Fast DPA predictions overlapped the true binding site 94% of the time, compared to 87% of the time for original DPA. In addition, per-protein recall statistics (fraction of binding-site residues that are among predicted residues were slightly better for Fast DPA. On the other hand, per-protein precision statistics (fraction of predicted residues that are among binding-site residues were slightly better using original DPA. Overall, the performance of Fast DPA in predicting ligand-binding-site residues was comparable to that of the original DPA algorithm. Conclusion Compared to the original DPA algorithm, the decreased run time with comparable performance makes Fast DPA well-suited for implementation on a web server and for high-throughput analysis.

  17. CRITICAL RADIONUCLIDE AND PATHWAY ANALYSIS FOR THE SAVANNAH RIVER SITE

    Jannik, T.

    2011-08-30

    This report is an update to the analysis, Assessment of SRS Radiological Liquid and Airborne Contaminants and Pathways, that was performed in 1997. An electronic version of this large original report is included in the attached CD to this report. During the operational history (1954 to the present) of the Savannah River Site (SRS), many different radionuclides have been released to the environment from the various production facilities. However, as will be shown by this updated radiological critical contaminant/critical pathway analysis, only a small number of the released radionuclides have been significant contributors to potential doses and risks to offsite people. The analysis covers radiological releases to the atmosphere and to surface waters, the principal media that carry contaminants offsite. These releases potentially result in exposure to offsite people. The groundwater monitoring performed at the site shows that an estimated 5 to 10% of SRS has been contaminated by radionuclides, no evidence exists from the extensive monitoring performed that groundwater contaminated with these constituents has migrated off the site (SRS 2011). Therefore, with the notable exception of radiological source terms originating from shallow surface water migration into site streams, onsite groundwater was not considered as a potential exposure pathway to offsite people. In addition, in response to the Department of Energy's (DOE) Order 435.1, several Performance Assessments (WSRC 2008; LWO 2009; SRR 2010; SRR 2011) and a Comprehensive SRS Composite Analysis (SRNO 2010) have recently been completed at SRS. The critical radionuclides and pathways identified in these extensive reports are discussed and, where applicable, included in this analysis.

  18. A systems analysis approach to nuclear facility siting

    Gros, J.G.; Avenhaus, R.; Linnerooth, J.; Pahner, P.D.; Otway, H.J.

    1975-01-01

    An attempt is made to demonstrate an application of the techniques of systems analysis, which have been successful in solving a variety of problems, to nuclear facility siting. Within the framework of an overall regional land-use plan, a methodology for establishing the acceptability of a combination of site and facility is discussed. The consequences (e.g. the energy produced, thermal and chemical discharges, radioactive releases, aeshetic values, etc.) of the site-facility combination are identified and compared with formalized criteria in order to ensure 'legal acceptability'. Failure of any consequences to satisfy standard requirements results in a feedback channel which works to effect design changes in the facility. When 'legal acceptability' has been assured, the project enters the public sector for consideration. The responses of individuals and of various interested groups to the external attributes of the nuclear facility gradually emerge. The criteria by which interest groups judge technological advances reflect both their rational assessment and unconscious motivations. This process operates on individual, group, societal and international levels and may result in two basic feedback loops: one which might act to change regulatory criteria; the other which might influence facility design or site selection. Such reactions and responses on these levels result in a continuing process of confrontation, collaborative interchange and possible resolution in the direction of an acceptable solution. Finally, a Paretian approach to optimizing the site-facility combination is presented for the case where there are several possible combinations of site and facility. A hypothetical example of the latter is given, based upon typical preference functions determined for four interest groups. The research effort of the IIASA Energy Systems Project and the Joint IAEA/IIASA Research Project in the area of nuclear siting is summarized. (author)

  19. Modular risk analysis for assessing multiple waste sites

    Whelan, G.; Buck, J.W.; Nazarali, A.

    1994-06-01

    Human-health impacts, especially to the surrounding public, are extremely difficult to assess at installations that contain multiple waste sites and a variety of mixed-waste constituents (e.g., organic, inorganic, and radioactive). These assessments must address different constituents, multiple waste sites, multiple release patterns, different transport pathways (i.e., groundwater, surface water, air, and overland soil), different receptor types and locations, various times of interest, population distributions, land-use patterns, baseline assessments, a variety of exposure scenarios, etc. Although the process is complex, two of the most important difficulties to overcome are associated with (1) establishing an approach that allows for modifying the source term, transport, or exposure component as an individual module without having to re-evaluate the entire installation-wide assessment (i.e., all modules simultaneously), and (2) displaying and communicating the results in an understandable and useable maimer to interested parties. An integrated, physics-based, compartmentalized approach, which is coupled to a Geographical Information System (GIS), captures the regional health impacts associated with multiple waste sites (e.g., hundreds to thousands of waste sites) at locations within and surrounding the installation. Utilizing a modular/GIS-based approach overcomes difficulties in (1) analyzing a wide variety of scenarios for multiple waste sites, and (2) communicating results from a complex human-health-impact analysis by capturing the essence of the assessment in a relatively elegant manner, so the meaning of the results can be quickly conveyed to all who review them

  20. Analysis of correlations between sites in models of protein sequences

    Giraud, B.G.; Lapedes, A.; Liu, L.C.

    1998-01-01

    A criterion based on conditional probabilities, related to the concept of algorithmic distance, is used to detect correlated mutations at noncontiguous sites on sequences. We apply this criterion to the problem of analyzing correlations between sites in protein sequences; however, the analysis applies generally to networks of interacting sites with discrete states at each site. Elementary models, where explicit results can be derived easily, are introduced. The number of states per site considered ranges from 2, illustrating the relation to familiar classical spin systems, to 20 states, suitable for representing amino acids. Numerical simulations show that the criterion remains valid even when the genetic history of the data samples (e.g., protein sequences), as represented by a phylogenetic tree, introduces nonindependence between samples. Statistical fluctuations due to finite sampling are also investigated and do not invalidate the criterion. A subsidiary result is found: The more homogeneous a population, the more easily its average properties can drift from the properties of its ancestor. copyright 1998 The American Physical Society

  1. The LivePhoto Physics videos and video analysis site

    Abbott, David

    2009-09-01

    The LivePhoto site is similar to an archive of short films for video analysis. Some videos have Flash tools for analyzing the video embedded in the movie. Most of the videos address mechanics topics with titles like Rolling Pencil (check this one out for pedagogy and content knowledge—nicely done!), Juggler, Yo-yo, Puck and Bar (this one is an inelastic collision with rotation), but there are a few titles in other areas (E&M, waves, thermo, etc.).

  2. Portable Infrared Laser Spectroscopy for On-site Mycotoxin Analysis

    Sieger, Markus; Kos, Gregor; Sulyok, Michael; Godejohann, Matthias; Krska, Rudolf; Mizaikoff, Boris

    2017-03-01

    Mycotoxins are toxic secondary metabolites of fungi that spoil food, and severely impact human health (e.g., causing cancer). Therefore, the rapid determination of mycotoxin contamination including deoxynivalenol and aflatoxin B1 in food and feed samples is of prime interest for commodity importers and processors. While chromatography-based techniques are well established in laboratory environments, only very few (i.e., mostly immunochemical) techniques exist enabling direct on-site analysis for traders and manufacturers. In this study, we present MYCOSPEC - an innovative approach for spectroscopic mycotoxin contamination analysis at EU regulatory limits for the first time utilizing mid-infrared tunable quantum cascade laser (QCL) spectroscopy. This analysis technique facilitates on-site mycotoxin analysis by combining QCL technology with GaAs/AlGaAs thin-film waveguides. Multivariate data mining strategies (i.e., principal component analysis) enabled the classification of deoxynivalenol-contaminated maize and wheat samples, and of aflatoxin B1 affected peanuts at EU regulatory limits of 1250 μg kg-1 and 8 μg kg-1, respectively.

  3. Caspase activation increases beta-amyloid generation independently of caspase cleavage of the beta-amyloid precursor protein (APP).

    Tesco, Giuseppina; Koh, Young Ho; Tanzi, Rudolph E

    2003-11-14

    The amyloid precursor protein (APP) undergoes "alternative" proteolysis mediated by caspases. Three major caspase recognition sites have been identified in the APP, i.e. one at the C terminus (Asp720) and two at the N terminus (Asp197 and Asp219). Caspase cleavage at Asp720 has been suggested as leading to increased production of Abeta. Thus, we set out to determine which putative caspase sites in APP, if any, are cleaved in Chinese hamster ovary cell lines concurrently with the increased Abeta production that occurs during apoptosis. We found that cleavage at Asp720 occurred concurrently with caspase 3 activation and the increased production of total secreted Abeta and Abeta1-42 in association with staurosporine- and etoposide-induced apoptosis. To investigate the contribution of caspase cleavage of APP to Abeta generation, we expressed an APP mutant truncated at Asp720 that mimics APP caspase cleavage at the C-terminal site. This did not increase Abeta generation but, in contrast, dramatically decreased Abeta production in Chinese hamster ovary cells. Furthermore, the ablation of caspase-dependent cleavage at Asp720, Asp197, and Asp219 (by site-directed mutagenesis) did not prevent enhanced Abeta production following etoposide-induced apoptosis. These findings indicate that the enhanced Abeta generation associated with apoptosis does not require cleavage of APP at its C-terminal (Asp720) and/or N-terminal caspase sites.

  4. Yeast ribonuclease III uses a network of multiple hydrogen bonds for RNA binding and cleavage.

    Lavoie, Mathieu; Abou Elela, Sherif

    2008-08-19

    Members of the bacterial RNase III family recognize a variety of short structured RNAs with few common features. It is not clear how this group of enzymes supports high cleavage fidelity while maintaining a broad base of substrates. Here we show that the yeast orthologue of RNase III (Rnt1p) uses a network of 2'-OH-dependent interactions to recognize substrates with different structures. We designed a series of bipartite substrates permitting the distinction between binding and cleavage defects. Each substrate was engineered to carry a single or multiple 2'- O-methyl or 2'-fluoro ribonucleotide substitutions to prevent the formation of hydrogen bonds with a specific nucleotide or group of nucleotides. Interestingly, introduction of 2'- O-methyl ribonucleotides near the cleavage site increased the rate of catalysis, indicating that 2'-OH are not required for cleavage. Substitution of nucleotides in known Rnt1p binding site with 2'- O-methyl ribonucleotides inhibited cleavage while single 2'-fluoro ribonucleotide substitutions did not. This indicates that while no single 2'-OH is essential for Rnt1p cleavage, small changes in the substrate structure are not tolerated. Strikingly, several nucleotide substitutions greatly increased the substrate dissociation constant with little or no effect on the Michaelis-Menten constant or rate of catalysis. Together, the results indicate that Rnt1p uses a network of nucleotide interactions to identify its substrate and support two distinct modes of binding. One mode is primarily mediated by the dsRNA binding domain and leads to the formation of stable RNA/protein complex, while the other requires the presence of the nuclease and N-terminal domains and leads to RNA cleavage.

  5. Value of Information Analysis Project Gnome Site, New Mexico

    Pohll, Greg; Chapman, Jenny

    2010-01-01

    The Project Gnome site in southeastern New Mexico was the location of an underground nuclear detonation in 1961 and a hydrologic tracer test using radionuclides in 1963. The tracer test is recognized as having greater radionuclide migration potential than the nuclear test because the tracer test radionuclides (tritium, 90Sr, 131I, and 137Cs) are in direct contact with the Culebra Dolomite aquifer, whereas the nuclear test is within a bedded salt formation. The tracer test is the topic here. Recognizing previous analyses of the fate of the Gnome tracer test contaminants (Pohll and Pohlmann, 1996; Pohlmann and Andricevic, 1994), and the existence of a large body of relevant investigations and analyses associated with the nearby Waste Isolation Pilot Plant (WIPP) site (summarized in US DOE, 2009), the Gnome Site Characterization Work Plan (U.S. DOE, 2002) called for a Data Decision Analysis to determine whether or not additional characterization data are needed prior to evaluating existing subsurface intrusion restrictions and determining long-term monitoring for the tracer test. Specifically, the Work Plan called for the analysis to weigh the potential reduction in uncertainty from additional data collection against the cost of such field efforts.

  6. LOG FILE ANALYSIS AND CREATION OF MORE INTELLIGENT WEB SITES

    Mislav Šimunić

    2012-07-01

    Full Text Available To enable successful performance of any company or business system, both inthe world and in the Republic of Croatia, among many problems relating to its operationsand particularly to maximum utilization and efficiency of the Internet as a media forrunning business (especially in terms of marketing, they should make the best possible useof the present-day global trends and advantages of sophisticated technologies andapproaches to running a business. Bearing in mind the fact of daily increasing competitionand more demanding market, this paper addresses certain scientific and practicalcontribution to continuous analysis of demand market and adaptation thereto by analyzingthe log files and by retroactive effect on the web site. A log file is a carrier of numerousdata and indicators that should be used in the best possible way to improve the entirebusiness operations of a company. However, this is not always simple and easy. The websites differ in size, purpose, and technology used for designing them. For this very reason,the analytic analysis frameworks should be such that can cover any web site and at thesame time leave some space for analyzing and investigating the specific characteristicof each web site and provide for its dynamics by analyzing the log file records. Thoseconsiderations were a basis for this paper

  7. Corporate Social Responsibility In Turkey: An Analysis Through Web Sites

    Burcu EKER AKGÖZ

    2016-06-01

    Full Text Available In today’s business environment, there is no chance for a corporation to be succeeded simply by doing profit oriented business. Corporations are the living entities along with their stakeholders therefore corporations are expected to be responsive on the cultural, environmental and economic issues of society. Socially responsible programs that corporations execute would contribute the corporate image and reputation. Furthermore, these programs benefit both corporations and society. The aim of this study is to examine the presence and organization of information on corporate responsibility present on the corporate web sites of companies which are in the Capital top 500 list. We will choose the top 100 companies on the top 500 list. By using content analysis, identified corporate responsibility issues will be analyzed. In this study, the information given in the web sites of these top 100 companies will be analyzed. We will be examining how the information is organized, presented, and related to the corporate responsibilities.

  8. Engineering, Analysis and Technology FY 1995 Site Support Program Plan

    Suyama, R.M.

    1994-09-01

    The vision of the Engineering, Analysis and Technology organization is to be recognized as the cost-effective supplier of specialized, integrated, multi-disciplined engineering teams to support Hanford missions. The mission of the Engineering, Analysis and Technology organization is to provide centralized engineering services. These services are focused on supplying technical design, analytical engineering and related support services that support Hanford's environmental restoration mission. These services include engineering analysis, design and development of systems and engineered equipment, supplying multi-disciplined engineering teams to all Hanford programs and project organizations, engineering document release, and site-wide leadership in the development and implementation of engineering standards, engineering practices, and configuration management processes

  9. Monoclonal antibodies to the reactive centre loop (RCL) of human corticosteroid-binding globulin (CBG) can protect against proteolytic cleavage.

    Lewis, John G; Elder, Peter A

    2017-07-01

    Corticosteroid-binding globulin (CBG) binds most of the cortisol in circulation and is a non-functional member of the family of serine protease inhibitors (serpins) with an exposed elastase sensitive reactive centre loop (RCL). The RCL can be cleaved by human neutrophil elastase, released from activated neutrophils, and can also be cleaved at nearby site(s) by elastase released by Pseudomonas aeruginosa, and at two further sites, also within the RCL, by bovine chymotrypsin. Cleavage of the RCL results in a conformational change accompanied by a marked decrease in affinity for cortisol and hence its release at the site of proteolysis. These cleavages are irreversible and the similar half-lives of cleaved and intact CBG could mean that there may be some advantage in slowing the rate of CBG cleavage in acute inflammation thereby increasing the proportion of intact CBG in circulation. Here we show, for the first time, that pre-incubation of tethered human CBG with two monoclonal antibodies to the RCL of CBG protects against cleavage by all three enzymes. Furthermore, in plasma, pre-incubation with both RCL monoclonal antibodies delays neutrophil elastase cleavage of the RCL and one of these RCL monoclonal antibodies also delays bovine chymotrypsin cleavage of the RCL. These findings may provide a basis and rationale for the concept of the use of RCL antibodies as therapeutic agents to effectively increase the proportion of intact CBG in circulation which may be of benefit in acute inflammation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. On-site vs off-site management of environmental restoration waste: A cost effectiveness analysis

    Morse, M.A.; Aamodt, P.L.; Cox, W.B.

    1996-01-01

    The Sandia National Laboratories Environmental Restoration Project is expected to generate relatively large volumes of hazardous waste as a result of cleanup operations. These volumes will exceed the Laboratories existing waste management capacity. This paper presents four options for managing remediation wastes, including three alternatives for on-site waste management utilizing a corrective action management unit (CAMU). Costs are estimated for each of the four options based on current volumetric estimates of hazardous waste. Cost equations are derived for each of the options with the variables being waste volumes, the major unknowns in the analysis. These equations provide a means to update cost estimates as volume estimates change. This approach may be helpful to others facing similar waste management decisions

  11. Flood risk analysis of Cold Creek near the Hanford Site

    Skaggs, R.L.; Walters, W.H.

    1981-01-01

    The Pacific Northwest Laboratory has analyzed the flood potential at the reference repository location located on the Hanford Site near Richland, Washington. It is emphasized that this work is not intended as a basis for engineering design, but rather as an initial, regional appraisal of whether detailed engineering design analysis will be required. In order to achieve the detail required for engineering design specifications, the study results should be refined using more detailed channel geometry data, and the topography of the western portion of the reference repository location should be mapped using a contour interval of not less than 2 ft. 19 refs., 15 figs., 5 tabs

  12. Predicting proteasomal cleavage sites: a comparison of available methods

    Saxova, P.; Buus, S.; Brunak, Søren

    2003-01-01

    -terminal, in particular, of CTL epitopes is cleaved precisely by the proteasome, whereas the N-terminal is produced with an extension, and later trimmed by peptidases in the cytoplasm and in the endoplasmic reticulum. Recently, three publicly available methods have been developed for prediction of the specificity...

  13. [Cleavage of DNA fragments induced by UV nanosecond laser excitation at 193 nm].

    Vtiurina, N N; Grokhovskiĭ, S L; Filimonov, I V; Medvedkov, O I; Nechipurenko, D Iu; Vasil'ev, S A; Nechipurenko, Iu D

    2011-01-01

    The cleavage of dsDNA fragments in aqueous solution after irradiation with UV laser pulses at 193 nm has been studied. Samples were investigated using polyacrylamide gel electrophoresis. The intensity of damage of particular phosphodiester bond after hot alkali treatment was shown to depend on the base pair sequence. It was established that the probability of cleavage is twice higher for sites of DNA containing two or more successively running guanine residues. A possible mechanism of damage to the DNA molecule connected with the migration of holes along the helix is discussed.

  14. Statistical Analysis of Environmental Tritium around Wolsong Site

    Kim, Ju Youl [FNC Technology Co., Yongin (Korea, Republic of)

    2010-04-15

    To find the relationship among airborne tritium, tritium in rainwater, TFWT (Tissue Free Water Tritium) and TBT (Tissue Bound Tritium), statistical analysis is conducted based on tritium data measured at KHNP employees' house around Wolsong nuclear power plants during 10 years from 1999 to 2008. The results show that tritium in such media exhibits a strong seasonal and annual periodicity. Tritium concentration in rainwater is observed to be highly correlated with TFWT and directly transmitted to TFWT without delay. The response of environmental radioactivity of tritium around Wolsong site is analyzed using time-series technique and non-parametric trend analysis. Tritium in the atmosphere and rainwater is strongly auto-correlated by seasonal and annual periodicity. TFWT concentration in pine needle is proven to be more sensitive to rainfall phenomenon than other weather variables. Non-parametric trend analysis of TFWT concentration within pine needle shows a increasing slope in terms of confidence level of 95%. This study demonstrates a usefulness of time-series and trend analysis for the interpretation of environmental radioactivity relationship with various environmental media.

  15. Stimulation of topoisomerase II mediated DNA cleavage at specific sequence elements by the 2-nitroimidazole Ro 15-0216

    Sorensen, B.S.; Jensen, P.S.; Andersen, A.H.; Christiansen, K.; Alsner, J.; Thomsen, B.; Westergaard, O.

    1990-01-01

    The effect of the 2-nitroimidazole Ro 15-0216 upon the interaction between purified topoisomerase II and its DNA substrate was investigated. The cleavage reaction in the presence of this DNA-nonintercalative drug took place with the hallmarks of a regular topoisomerase II mediated cleavage reaction, including covalent linkage of the enzyme to the cleaved DNA. In the presence of Ro 15-0216, topoisomerase II mediated cleavage was extensively stimulated at major cleavage sites of which only one existed in the 4363 base pair pBR322 molecule. The sites stimulated by Ro 15-0216 shared a pronounced sequence homology, indicating that a specific nucleotide sequence is crucial for the action of this drug. The effect of Ro 15-0216 thus differs from that of the clinically important topoisomerase II targeted agents such as mAMSA, VM26, and VP16, which enhance enzyme-mediated cleavage at a multiple number of sites. In contrast to the previous described drugs, Ro 15-0216 did not exert any inhibitory effect on the enzyme's catalytic activity. This observation might be ascribed to the low stability of the cleavage complexes formed in the presence of Ro 15-0216 as compared to the stability of the ones formed in the presence of traditional topoisomerase II targeted drugs

  16. Abnormal early cleavage events predict early embryo demise: sperm oxidative stress and early abnormal cleavage.

    Burruel, Victoria; Klooster, Katie; Barker, Christopher M; Pera, Renee Reijo; Meyers, Stuart

    2014-10-13

    Human embryos resulting from abnormal early cleavage can result in aneuploidy and failure to develop normally to the blastocyst stage. The nature of paternal influence on early embryo development has not been directly demonstrated although many studies have suggested effects from spermatozoal chromatin packaging, DNA damage, centriolar and mitotic spindle integrity, and plasma membrane integrity. The goal of this study was to determine whether early developmental events were affected by oxidative damage to the fertilizing sperm. Survival analysis was used to compare patterns of blastocyst formation based on P2 duration. Kaplan-Meier survival curves demonstrate that relatively few embryos with short (P2 times reached blastocysts, and the two curves diverged beginning on day 4, with nearly all of the embryos with longer P2 times reaching blastocysts by day 6 (p < .01). We determined that duration of the 2nd to 3rd mitoses were sensitive periods in the presence of spermatozoal oxidative stress. Embryos that displayed either too long or too short cytokineses demonstrated an increased failure to reach blastocyst stage and therefore survive for further development. Although paternal-derived gene expression occurs later in development, this study suggests a specific role in early mitosis that is highly influenced by paternal factors.

  17. Controllable laser thermal cleavage of sapphire wafers

    Xu, Jiayu; Hu, Hong; Zhuang, Changhui; Ma, Guodong; Han, Junlong; Lei, Yulin

    2018-03-01

    Laser processing of substrates for light-emitting diodes (LEDs) offers advantages over other processing techniques and is therefore an active research area in both industrial and academic sectors. The processing of sapphire wafers is problematic because sapphire is a hard and brittle material. Semiconductor laser scribing processing suffers certain disadvantages that have yet to be overcome, thereby necessitating further investigation. In this work, a platform for controllable laser thermal cleavage was constructed. A sapphire LED wafer was modeled using the finite element method to simulate the thermal and stress distributions under different conditions. A guide groove cut by laser ablation before the cleavage process was observed to guide the crack extension and avoid deviation. The surface and cross section of sapphire wafers processed using controllable laser thermal cleavage were characterized by scanning electron microscopy and optical microscopy, and their morphology was compared to that of wafers processed using stealth dicing. The differences in luminous efficiency between substrates prepared using these two processing methods are explained.

  18. Pressure modulates the self-cleavage step of the hairpin ribozyme

    Schuabb, Caroline; Kumar, Narendra; Pataraia, Salome; Marx, Dominik; Winter, Roland

    2017-03-01

    The ability of certain RNAs, denoted as ribozymes, to not only store genetic information but also catalyse chemical reactions gave support to the RNA world hypothesis as a putative step in the development of early life on Earth. This, however, might have evolved under extreme environmental conditions, including the deep sea with pressures in the kbar regime. Here we study pressure-induced effects on the self-cleavage of hairpin ribozyme by following structural changes in real-time. Our results suggest that compression of the ribozyme leads to an accelerated transesterification reaction, being the self-cleavage step, although the overall process is retarded in the high-pressure regime. The results reveal that favourable interactions between the reaction site and neighbouring nucleobases are strengthened under pressure, resulting therefore in an accelerated self-cleavage step upon compression. These results suggest that properly engineered ribozymes may also act as piezophilic biocatalysts in addition to their hitherto known properties.

  19. Systematic analysis of transcription start sites in avian development.

    Marina Lizio

    2017-09-01

    Full Text Available Cap Analysis of Gene Expression (CAGE in combination with single-molecule sequencing technology allows precision mapping of transcription start sites (TSSs and genome-wide capture of promoter activities in differentiated and steady state cell populations. Much less is known about whether TSS profiling can characterize diverse and non-steady state cell populations, such as the approximately 400 transitory and heterogeneous cell types that arise during ontogeny of vertebrate animals. To gain such insight, we used the chick model and performed CAGE-based TSS analysis on embryonic samples covering the full 3-week developmental period. In total, 31,863 robust TSS peaks (>1 tag per million [TPM] were mapped to the latest chicken genome assembly, of which 34% to 46% were active in any given developmental stage. ZENBU, a web-based, open-source platform, was used for interactive data exploration. TSSs of genes critical for lineage differentiation could be precisely mapped and their activities tracked throughout development, suggesting that non-steady state and heterogeneous cell populations are amenable to CAGE-based transcriptional analysis. Our study also uncovered a large set of extremely stable housekeeping TSSs and many novel stage-specific ones. We furthermore demonstrated that TSS mapping could expedite motif-based promoter analysis for regulatory modules associated with stage-specific and housekeeping genes. Finally, using Brachyury as an example, we provide evidence that precise TSS mapping in combination with Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR-on technology enables us, for the first time, to efficiently target endogenous avian genes for transcriptional activation. Taken together, our results represent the first report of genome-wide TSS mapping in birds and the first systematic developmental TSS analysis in any amniote species (birds and mammals. By facilitating promoter-based molecular analysis and genetic

  20. Fetal hemoglobin is much less prone to DNA cleavage compared to the adult protein

    Sandeep Chakane

    2017-08-01

    Full Text Available Hemoglobin (Hb is well protected inside the red blood cells (RBCs. Upon hemolysis and when free in circulation, Hb can be involved in a range of radical generating reactions and may thereby attack several different biomolecules. In this study, we have examined the potential damaging effects of cell-free Hb on plasmid DNA (pDNA. Hb induced cleavage of supercoiled pDNA (sc pDNA which was proportional to the concentration of Hb applied. Almost 70% of sc pDNA was converted to open circular or linear DNA using 10 µM of Hb in 12 h. Hb can be present in several different forms. The oxy (HbO2 and met forms are most reactive, while the carboxy-protein shows only low hydrolytic activity. Hemoglobin A (HbA could easily induce complete pDNA cleavage while fetal hemoglobin (HbF was three-fold less reactive. By inserting, a redox active cysteine residue on the surface of the alpha chain of HbF by site-directed mutagenesis, the DNA cleavage reaction was enhanced by 82%. Reactive oxygen species were not directly involved in the reaction since addition of superoxide dismutase and catalase did not prevent pDNA cleavage. The reactivity of Hb with pDNA can rather be associated with the formation of protein based radicals. Keywords: Adult hemoglobin, Fetal hemoglobin, Supercoiled plasmid DNA, DNA cleavage, Cysteine, Protein radicals

  1. Characterization of cleavage events in the multifunctional cilium adhesin Mhp684 (P146) reveals a mechanism by which Mycoplasma hyopneumoniae regulates surface topography.

    Bogema, Daniel R; Deutscher, Ania T; Woolley, Lauren K; Seymour, Lisa M; Raymond, Benjamin B A; Tacchi, Jessica L; Padula, Matthew P; Dixon, Nicholas E; Minion, F Chris; Jenkins, Cheryl; Walker, Mark J; Djordjevic, Steven P

    2012-01-01

    Mycoplasma hyopneumoniae causes enormous economic losses to swine production worldwide by colonizing the ciliated epithelium in the porcine respiratory tract, resulting in widespread damage to the mucociliary escalator, prolonged inflammation, reduced weight gain, and secondary infections. Protein Mhp684 (P146) comprises 1,317 amino acids, and while the N-terminal 400 residues display significant sequence identity to the archetype cilium adhesin P97, the remainder of the molecule is novel and displays unusual motifs. Proteome analysis shows that P146 preprotein is endogenously cleaved into three major fragments identified here as P50(P146), P40(P146), and P85(P146) that reside on the cell surface. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) identified a semitryptic peptide that delineated a major cleavage site in Mhp684. Cleavage occurred at the phenylalanine residue within sequence (672)ATEF↓QQ(677), consistent with a cleavage motif resembling S/T-X-F↓X-D/E recently identified in Mhp683 and other P97/P102 family members. Biotinylated surface proteins recovered by avidin chromatography and separated by two-dimensional gel electrophoresis (2-D GE) showed that more-extensive endoproteolytic cleavage of P146 occurs. Recombinant fragments F1(P146)-F3(P146) that mimic P50(P146), P40(P146), and P85(P146) were constructed and shown to bind porcine epithelial cilia and biotinylated heparin with physiologically relevant affinity. Recombinant versions of F3(P146) generated from M. hyopneumoniae strain J and 232 sequences strongly bind porcine plasminogen, and the removal of their respective C-terminal lysine and arginine residues significantly reduces this interaction. These data reveal that P146 is an extensively processed, multifunctional adhesin of M. hyopneumoniae. Extensive cleavage coupled with variable cleavage efficiency provides a mechanism by which M. hyopneumoniae regulates protein topography. Vaccines used to control Mycoplasma

  2. Seismic hazard analysis of the NPP Kozloduy site

    Petrovski, D.; Stamatovska, S.; Arsovski, M.; Hadzievski, D.; Sokerova, D.; Solakov, D.; Vaptzarov, I.; Satchanski, S.

    1993-01-01

    The principal objective of this study is to define the seismic hazard for the NPP Kozloduy site. Seismic hazard is by rule defined by the probability distribution function of the peak value of the chosen ground motion parameter in a defined time interval. The overall study methodology consists of reviewing the existing geological, seismological and tectonic information to formulate this information into a mathematical model of seismic activity of the region and using this assess earthquake ground motion in terms of probability. Detailed regional and local seismological investigations have been performed. Regional investigations encompass the area within a radius of 320 km from the NPP Kozloduy site. The results of these investigations include all seismological parameters that are necessary for determination of the mathematical model of the seismicity of the region needed for the seismic hazard analysis. Regional geological and neotectonic investigations were also performed for the wider area including almost the whole territory of Bulgaria, a large part of Serbia, part of Macedonia and almost the whole south part of Romania

  3. Fractal analysis of striatal dopamine re-uptake sites

    Kuikka, J.T.; Bergstroem, K.A.; Tiihonen, J.; Raesaenen, P.; Karhu, J.

    1997-01-01

    Spatial variation in regional blood flow, metabolism and receptor density within the brain and in other organs is measurable even with a low spatial resolution technique such as emission tomography. It has been previously shown that the observed variance increases with increasing number of subregions in the organ/tissue studied. This resolution-dependent variance can be described by fractal analysis. We studied striatal dopamine re-uptake sites in 39 healthy volunteers with high-resolution single-photon emission tomography using iodine-123 labelled 2β-carbomethoxy-3β-(4-iodophenyl)tropane ([ 123 I]β-CIT). The mean fractal dimension was 1.15±0.07. The results indicate that regional striatal dopamine re-uptake sites involve considerable spatial heterogeneity which is higher than the uniform density (dimension=1.00) but much lower than complete randomness (dimension=1.50). There was a gender difference, with females having a higher heterogeneity in both the left and the right striatum. In addition, we found striatal asymmetry (left-to-right heterogeneity ratio of 1.19±0.15; P<0.001), suggesting functional hemispheric lateralization consistent with the control of motor behaviour and integrative functions. (orig.). With 5 figs., 1 tab

  4. Fractal analysis of striatal dopamine re-uptake sites

    Kuikka, J.T.; Bergstroem, K.A. [Department of Clinical Physiology, Kuopio University Hospital, Kuopio (Finland); Tiihonen, J.; Raesaenen, P. [Department of Forensic Psychiatry, University of Kuopio and Niuvanniemi Hospital, Kuopio (Finland); Karhu, J. [Department of Clinical Neurophysiology, Kuopio University Hospital, Kuopio (Finland)

    1997-09-01

    Spatial variation in regional blood flow, metabolism and receptor density within the brain and in other organs is measurable even with a low spatial resolution technique such as emission tomography. It has been previously shown that the observed variance increases with increasing number of subregions in the organ/tissue studied. This resolution-dependent variance can be described by fractal analysis. We studied striatal dopamine re-uptake sites in 39 healthy volunteers with high-resolution single-photon emission tomography using iodine-123 labelled 2{beta}-carbomethoxy-3{beta}-(4-iodophenyl)tropane ([{sup 123}I]{beta}-CIT). The mean fractal dimension was 1.15{+-}0.07. The results indicate that regional striatal dopamine re-uptake sites involve considerable spatial heterogeneity which is higher than the uniform density (dimension=1.00) but much lower than complete randomness (dimension=1.50). There was a gender difference, with females having a higher heterogeneity in both the left and the right striatum. In addition, we found striatal asymmetry (left-to-right heterogeneity ratio of 1.19{+-}0.15; P<0.001), suggesting functional hemispheric lateralization consistent with the control of motor behaviour and integrative functions. (orig.). With 5 figs., 1 tab.

  5. External events analysis for the Savannah River Site K reactor

    Brandyberry, M.D.; Wingo, H.E.

    1990-01-01

    The probabilistic external events analysis performed for the Savannah River Site K-reactor PRA considered many different events which are generally perceived to be ''external'' to the reactor and its systems, such as fires, floods, seismic events, and transportation accidents (as well as many others). Events which have been shown to be significant contributors to risk include seismic events, tornados, a crane failure scenario, fires and dam failures. The total contribution to the core melt frequency from external initiators has been found to be 2.2 x 10 -4 per year, from which seismic events are the major contributor (1.2 x 10 -4 per year). Fire initiated events contribute 1.4 x 10 -7 per year, tornados 5.8 x 10 -7 per year, dam failures 1.5 x 10 -6 per year and the crane failure scenario less than 10 -4 per year to the core melt frequency. 8 refs., 3 figs., 5 tabs

  6. Hanford Site Composite Analysis Technical Approach Description: Hanford Site Disposition Baseline.

    Cobb, M. A. [CH2M HILL Plateau Remediation Company, Richland, WA (United States); Dockter, R. E. [CH2M HILL Plateau Remediation Company, Richland, WA (United States)

    2017-10-02

    The permeability of ground surfaces within the U.S. Department of Energy’s (DOE) Hanford Site strongly influences boundary conditions when simulating the movement of groundwater using the Subsurface Transport Over Multiple Phases model. To conduct site-wide modeling of cumulative impacts to groundwater from past, current, and future waste management activities, a site-wide assessment of the permeability of surface conditions is needed. The surface condition of the vast majority of the Hanford Site has been and continues to be native soils vegetated with dryland grasses and shrubs.

  7. Ecological restoration of Central European mining sites: a summary of a multi-site analysis

    Prach, K.; Rehounkova, K.; Rehounek, J.; Konvalinkova, P. [University of South Bohemia, Ceske Budejovice (Czech Republic)

    2011-07-01

    Sites disturbed by mining were surveyed in the Czech Republic, central Europe. The sites included spoil heaps from coal mining, sand and gravel pits, extracted peatlands and stone quarries. The following main conclusions emerged: I) potential for spontaneous succession to be used in restoration projects is between 95 and 100% of the total area disturbed; ii) mining sites, if mining is properly designed and then the sites are left to spontaneous succession, often act as refugia for endangered and retreating organisms, and may contribute substantially to local biodiversity.

  8. Confirmation Sampling and Analysis Plan for Spill Site Number 1

    1998-01-01

    ... No. 1 to document the effectiveness of bioventing for the remediation of petroleum-hydrocarbon-contaminated soils and to provide data for a risk-based assessment of contaminants remaining in site soils and groundwater. Spill Site...

  9. Site-specific meteorology identification for DOE facility accident analysis

    Rabin, S.B.

    1995-01-01

    Currently, chemical dispersion calculations performed for safety analysis of DOE facilities assume a Pasquill D-Stability Class with a 4.5 m/s windspeed. These meteorological conditions are assumed to conservatively address the source term generation mechanism as well as the dispersion mechanism thereby resulting in a net conservative downwind consequence. While choosing this Stability Class / Windspeed combination may result in an overall conservative consequence, the level of conservative can not be quantified. The intent of this paper is to document a methodology which incorporates site-specific meteorology to determine a quantifiable consequence of a chemical release. A five-year meteorological database, appropriate for the facility location, is utilized for these chemical consequence calculations, and is consistent with the approach used for radiological releases. The hourly averages of meteorological conditions have been binned into 21 groups for the chemical consequence calculations. These 21 cases each have a probability of occurrence based on the number of times each case has occurred over the five year sampling period. A code has been developed which automates the running of all the cases with a commercially available air modeling code. The 21 cases are sorted by concentration. A concentration may be selected by the user for a quantified level of conservatism. The methodology presented is intended to improve the technical accuracy and defensability of Chemical Source Term / Dispersion Safety Analysis work. The result improves the quality of safety analyses products without significantly increasing the cost

  10. Molecular cloning and characterization of cDNAs encoding carotenoid cleavage dioxygenase in bitter melon (Momordica charantia).

    Tuan, Pham Anh; Park, Sang Un

    2013-01-01

    Carotenoid cleavage dioxygenases (CCDs) are a family of enzymes that catalyze the oxidative cleavage of carotenoids at various chain positions to form a broad spectrum of apocarotenoids, including aromatic substances, pigments and phytohormones. Using the rapid amplification of cDNA ends (RACE) PCR method, we isolated three cDNA-encoding CCDs (McCCD1, McCCD4, and McNCED) from Momordica charantia. Amino acid sequence alignments showed that they share high sequence identity with other orthologous genes. Quantitative real-time RT PCR (reverse transcriptase PCR) analysis revealed that the expression of McCCD1 and McCCD4 was highest in flowers, and lowest in roots and old leaves (O-leaves). During fruit maturation, the two genes displayed differential expression, with McCCD1 peaking at mid-stage maturation while McCCD4 showed the lowest expression at that stage. The mRNA expression level of McNCED, a key enzyme involved in abscisic acid (ABA) biosynthesis, was high during fruit maturation and further increased at the beginning of seed germination. When first-leaf stage plants of M. charantia were exposed to dehydration stress, McNCED mRNA expression was induced primarily in the leaves and, to a lesser extend, in roots and stems. McNCED expression was also induced by high temperature and salinity, while treatment with exogenous ABA led to a decrease. These results should be helpful in determining the substrates and cleavage sites catalyzed by CCD genes in M. charantia, and also in defining the roles of CCDs in growth and development, and in the plant's response to environmental stress. Copyright © 2012 Elsevier GmbH. All rights reserved.

  11. Library Web Sites in Pakistan: An Analysis of Content

    Qutab, Saima; Mahmood, Khalid

    2009-01-01

    Purpose: The purpose of this paper is to investigate library web sites in Pakistan, to analyse their content and navigational strengths and weaknesses and to give recommendations for developing better web sites and quality assessment studies. Design/methodology/approach: Survey of web sites of 52 academic, special, public and national libraries in…

  12. From Site Data to Safety Assessment: Analysis of Present and Future Hydrological Conditions at a Coastal Site in Sweden

    Berglund, Sten; Bosson, Emma; Sassner, Mona

    2013-01-01

    This paper presents an analysis of present and future hydrological conditions at the Forsmark site in Sweden, which has been proposed as the site for a geological repository for spent nuclear fuel. Forsmark is a coastal site that changes in response to shoreline displacement. In the considered time frame (until year 10 000 ad), the hydrological system will be affected by landscape succession associated with shoreline displacement and changes in vegetation, regolith stratigraphy, and climate. Based on extensive site investigations and modeling of present hydrological conditions, the effects of different processes on future site hydrology are quantified. As expected, shoreline displacement has a strong effect on local hydrology (e.g., groundwater flow) in areas that change from sea to land. The comparison between present and future land areas emphasizes the importance of climate variables relative to other factors for main hydrological features such as water balances

  13. Can laccases catalyze bond cleavage in lignin?

    Munk, Line; Sitarz, Anna Katarzyna; Kalyani, Dayanand

    2015-01-01

    illustrations of the putative laccase catalyzed reactions, including the possible reactions of the reactive radical intermediates taking place after the initial oxidation of the phenol-hydroxyl groups, we show that i) Laccase activity is able to catalyze bond cleavage in low molecular weight phenolic lignin......-substituted phenols, benzenethiols, polyphenols, and polyamines, which may be oxidized. In addition, the currently available analytical methods that can be used to detect enzyme catalyzed changes in lignin are summarized, and an improved nomenclature for unequivocal interpretation of the action of laccases on lignin...

  14. Transcriptional down-regulation and rRNA cleavage in Dictyostelium discoideum mitochondria during Legionella pneumophila infection.

    Chenyu Zhang

    2009-05-01

    Full Text Available Bacterial pathogens employ a variety of survival strategies when they invade eukaryotic cells. The amoeba Dictyostelium discoideum is used as a model host to study the pathogenic mechanisms that Legionella pneumophila, the causative agent of Legionnaire's disease, uses to kill eukaryotic cells. Here we show that the infection of D. discoideum by L. pneumophila results in a decrease in mitochondrial messenger RNAs, beginning more than 8 hours prior to detectable host cell death. These changes can be mimicked by hydrogen peroxide treatment, but not by other cytotoxic agents. The mitochondrial large subunit ribosomal RNA (LSU rRNA is also cleaved at three specific sites during the course of infection. Two LSU rRNA fragments appear first, followed by smaller fragments produced by additional cleavage events. The initial LSU rRNA cleavage site is predicted to be on the surface of the large subunit of the mitochondrial ribosome, while two secondary sites map to the predicted interface with the small subunit. No LSU rRNA cleavage was observed after exposure of D. discoideum to hydrogen peroxide, or other cytotoxic chemicals that kill cells in a variety of ways. Functional L. pneumophila type II and type IV secretion systems are required for the cleavage, establishing a correlation between the pathogenesis of L. pneumophila and D. discoideum LSU rRNA destruction. LSU rRNA cleavage was not observed in L. pneumophila infections of Acanthamoeba castellanii or human U937 cells, suggesting that L. pneumophila uses distinct mechanisms to interrupt metabolism in different hosts. Thus, L. pneumophila infection of D. discoideum results in dramatic decrease of mitochondrial RNAs, and in the specific cleavage of mitochondrial rRNA. The predicted location of the cleavage sites on the mitochondrial ribosome suggests that rRNA destruction is initiated by a specific sequence of events. These findings suggest that L. pneumophila specifically disrupts mitochondrial

  15. Food marketing on popular children's web sites: a content analysis.

    Alvy, Lisa M; Calvert, Sandra L

    2008-04-01

    In 2006 the Institute of Medicine (IOM) concluded that food marketing was a contributor to childhood obesity in the United States. One recommendation of the IOM committee was for research on newer marketing venues, such as Internet Web sites. The purpose of this cross-sectional study was to answer the IOM's call by examining food marketing on popular children's Web sites. Ten Web sites were selected based on market research conducted by KidSay, which identified favorite sites of children aged 8 to 11 years during February 2005. Using a standardized coding form, these sites were examined page by page for the existence, type, and features of food marketing. Web sites were compared using chi2 analyses. Although food marketing was not pervasive on the majority of the sites, seven of the 10 Web sites contained food marketing. The products marketed were primarily candy, cereal, quick serve restaurants, and snacks. Candystand.com, a food product site, contained a significantly greater amount of food marketing than the other popular children's Web sites. Because the foods marketed to children are not consistent with a healthful diet, nutrition professionals should consider joining advocacy groups to pressure industry to reduce online food marketing directed at youth.

  16. Draft site characterization analysis of the site characterization report for the Basalt Waste Isolation Project, Hanford, Washington Site. Main report and Appendices A through D

    1983-03-01

    On November 12, 1982, the US Department of Energy submitted to the US Nuclear Regulatory Commission the Site Characterization Report for the Basalt Waste Isolation Project (DOE/RL 82-3). The Basalt Waste Isolation Project is located on DOE's Hanford Reservation in the State of Washington. NUREG-0960 contains the detailed analysis, by the NRC staff, of the site characterization report. Supporting technical material is contained in Appendices A through W

  17. Heat integration and analysis of decarbonised IGCC sites

    Ng, K.S.; Lopez, Y.; Campbell, G.M.; Sadhukhan, J. [University of Manchester, Manchester (United Kingdom). School of Chemical Engineering & Analytical Science

    2010-02-15

    Integrated gasification combined cycle (IGCC) power generation systems have become of interest due to their high combined heat and power (CHP) generation efficiency and flexibility to include carbon capture and storage (CCS) in order to reduce CO{sub 2} emissions. However, IGCC's biggest challenge is its high cost of energy production. In this study, decarbonised coal IGCC sites integrated with CCS have been investigated for heat integration and economic value analyses. It is envisaged that the high energy production cost of an IGCC site can be offset by maximising site-wide heat recovery and thereby improving the cost of electricity (COE) of CHP generation. Strategies for designing high efficiency CHP networks have been proposed based on thermodynamic heuristics and pinch theory. Additionally, a comprehensive methodology to determine the COE from a process site has been developed. In this work, we have established thermodynamic and economic comparisons between IGCC sites with and without CCS and a trade-off between the degree of decarbonisation and the COE from the heat integrated IGCC sites. The results show that the COE from the heat integrated decarbonised IGCC sites is significantly lower compared to IGCC sites without heat integration making application of CCS in IGCC sites economically competitive.

  18. Cell Death-Associated Ribosomal RNA Cleavage in Postmortem Tissues and Its Forensic Applications.

    Kim, Ji Yeon; Kim, Yunmi; Cha, Hyo Kyeong; Lim, Hye Young; Kim, Hyungsub; Chung, Sooyoung; Hwang, Juck-Joon; Park, Seong Hwan; Son, Gi Hoon

    2017-06-30

    Estimation of postmortem interval (PMI) is a key issue in the field of forensic pathology. With the availability of quantitative analysis of RNA levels in postmortem tissues, several studies have assessed the postmortem degradation of constitutively expressed RNA species to estimate PMI. However, conventional RNA quantification as well as biochemical and physiological changes employed thus far have limitations related to standardization or normalization. The present study focuses on an interesting feature of the subdomains of certain RNA species, in which they are site-specifically cleaved during apoptotic cell death. We found that the D8 divergent domain of ribosomal RNA (rRNA) bearing cell death-related cleavage sites was rapidly removed during postmortem RNA degradation. In contrast to the fragile domain, the 5' terminal region of 28S rRNA was remarkably stable during the postmortem period. Importantly, the differences in the degradation rates between the two domains in mammalian 28S rRNA were highly proportional to increasing PMI with a significant linear correlation observed in mice as well as human autopsy tissues. In conclusion, we demonstrate that comparison of the degradation rates between domains of a single RNA species provides quantitative information on postmortem degradation states, which can be applied for the estimation of PMI.

  19. Regioselectivity in the Reductive Bond Cleavage of Diarylalkylsulfonium Salts

    Kampmeier, Jack; Mansurul Hoque, AKM; D. Saeva, Franklin

    2009-01-01

    products vary from regiospecific alkyl cleavage to predominant aryl cleavage as a function of the potential of the reducing agent. We conclude that differences between the reductive cleavages of mono- and diarylsulfonium salts are direct consequences of the structures of the sulfuranyl radical......- tolylethylsulfonium and di-4-tolyl-2-phenylethylsulfonium salts by a variety of one-electron reducing agents ranging in potential from -0.77 to +2.5 eV (vs SCE) and including thermal reductants, indirect electrolyses mediated by a series of cyanoaromatics, and excited singlet states. We report that the cleavage...... intermediates and the bond dissociation energies of the alkyl and aryl bonds. Competitions between the rates of cleavage and oxidation of the intermediate sulfuranyl radicals and between concerted and stepwise mechanisms are discussed to explain the variations in bond cleavage products as a function...

  20. Analysis of safety culture components based on site interviews

    Ueno, Akira; Nagano, Yuko; Matsuura, Shojiro

    2002-01-01

    Safety culture of an organization is influenced by many factors such as employee's moral, safety policy of top management and questioning attitude among site staff. First this paper analyzes key factors of safety culture on the basis of site interviews. Then the paper presents a safety culture composite model and its applicability in various contexts. (author)

  1. Site and stand analysis for growth prediction of Eucalyptus grandis ...

    The integration of site information with that of tree growth is of special importance in Zululand, where sustainable supply of timber is essential for local processing and export commitments. Site prediction growth models need to be based on easily attainable input variables that are suitable for operational implementation by ...

  2. Solid waste dumping site suitability analysis using geographic ...

    Solid waste dumping is a serious problem in the urban areas because most solid wastes are not dumped in the suitable areas. Bahir Dar Town has the problem of solid waste dumping site identification. The main objective of this study was to select potential areas for suitable solid waste dumping sites for Bahir Dar Town, ...

  3. Wind resource assessment and siting analysis in Italy

    Ricci, A.; Mizzoni, G.; Rossi, E.

    1992-01-01

    Recently, the wind power industry has matured; consequently, in many countries a lot of wind energy applications have been programmed. Many of them are already realized and running. As such, there is a direct necessity to identify a sizeable number of wind power plant sites. Choosing the right sites to match specific Wind Energy Conversion Systems (WECS) is also needed to harness this clean energy from the points of view of industrial viability and project financing. As a pre-requisite to install a wind turbine at a particular site, it is necessary to have knowledge of the theoretical available wind energy at the site, as well as, of the practicability of the design in matching the characteristics of the WECS. In this paper, ENEA (Italian National Agency for New Technology, Energy and Environment) wind siting and resource assessment activities, currently on-going in different regions in Italy, along with the present status and future prospects of the wind power industry

  4. Geoscientific long-term prognosis. Preliminary safety analysis for the site Gorleben

    Mrugalla, Sabine

    2011-07-01

    The preliminary safety analysis of the site Gorleben includes the following chapters: (1) Introduction; (2) Aim and content of the geoscientific long-term prognosis for the site Gorleben; (3) Boundary conditions at the site Gorleben: climate; geomorphology; overlying rocks and adjoining rocks; hydrogeology; salt deposit Gorleben. (4) Probable future geological developments at the site Gorleben: supraregional developments with effects on the site Gorleben; glacial period developments; developments of the geomorphology, overlying and adjoining rocks; future developments of the hydrological systems at the site Gorleben; future saliniferous specific developments of the salt deposit Gorleben. (5) Commentary on the unlikely or excludable developments of the site Gorleben.

  5. Analysis of the Monitoring Network at the Salmon, Mississippi, Site

    None

    2013-08-01

    The Salmon site in southern Mississippi was the location of two underground nuclear tests and two methane-oxygen gas explosion tests conducted in the Tatum Salt Dome at a depth of 2,715 feet below ground surface. The U.S. Atomic Energy Commission (a predecessor agency of the U.S. Department of Energy [DOE]) and the U.S. Department of Defense jointly conducted the tests between 1964 and 1970. The testing operations resulted in surface contamination at multiple locations on the site and contamination of shallow aquifers. No radionuclides from the nuclear tests were released to the surface or to groundwater, although radionuclide-contaminated drill cuttings were brought to the surface during re-entry drilling. Drilling operations generated the largest single volume of waste materials, including radionuclide-contaminated drill cuttings and drilling fluids. Nonradioactive wastes were also generated as part of the testing operations. Site cleanup and decommissioning began in 1971 and officially ended in 1972. DOE conducted additional site characterization between 1992 and 1999. The historical investigations have provided a reasonable understanding of current surface and shallow subsurface conditions at the site, although some additional investigation is desirable. For example, additional hydrologic data would improve confidence in assigning groundwater gradients and flow directions in the aquifers. The U.S. Environmental Protection Agency monitored groundwater at the site as part of its Long-Term Hydrologic Monitoring Program from 1972 through 2007, when DOE's Office of Legacy Management (LM) assumed responsibility for site monitoring. The current monitoring network consists of 28 monitoring wells and 11 surface water locations. Multiple aquifers which underlie the site are monitored. The current analyte list includes metals, radionuclides, and volatile organic compounds (VOCs).

  6. Restriction enzyme cleavage of ultraviolet-damaged Simian virus 40 and pBR322 DNA

    Cleaver, J.E.

    1983-01-01

    Cleavage of specific DNA sequences by the restriction enzymes EcoRI, HindIII and TaqI was prevented when the DNA was irradiated with ultraviolet light. Most of the effects were attributed to cyclobutane pyrimidine dimers in the recognition sequences; the effectiveness of irradiation was directly proportional to the number of potential dimer sites in the DNA. Combining EcoRI with dimer-specific endonuclease digestion revealed that pyrimidine dimers blocked cleavage within one base-pair on the strand opposite to the dimer but did not block cleavage three to four base-pairs away on the same strand. These are the probable limits for the range of influence of pyrimidine dimers along the DNA, at least for this enzyme. The effect of irradiation on cleavage by TaqI seemed far greater than expected for the cyclobutane dimer yield, possibly because of effects from photoproducts flanking the tetranucleotide recognition sequence and the effect of non-cyclobutane (6-4)pyrimidine photoproducts involving adjacent T and C bases. (author)

  7. 40 CFR 1400.5 - Internet access to certain off-site consequence analysis data elements.

    2010-07-01

    ... consequence analysis data elements. 1400.5 Section 1400.5 Protection of Environment ENVIRONMENTAL PROTECTION... UNDER THE CLEAN AIR ACT SECTION 112(r)(7); DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION Public Access § 1400.5 Internet access to certain off...

  8. 40 CFR 60.1125 - What must I include in my siting analysis?

    2010-07-01

    ... 40 Protection of Environment 6 2010-07-01 2010-07-01 false What must I include in my siting... § 60.1125 What must I include in my siting analysis? (a) Include an analysis of how your municipal...) Vegetation. (b) Include an analysis of alternatives for controlling air pollution that minimize potential...

  9. Beta-scission of alkoxyl radicals on peptides and proteins can give rise to backbone cleavage and loss of side-chains

    Headlam, H.A.; Davies, M.J.; Mortimer, A.; Easton, C.J.

    2000-01-01

    reaction, and loss of the side-chain as formaldehyde. The latter material has been quantified by product analysis. Experiments with other hydroperoxides and nitrate esters suggest that this type of reaction is a common process with many C-3 alkoxyl radicals, with the driving force for sidechain loss being the stability of the resultant α-carbon radicals; these species give rise to subsequent backbone cleavage. This pathway provides a novel mechanism by which initial reaction at side-chain sites can give rise to both backbone cleavage, and loss of side-chain groups on peptides and proteins. These reactions are also chain propagation steps as they result in damage to one amino acid and the release of a second, potentially damaging species. The significance and quantification of this pathway, compared to other routes that lead to backbone cleavage, are currently being explored

  10. Determinants of the VP1/2A junction cleavage by the 3C protease in foot-and-mouth disease virus-infected cells.

    Kristensen, Thea; Normann, Preben; Gullberg, Maria; Fahnøe, Ulrik; Polacek, Charlotta; Rasmussen, Thomas Bruun; Belsham, Graham J

    2017-03-01

    The foot-and-mouth disease virus (FMDV) capsid precursor, P1-2A, is cleaved by FMDV 3C protease to yield VP0, VP3, VP1 and 2A. Cleavage of the VP1/2A junction is the slowest. Serotype O FMDVs with uncleaved VP1-2A (having a K210E substitution in VP1; at position P2 in cleavage site) have been described previously and acquired a second site substitution (VP1 E83K) during virus rescue. Furthermore, introduction of the VP1 E83K substitution alone generated a second site change at the VP1/2A junction (2A L2P, position P2' in cleavage site). These virus adaptations have now been analysed using next-generation sequencing to determine sub-consensus level changes in the virus; this revealed other variants within the E83K mutant virus population that changed residue VP1 K210. The construction of serotype A viruses with a blocked VP1/2A cleavage site (containing K210E) has now been achieved. A collection of alternative amino acid substitutions was made at this site, and the properties of the mutant viruses were determined. Only the presence of a positively charged residue at position P2 in the cleavage site permitted efficient cleavage of the VP1/2A junction, consistent with analyses of diverse FMDV genome sequences. Interestingly, in contrast to the serotype O virus results, no second site mutations occurred within the VP1 coding region of serotype A viruses with the blocked VP1/2A cleavage site. However, some of these viruses acquired changes in the 2C protein that is involved in enterovirus morphogenesis. These results have implications for the testing of potential antiviral agents targeting the FMDV 3C protease.

  11. Improving Sampling, Analysis, and Data Management for Site Investigation and Cleanup

    The United States Environmental Protection Agency (EPA) supports the adoption of streamlined approaches to sampling, analysis, and data management activities conducted during site assessment, characterization, and cleanup.

  12. Modelling of ductile and cleavage fracture by local approach

    Samal, M.K.; Dutta, B.K.; Kushwaha, H.S.

    2000-08-01

    This report describes the modelling of ductile and cleavage fracture processes by local approach. It is now well known that the conventional fracture mechanics method based on single parameter criteria is not adequate to model the fracture processes. It is because of the existence of effect of size and geometry of flaw, loading type and rate on the fracture resistance behaviour of any structure. Hence, it is questionable to use same fracture resistance curves as determined from standard tests in the analysis of real life components because of existence of all the above effects. So, there is need to have a method in which the parameters used for the analysis will be true material properties, i.e. independent of geometry and size. One of the solutions to the above problem is the use of local approaches. These approaches have been extensively studied and applied to different materials (including SA33 Gr.6) in this report. Each method has been studied and reported in a separate section. This report has been divided into five sections. Section-I gives a brief review of the fundamentals of fracture process. Section-II deals with modelling of ductile fracture by locally uncoupled type of models. In this section, the critical cavity growth parameters of the different models have been determined for the primary heat transport (PHT) piping material of Indian pressurised heavy water reactor (PHWR). A comparative study has been done among different models. The dependency of the critical parameters on stress triaxiality factor has also been studied. It is observed that Rice and Tracey's model is the most suitable one. But, its parameters are not fully independent of triaxiality factor. For this purpose, a modification to Rice and Tracery's model is suggested in Section-III. Section-IV deals with modelling of ductile fracture process by locally coupled type of models. Section-V deals with the modelling of cleavage fracture process by Beremins model, which is based on Weibulls

  13. Analysis and recognition of 5 ' UTR intron splice sites in human pre-mRNA

    Eden, E.; Brunak, Søren

    2004-01-01

    Prediction of splice sites in non-coding regions of genes is one of the most challenging aspects of gene structure recognition. We perform a rigorous analysis of such splice sites embedded in human 5' untranslated regions (UTRs), and investigate correlations between this class of splice sites and...

  14. Management of radiation contaminated sites: public policy analysis

    Massuelle, M.H.; Brenot, J.; Hubert, P.

    1999-01-01

    The present document is interested in the management of radio-contaminated sites whom pollution comes essentially from some leftover old controlled practices and more precisely from mining activities relative to uranium and radium and thorium industries. The contaminations coming from atmospheric nuclear explosions and post accidental situations do not come into the field of this document. The management of contaminated sites is tackled in the aspect of radiation protection of populations living near or that could be there in the future. (N.C.)

  15. Full Length Research Paper Curcumin induces cleavage of -catenin ...

    β-Catenin/Tcf-4 signaling pathway plays important roles in colorectal tumorigenesis. RT-PCR, western blotting and immunoprecipitation were used to study the effects of curcumin on β-catenin/Tcf-4 signaling pathway in HT-29 cells. Treatment of curcumin could induce cleavage of β-catenin and the cleavage could be ...

  16. Modeling and inferring cleavage patterns in proliferating epithelia.

    Ankit B Patel

    2009-06-01

    Full Text Available The regulation of cleavage plane orientation is one of the key mechanisms driving epithelial morphogenesis. Still, many aspects of the relationship between local cleavage patterns and tissue-level properties remain poorly understood. Here we develop a topological model that simulates the dynamics of a 2D proliferating epithelium from generation to generation, enabling the exploration of a wide variety of biologically plausible cleavage patterns. We investigate a spectrum of models that incorporate the spatial impact of neighboring cells and the temporal influence of parent cells on the choice of cleavage plane. Our findings show that cleavage patterns generate "signature" equilibrium distributions of polygonal cell shapes. These signatures enable the inference of local cleavage parameters such as neighbor impact, maternal influence, and division symmetry from global observations of the distribution of cell shape. Applying these insights to the proliferating epithelia of five diverse organisms, we find that strong division symmetry and moderate neighbor/maternal influence are required to reproduce the predominance of hexagonal cells and low variability in cell shape seen empirically. Furthermore, we present two distinct cleavage pattern models, one stochastic and one deterministic, that can reproduce the empirical distribution of cell shapes. Although the proliferating epithelia of the five diverse organisms show a highly conserved cell shape distribution, there are multiple plausible cleavage patterns that can generate this distribution, and experimental evidence suggests that indeed plants and fruitflies use distinct division mechanisms.

  17. Pro-eating disorder communities on social networking sites: a content analysis.

    Juarascio, Adrienne S; Shoaib, Amber; Timko, C Alix

    2010-01-01

    The purpose of this study was to assess the number of pro-ana groups on social networking sites and to analyze their content. A general inductive approach was used to analyze the content. Two main themes emerged from the content analysis: social support and eating disorder specific content. Themes were similar across all groups; however, a linguistic analysis indicated differences between groups on the two different networking sites. There was an absence of content typically found on Internet sites. Pro-ana groups on social networking sites are focused on social interactions, and lack eating disorder specific content found on Internet sites.

  18. Marketing Research of Construction Sites based on ABC-XYZ Analysis and Relational Data

    Konikov Aleksandr

    2017-01-01

    Full Text Available ABC-XYZ analysis is well known in marketing. It allows identifying sites that yield maximum profits when sold, sites that enjoy stable demand, or sites have both qualities specified above. However, the methods are quite abstract and are not designed to study specific factors that impact the results of ABC-XYZ analysis. Meanwhile, for some applications, particularly for marketing research of construction sites, it is critical not only to identify high-profit and stable sites but also to find out what combination of technical parameters, factors related to their location, transport accessibility, etc. are typical of them. This work suggests an approach to address the issue.

  19. Systematic Analysis of Splice-Site-Creating Mutations in Cancer

    Reyka G. Jayasinghe

    2018-04-01

    Full Text Available Summary: For the past decade, cancer genomic studies have focused on mutations leading to splice-site disruption, overlooking those having splice-creating potential. Here, we applied a bioinformatic tool, MiSplice, for the large-scale discovery of splice-site-creating mutations (SCMs across 8,656 TCGA tumors. We report 1,964 originally mis-annotated mutations having clear evidence of creating alternative splice junctions. TP53 and GATA3 have 26 and 18 SCMs, respectively, and ATRX has 5 from lower-grade gliomas. Mutations in 11 genes, including PARP1, BRCA1, and BAP1, were experimentally validated for splice-site-creating function. Notably, we found that neoantigens induced by SCMs are likely several folds more immunogenic compared to missense mutations, exemplified by the recurrent GATA3 SCM. Further, high expression of PD-1 and PD-L1 was observed in tumors with SCMs, suggesting candidates for immune blockade therapy. Our work highlights the importance of integrating DNA and RNA data for understanding the functional and the clinical implications of mutations in human diseases. : Jayasinghe et al. identify nearly 2,000 splice-site-creating mutations (SCMs from over 8,000 tumor samples across 33 cancer types. They provide a more accurate interpretation of previously mis-annotated mutations, highlighting the importance of integrating data types to understand the functional and the clinical implications of splicing mutations in human disease. Keywords: splicing, RNA, mutations of clinical relevance

  20. Critical cleavage fracture stress characterization of A508 nuclear pressure vessel steels

    Wu, Sujun; Jin, Huijin; Sun, Yanbin; Cao, Luowei

    2014-01-01

    The critical cleavage fracture stress of SA508 Gr.4N and SA508 Gr.3 low alloy reactor pressure vessel (RPV) steels was studied through the combination of experiments and finite element method (FEM) analysis. The results showed that the value of the local cleavage fracture stress, σ F , of SA508 Gr.4N steel was significantly higher than that of SA508 Gr.3 steel. Detailed microstructural analysis was carried out using FEGSEM which revealed much smaller grains, finer and more homogenous carbide particles formed in SA508 Gr.4N steel. Compared with the SA508 Gr.3 steel currently used in the nuclear industry, the SA508 Gr.4N steel possesses higher strength and notch toughness as well as improved cleavage fracture behavior, and is considered a better candidate RPV steel for the next generation nuclear reactors. - Highlights: • Critical cleavage fracture stress was calculated through experiments and FEM. • Effects of both grain and carbide particle sizes on σ F were discussed. • The SA508 Gr.4N steel is a better candidate for the next generation nuclear reactors

  1. The Generation of Dehydroalanine Residues in Protonated Polypeptides: Ion/Ion Reactions for Introducing Selective Cleavages

    Peng, Zhou; Bu, Jiexun; McLuckey, Scott A.

    2017-09-01

    We examine a gas-phase approach for converting a subset of amino acid residues in polypeptide cations to dehydroalanine (Dha). Subsequent activation of the modified polypeptide ions gives rise to specific cleavage N-terminal to the Dha residue. This process allows for the incorporation of selective cleavages in the structural characterization of polypeptide ions. An ion/ion reaction within the mass spectrometer between a multiply protonated polypeptide and the sulfate radical anion introduces a radical site into the multiply protonated polypeptide reactant. Subsequent collisional activation of the polypeptide radical cation gives rise to radical side chain loss from one of several particular amino acid side chains (e.g., leucine, asparagine, lysine, glutamine, and glutamic acid) to yield a Dha residue. The Dha residues facilitate preferential backbone cleavages to produce signature c- and z-ions, demonstrated with cations derived from melittin, mechano growth factor (MGF), and ubiquitin. The efficiencies for radical side chain loss and for subsequent generation of specific c- and z-ions have been examined as functions of precursor ion charge state and activation conditions using cations of ubiquitin as a model for a small protein. It is noted that these efficiencies are not strongly dependent on ion trap collisional activation conditions but are sensitive to precursor ion charge state. Moderate to low charge states show the greatest overall yields for the specific Dha cleavages, whereas small molecule losses (e.g., water/ammonia) dominate at the lowest charge states and proton catalyzed amide bond cleavages that give rise to b- and y-ions tend to dominate at high charge states. [Figure not available: see fulltext.

  2. Ultrarapid mutation detection by multiplex, solid-phase chemical cleavage

    Rowley, G.; Saad, S.; Giannelli, F.; Green, P.M. [Guy`s & St. Thomas`s Hospitals, London (United Kingdom)

    1995-12-10

    The chemical cleavage of mismatches in heteroduplexes formed by probe and test DNA detects and locates any sequence change in long DNA segments ({approximately}1.8 kb), and its efficiency has been well tested in the analysis of both average (e.g., coagulation factor IX) and large, complex genes (e.g., coagulation factor VIII and dystrophin). In the latter application RT/PCR products allow the examination of all essential sequences of the gene in a minimum number of reactions. We use two specific chemical reactants (hydroxylamine and osmium tetroxide) and piperidine cleavage of the above procedure to develop a very fast mutation screening method. This is based on: (1) 5{prime} or internal fluorescent labeling to allow concurrent screening of three to four DNA fragments and (2) solid-phase chemistry to use a microliter format and reduce the time required for the procedure, from amplification of sequence to gel loading inclusive, to one person-working-day. We test the two variations of the method, one entailing 5{prime} labeling of probe DNA and the other uniform labeling of both probe and target DNA, by detecting 114 known hemophilia B (coagulation factor IX) mutations and by analyzing 129 new patients. Uniform labeling of both probe and target DNA prior to formation of the heteroduplexes leads to almost twofold redundancy in the ability to detect mutations. Alternatively, the latter procedure may offer very efficient though less than 100% screening for sequence changes with only hydroxylamine. The full method with two chemical reactions (hydroxylamine and osmium tetroxide) should allow one person to screen with virtually 100% accuracy more than 300 kb of sequence in three ABI 373 gels in 1 day. 26 refs., 7 figs., 1 tab.

  3. Micromechanisms and toughness for cleavage fracture of steel

    Rosenfield, A.R.; Majumdar, B.S.

    1987-01-01

    A complete understanding of the fracture mechanisms of steel in the ductile/brittle transition region requires analysis not only of crack initiation, but also of crack propagation. This paper reviews micrographic and fractographic experiments that give insight into both phenomena, and suggests a frame-work through which both may be related. Unstable cleavage crack initiation can occur after some blunting of the original fatigue precrack or after some stable crack growth. In either event, instability appears to be triggered by the fracture of a brittle micro-constituent ahead of the precrack. The large scatter in reported K IC values within the transition region reflects the size distribution and relative scarcity of these 'trigger' particles. While a large number of models have attempted to correlate toughness in the ductile/brittle transition regime to events occurring ahead of the crack tip, surprisingly little attention has been paid to events occurring behind the crack front. Fractographic evidence as well as metallographic sectioning of arrested cracks show that the mechanism of rapid crack propagation by cleavage is affected strongly by partial crack-plane deflection which leaves unbroken ligaments in its wake. The tearing of these ligaments by dimple-rupture is the dominant energy-absorbing mechanism. Etch-pit experiments using an Fe-Si alloy show that the crack-tip stress intensity based on plastic zone size is extremely low. It is suggested that the mechanism of crack arrest should be modeled using a sharp crack which is restrained by a distribution of discrete pinching forces along its faces. The same model is applied to crack initiation. (orig.)

  4. 40 CFR 1400.9 - Access to off-site consequence analysis information by State and local government officials.

    2010-07-01

    ... 40 Protection of Environment 32 2010-07-01 2010-07-01 false Access to off-site consequence... CONSEQUENCE ANALYSIS INFORMATION DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION Access to Off-Site Consequence Analysis Information by Government Officials. § 1400.9 Access to off-site consequence analysis...

  5. Multiattribute utility analysis of alternative sites for the disposal of nuclear waste

    Merkhofer, M.W.; Keeney, R.L.

    1987-01-01

    Five potential sites nominated for the Nation's first geologic repository for disposing of nuclear waste are evaluated using multiattribute utility analysis. The analysis was designed to aid the Department of Energy in its selection of 3 sites for characterization, a detailed data-gathering process that will involve the construction of exploratory shafts for underground testing and that may cost as much as $1 billion per site. The analysis produced insights into the relative advantages and disadvantages of the nominated sites and clarified current uncertainties regarding repository performance

  6. Critical Radionuclide and Pathway Analysis for the Savannah River Site, 2016 Update

    Jannik, Tim [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL); Hartman, Larry [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL)

    2016-09-08

    During the operational history of Savannah River Site, many different radionuclides have been released from site facilities. However, as shown in this analysis, only a relatively small number of the released radionuclides have been significant contributors to doses to the offsite public. This report is an update to the 2011 analysis, Critical Radionuclide and Pathway Analysis for the Savannah River Site. SRS-based Performance Assessments for E-Area, Saltstone, F-Tank Farm, H-Tank Farm, and a Comprehensive SRS Composite Analysis have been completed. The critical radionuclides and pathways identified in those extensive reports are also detailed and included in this analysis.

  7. Risk analysis and solving the nuclear waste siting problem

    Inhaber, H.

    1994-01-01

    In spite of millions of dollars and countless human resources being expended on finding nuclear wastes sites, the search has proved extremely difficult for the nuclear industry. This may be due to the approach followed, rather than inadequacies in research or funding. A new approach to the problem, the reverse Dutch auction, is suggested. It retains some of the useful elements of the present system, but it also adds new ones. It allows natural market forces to set the level of compensation, rather than relying on close-door negotiations or theoretical calculations. Two flow charts show the pre-bid and post-bid steps of the reverse Duch auction system of Inhaber. It is assumed that a state wishes to site a waste facility somewhere in its boundaries. 22 refs., 3 figs

  8. Synthesis, characterization, anti-microbial, DNA binding and cleavage studies of Schiff base metal complexes

    Poomalai Jayaseelan

    2016-09-01

    Full Text Available A novel Schiff base ligand has been prepared by the condensation between butanedione monoxime with 3,3′-diaminobenzidine. The ligand and metal complexes have been characterized by elemental analysis, UV, IR, 1H NMR, conductivity measurements, EPR and magnetic studies. The molar conductance studies of Cu(II, Ni(II, Co(II and Mn(II complexes showed non-electrolyte in nature. The ligand acts as dibasic with two N4-tetradentate sites and can coordinate with two metal ions to form binuclear complexes. The spectroscopic data of metal complexes indicated that the metal ions are complexed with azomethine nitrogen and oxyimino nitrogen atoms. The binuclear metal complexes exhibit octahedral arrangements. DNA binding properties of copper(II metal complex have been investigated by electronic absorption spectroscopy. Results suggest that the copper(II complex bind to DNA via an intercalation binding mode. The nucleolytic cleavage activities of the ligand and their complexes were assayed on CT-DNA using gel electrophoresis in the presence and absence of H2O2. The ligand showed increased nuclease activity when administered as copper complex and copper(II complex behave as efficient chemical nucleases with hydrogen peroxide activation. The anti-microbial activities and thermal studies have also been studied. In anti-microbial activity all complexes showed good anti-microbial activity higher than ligand against gram positive, gram negative bacteria and fungi.

  9. Chromosome fragility at FRAXA in human cleavage stage embryos at risk for fragile X syndrome.

    Verdyck, Pieter; Berckmoes, Veerle; De Vos, Anick; Verpoest, Willem; Liebaers, Inge; Bonduelle, Maryse; De Rycke, Martine

    2015-10-01

    Fragile X syndrome (FXS), the most common inherited intellectual disability syndrome, is caused by expansion and hypermethylation of the CGG repeat in the 5' UTR of the FMR1 gene. This expanded repeat, also known as the rare fragile site FRAXA, causes X chromosome fragility in cultured cells from patients but only when induced by perturbing pyrimidine synthesis. We performed preimplantation genetic diagnosis (PGD) on 595 blastomeres biopsied from 442 cleavage stage embryos at risk for FXS using short tandem repeat (STR) markers. In six blastomeres, from five embryos an incomplete haplotype was observed with loss of all alleles telomeric to the CGG repeat. In all five embryos, the incomplete haplotype corresponded to the haplotype carrying the CGG repeat expansion. Subsequent analysis of additional blastomeres from three embryos by array comparative genomic hybridization (aCGH) confirmed the presence of a terminal deletion with a breakpoint close to the CGG repeat in two blastomeres from one embryo. A blastomere from another embryo showed the complementary duplication. We conclude that a CGG repeat expansion at FRAXA causes X chromosome fragility in early human IVF embryos at risk for FXS. © 2015 Wiley Periodicals, Inc.

  10. Risk analysis and solving the nuclear waste siting problem

    Inhaber, H.

    1993-01-01

    In spite of millions of dollars and countless human resources being expended on finding nuclear wastes sites, the search has proved extremely difficult for the nuclear industry. This may be due to the approach followed, rather than inadequacies in research or funding. A new approach to the problem, the reverse Dutch auction, is suggested. It retains some of the useful elements of the present system, but it also adds new ones

  11. Impact analysis and community development needs at the salt site

    Gray, S.; Boryczka, M.; Hines, B.

    1984-01-01

    The Office of Nuclear Waste Isolation (ONWI) has developed a socioeconomic program for a nuclear waste repository constructed in salt. The program is comprised of three elements: impact assessment, impact mitigation, and impact monitoring. The first element, impact assessment, is the focus of ONWI's current activities. Socioeconomic data has been collected for seven salt sites in Texas, Utah, Mississippi and Louisiana. Demographic, economic, community service, governmental and social structure information has been assembled into data base reports for each site area. These socioeconomic reports will be the basis for analyzing community-related impacts. Socioeconomic effects are currently being evaluated for the environmental assessment document required by the Nuclear Waste Policy Act. The approach to evaluating socioeconomic impacts for the environmental assessment impact includes developing the data base necessary for evaluation; assessing impacts of baseline population projected by the states; assessing project-related impacts through the use of an inmigration model and responding to socioeconomic issues raised in public meetings and hearings. The siting, construction, and operation of nuclear repositories will involve an extended period of time and an increased workforce, which can result in some impacts similar to those of other large development projects. The communities affected by a repository site will face increased demands for housing, community services (transportation, sewer and water, schools, etc.) and land, as well as a desire to maintain the community's ''character''. The management of this expansion and other related community impacts should be structured to meet community needs and goals. The management process should include the formation of an impact management comment, a public participation program, and a technical assistance program

  12. Risk analysis and solving the nuclear waste siting problem

    Inhaber, H.

    1993-12-01

    In spite of millions of dollars and countless human resources being expended on finding nuclear wastes sites, the search has proved extremely difficult for the nuclear industry. This may be due to the approach followed, rather than inadequacies in research or funding. A new approach to the problem, the reverse Dutch auction, is suggested. It retains some of the useful elements of the present system, but it also adds new ones.

  13. Systematic Analysis of Splice-Site-Creating Mutations in Cancer.

    Jayasinghe, Reyka G; Cao, Song; Gao, Qingsong; Wendl, Michael C; Vo, Nam Sy; Reynolds, Sheila M; Zhao, Yanyan; Climente-González, Héctor; Chai, Shengjie; Wang, Fang; Varghese, Rajees; Huang, Mo; Liang, Wen-Wei; Wyczalkowski, Matthew A; Sengupta, Sohini; Li, Zhi; Payne, Samuel H; Fenyö, David; Miner, Jeffrey H; Walter, Matthew J; Vincent, Benjamin; Eyras, Eduardo; Chen, Ken; Shmulevich, Ilya; Chen, Feng; Ding, Li

    2018-04-03

    For the past decade, cancer genomic studies have focused on mutations leading to splice-site disruption, overlooking those having splice-creating potential. Here, we applied a bioinformatic tool, MiSplice, for the large-scale discovery of splice-site-creating mutations (SCMs) across 8,656 TCGA tumors. We report 1,964 originally mis-annotated mutations having clear evidence of creating alternative splice junctions. TP53 and GATA3 have 26 and 18 SCMs, respectively, and ATRX has 5 from lower-grade gliomas. Mutations in 11 genes, including PARP1, BRCA1, and BAP1, were experimentally validated for splice-site-creating function. Notably, we found that neoantigens induced by SCMs are likely several folds more immunogenic compared to missense mutations, exemplified by the recurrent GATA3 SCM. Further, high expression of PD-1 and PD-L1 was observed in tumors with SCMs, suggesting candidates for immune blockade therapy. Our work highlights the importance of integrating DNA and RNA data for understanding the functional and the clinical implications of mutations in human diseases. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

  14. PROBABILISTIC RISK ANALYSIS OF REMEDIATION EFFORTS IN NAPL SITES

    Fernandez-Garcia, D.; de Vries, L.; Pool, M.; Sapriza, G.; Sanchez-Vila, X.; Bolster, D.; Tartakovsky, D. M.

    2009-12-01

    The release of non-aqueous phase liquids (NAPLs) such as petroleum hydrocarbons and chlorinated solvents in the subsurface is a severe source of groundwater and vapor contamination. Because these liquids are essentially immiscible due to low solubility, these contaminants get slowly dissolved in groundwater and/or volatilized in the vadoze zone threatening the environment and public health over a long period. Many remediation technologies and strategies have been developed in the last decades for restoring the water quality properties of these contaminated sites. The failure of an on-site treatment technology application is often due to the unnoticed presence of dissolved NAPL entrapped in low permeability areas (heterogeneity) and/or the remaining of substantial amounts of pure phase after remediation efforts. Full understanding of the impact of remediation efforts is complicated due to the role of many interlink physical and biochemical processes taking place through several potential pathways of exposure to multiple receptors in a highly unknown heterogeneous environment. Due to these difficulties, the design of remediation strategies and definition of remediation endpoints have been traditionally determined without quantifying the risk associated with the failure of such efforts. We conduct a probabilistic risk assessment of the likelihood of success of an on-site NAPL treatment technology that easily integrates all aspects of the problem (causes, pathways, and receptors). Thus, the methodology allows combining the probability of failure of a remediation effort due to multiple causes, each one associated to several pathways and receptors.

  15. Analysis of Discussion Board Interaction in an Online Peer Mentoring Site

    Ruane, Regina; Lee, Vera J.

    2016-01-01

    This study uses Critical Discourse Analysis and Social Network Analysis to examine an online peer mentoring site created to unite first-year and third-year preservice teachers enrolled in an undergraduate teacher education program. The peer mentoring site was developed to provide both first-year preservice teachers and more experienced peers the…

  16. Composite Analysis for the Area 5 Radioactive Waste Management Site at the Nevada Test Site, Nye County, Nevada

    V. Yucel

    2001-09-01

    This report summarizes the results of a Composite Analysis (CA) for the Area 5 Radioactive Waste Management Site (RWMS). The Area 5 RWMS is a US Department of Energy (DOE)-operated low-level radioactive waste (LLW) management site located in northern Frenchman Flat on the Nevada Test Site (NTS). The Area 5 RWMS has disposed of low-level radioactive waste in shallow unlined pits and trenches since 1960. Transuranic waste (TRU) and high-specific activity waste was disposed in Greater Confinement Disposal (GCD) boreholes from 1983 to 1989. The purpose of this CA is to determine if continuing operation of the Area 5 RWMS poses an acceptable or unacceptable risk to the public considering the total waste inventory and all other interacting sources of radioactive material in the vicinity. Continuing operation of the Area 5 RWMS will be considered acceptable if the total effective dose equivalent (TEDE) is less than 100 mrem in a year. If the TEDE exceeds 30 mrem in a year, a cost-benefit options analysis must be performed to determine if cost-effective management options exist to reduce the dose further. If the TEDE is found to be less than 30 mrem in a year, an analysis may be performed if warranted to determine if doses are as low as reasonably achievable (ALARA).

  17. Composite Analysis for the Area 5 Radioactive Waste Management Site at the Nevada Test Site, Nye County, Nevada

    V. Yucel

    2001-01-01

    This report summarizes the results of a Composite Analysis (CA) for the Area 5 Radioactive Waste Management Site (RWMS). The Area 5 RWMS is a US Department of Energy (DOE)-operated low-level radioactive waste (LLW) management site located in northern Frenchman Flat on the Nevada Test Site (NTS). The Area 5 RWMS has disposed of low-level radioactive waste in shallow unlined pits and trenches since 1960. Transuranic waste (TRU) and high-specific activity waste was disposed in Greater Confinement Disposal (GCD) boreholes from 1983 to 1989. The purpose of this CA is to determine if continuing operation of the Area 5 RWMS poses an acceptable or unacceptable risk to the public considering the total waste inventory and all other interacting sources of radioactive material in the vicinity. Continuing operation of the Area 5 RWMS will be considered acceptable if the total effective dose equivalent (TEDE) is less than 100 mrem in a year. If the TEDE exceeds 30 mrem in a year, a cost-benefit options analysis must be performed to determine if cost-effective management options exist to reduce the dose further. If the TEDE is found to be less than 30 mrem in a year, an analysis may be performed if warranted to determine if doses are as low as reasonably achievable (ALARA)

  18. Porcine deltacoronavirus nsp5 inhibits interferon-β production through the cleavage of NEMO.

    Zhu, Xinyu; Fang, Liurong; Wang, Dang; Yang, Yuting; Chen, Jiyao; Ye, Xu; Foda, Mohamed Frahat; Xiao, Shaobo

    2017-02-01

    Porcine deltacoronavirus (PDCoV) causes acute enteric disease and mortality in seronegative neonatal piglets. Previously we have demonstrated that PDCoV infection suppresses the production of interferon-beta (IFN-β), while the detailed mechanisms are poorly understood. Here, we demonstrate that nonstructural protein 5 (nsp5) of PDCoV, the 3C-like protease, significantly inhibits Sendai virus (SEV)-induced IFN-β production by targeting the NF-κB essential modulator (NEMO), confirmed by the diminished function of NEMO cleaved by PDCoV. The PDCoV nsp5 cleavage site in the NEMO protein was identified as glutamine 231, and was identical to the porcine epidemic diarrhea virus nsp5 cleavage site, revealing the likelihood of a common target in NEMO for coronaviruses. Furthermore, this cleavage impaired the ability of NEMO to activate the IFN response and downstream signaling. Taken together, our findings reveal PDCoV nsp5 to be a newly identified IFN antagonist and enhance the understanding of immune evasion by deltacoronaviruses. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. Preliminary Safety Analysis of the Gorleben Site: Geological Database - 13300

    Weber, Jan Richard; Mrugalla, Sabine; Dresbach, Christian; Hammer, Joerg

    2013-01-01

    The Gorleben salt dome is 4 km wide and nearly 15 km long. It is composed of different salt rock types of the Zechstein (Upper Permian) series and extends to the Zechstein basis in a depth of more than 3 km. In the course of the salt dome formation the salt was moved several kilometers. During the uplift of the salt the initially plane-bedded strata of the Zechstein series were extensively folded. In this process anhydrite as a competent layer was broken to isolated blocks. In the core of the salt dome the Hauptsalz, which is characterized by a particularly high creeping capacity, forms a homogeneous halite body with a volume of several cubic kilometres. The Hauptsalz contains gaseous and liquid hydrocarbons in separated zones of decimeter to meter dimensions. The overall hydrocarbon content is far below 0.01 %. At the flanks the salt dome consists of salt rocks with lower creeping capacities. Brine reservoirs with fluid volumes in the range of liters to hundreds of cubic meters exist in certain regions of this part of the salt dome. The water content of the Hauptsalz is below 0.02 %. Interconnected pores do not exist in the salt rock outside of fluid bearing or fractured areas, i.e. the salt rock is impermeable. The exploration of the Gorleben site as a potential site for a HLW-repository started in 1979 and is still in progress. To date no scientific findings contest the suitability of the site for a safe HLW-repository. (authors)

  20. Quantification of DNA cleavage specificity in Hi-C experiments.

    Meluzzi, Dario; Arya, Gaurav

    2016-01-08

    Hi-C experiments produce large numbers of DNA sequence read pairs that are typically analyzed to deduce genomewide interactions between arbitrary loci. A key step in these experiments is the cleavage of cross-linked chromatin with a restriction endonuclease. Although this cleavage should happen specifically at the enzyme's recognition sequence, an unknown proportion of cleavage events may involve other sequences, owing to the enzyme's star activity or to random DNA breakage. A quantitative estimation of these non-specific cleavages may enable simulating realistic Hi-C read pairs for validation of downstream analyses, monitoring the reproducibility of experimental conditions and investigating biophysical properties that correlate with DNA cleavage patterns. Here we describe a computational method for analyzing Hi-C read pairs to estimate the fractions of cleavages at different possible targets. The method relies on expressing an observed local target distribution downstream of aligned reads as a linear combination of known conditional local target distributions. We validated this method using Hi-C read pairs obtained by computer simulation. Application of the method to experimental Hi-C datasets from murine cells revealed interesting similarities and differences in patterns of cleavage across the various experiments considered. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  1. Analysis of garnets from the archaeological sites in Slovenia

    Šmit, Ž., E-mail: ziga.smit@fmf.uni-lj.si [Faculty of Mathematics and Physics, University of Ljubljana, Ljubljana (Slovenia); Jožef Stefan Institute, Ljubljana (Slovenia); Fajfar, H. [Jožef Stefan Institute, Ljubljana (Slovenia); Jeršek, M. [Slovenian Museum of National History, Ljubljana (Slovenia); Knific, T. [National Museum of Slovenia, Ljubljana (Slovenia); Lux, J. [Institute for the Protection of Cultural Heritage of Slovenia, Ljubljana (Slovenia)

    2014-06-01

    Garnets (62 individual stones) originating from the Migration Period cemeteries and hilltop settlements in Slovenia were analyzed by the combined PIXE/PIGE method for their chemical composition. Typologically, the analyzed stones may be classified as almandines originating from the sites in India, belonging to types I and II according to Calligaro. A smaller group of pyraldines intermediate between almandines and pyropes was also determined; identified as type III, their source is most likely in Sri Lanka. No garnets from Bohemia (Czech Republic) have been discovered, which may be related to important political changes in the 7th c. AD, induced by Slavic and Avaric migrations.

  2. SOCIAL CLEAVAGES IN THE AMERICAN SOCIETY AS A FACTOR OF 2016 PRESIDENTIAL CAMPAIGN

    P. S. Kanevskiy

    2017-01-01

    Full Text Available Current article is dedicated to analysis of social cleavages in the American elections and the ways they influenced on presidential election in 2016. Originally developed by S. Rokkan and S.M. Lipset, social cleavages became a classic theme for contemporary political sociology. However, despite the fact that the theory has been developing primarily by Americans, it has been rarely used to analyze electoral system in the USA. Traditionally it’s been aimed at European and developing countries where electoral fragmentation is seen more clearly. But recent changes in the American society and the political system demonstrate the emergence of social cleavages that had not been inherent before. The article shows how American electoral space transformed since the 1980s and how it became more fragmented under the influence of social, economic and ideological factors. Elections in 2016 became a watershed for social cleavages that accumulated through time and aggravated even more considering internal crises in the Democratic and more so in the Republican parties. Donald Trump’s victory is an impersonation of the American party system crisis and of the mainstream politicians’ inability to find proper explanation of the changing electorate. Author shows that American society today is polarized even more than many European countries while group identification determines vectors of political change.

  3. DNA cleavage enzymes for treatment of persistent viral infections: Recent advances and the pathway forward

    Weber, Nicholas D., E-mail: nweber@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Department of Laboratory Medicine, University of Washington, Seattle, WA 98195 (United States); Aubert, Martine, E-mail: maubert@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Dang, Chung H., E-mail: cdang@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Stone, Daniel, E-mail: dstone2@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Jerome, Keith R., E-mail: kjerome@fhcrc.org [Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave N, E5-110, Seattle, WA 98109 (United States); Department of Laboratory Medicine, University of Washington, Seattle, WA 98195 (United States); Department of Microbiology, University of Washington, Seattle, WA 98195 (United States)

    2014-04-15

    Treatment for most persistent viral infections consists of palliative drug options rather than curative approaches. This is often because long-lasting viral DNA in infected cells is not affected by current antivirals, providing a source for viral persistence and reactivation. Targeting latent viral DNA itself could therefore provide a basis for novel curative strategies. DNA cleavage enzymes can be used to induce targeted mutagenesis of specific genes, including those of exogenous viruses. Although initial in vitro and even in vivo studies have been carried out using DNA cleavage enzymes targeting various viruses, many questions still remain concerning the feasibility of these strategies as they transition into preclinical research. Here, we review the most recent findings on DNA cleavage enzymes for human viral infections, consider the most relevant animal models for several human viral infections, and address issues regarding safety and enzyme delivery. Results from well-designed in vivo studies will ideally provide answers to the most urgent remaining questions, and allow continued progress toward clinical application. - Highlights: • Recent in vitro and in vivo results for DNA cleavage enzymes targeting persistent viral infections. • Analysis of the best animal models for testing enzymes for HBV, HSV, HIV and HPV. • Challenges facing in vivo delivery of therapeutic enzymes for persistent viral infections. • Safety issues to be addressed with proper animal studies.

  4. Proximity-activated nanoparticles: in vitro performance of specific structural modification by enzymatic cleavage

    Adam Smith, R; Sewell, Sarah L; Giorgio, Todd D

    2008-01-01

    The development and in vitro performance of a modular nanoscale system capable of specific structural modification by enzymatic activity is described in this work. Due to its small physical size and adaptable characteristics, this system has the potential for utilization in targeted delivery systems and biosensing. Nanoparticle probes were synthesized containing two distinct fluorescent species including a quantum dot base particle and fluorescently labeled cleavable peptide substrate. Activity of these probes was monitored by gel electrophoresis with quantitative cleavage measurements made by fluorometric analysis. The model proximity-activated nanoparticles studied here exhibit significant susceptibility to cleavage by matrix metalloprotease-7 (MMP-7) at physiologically relevant concentrations, with nearly complete cleavage of available substrate molecules after 24 hours. This response is specific to MMP-7 enzyme activity, as cleavage is completely inhibited with the addition of EDTA. Utilization of enzyme-specific modification is a sensitive approach with broad applications for targeted therapeutics and biosensing. The versatility of this nanoparticle system is highlighted in its modular design, as it has the capability to integrate characteristics for detection, biosensing, targeting, and payload delivery into a single, multifunctional nanoparticle structure. PMID:18488420

  5. Status report on experiments and modelling of the cleavage fracture behaviour of F82Hmod using local fracture grid. Task TTMS-005

    Riesch-Oppermann, H.; Walter, M.

    2001-09-01

    Within the European Fusion Technology Programme framework, a fracture mechanics description of the material behaviour in the ductile to brittle transition-regime is developed using local fracture criteria. Based on experimental results using axisymmetrically notched and pre-cracked specimens together with a numerical stress analysis at fracture load, a statistical evaluation of cleavage fracture parameters can be performed along the lines described in various code schemes such as the British Energy R6-Code or the ESIS P6 procedure. The report contains results of the experimental characterization of the deformation and fracture behaviour of the fusion candidate RAFM steel variant F82Hmod, details and background of the numerical procedure for cleavage fracture parameter determination as well as additional statistical inference methods for transferability analysis. Fractographic results give important information about fracture mode and fracture origin sites and their location. Numerical prediction of fracture origin distribution is an important tool for transferability assessment. Future issues comprise constraint effect and ductile damage as well as incorporation of irradiation effects, which are topically addressed. The methodology developed and described in the present report will be applied to characterize material behaviour of future RAFM variants as the EUROFER 97, for which analysis is currently under way. (orig.)

  6. Bouguer density analysis using nettleton method at Banten NPP site

    Yuliastuti; Hadi Suntoko; Yarianto SBS

    2017-01-01

    Sub-surface information become crucial in determining a feasible NPP site that safe from external hazards. Gravity survey which result as density information, is essential to understand the sub-surface structure. Nevertheless, overcorrected or under corrected will lead to a false interpretation. Therefore, density correction in term of near-surface average density or Bouguer density is necessary to be calculated. The objective of this paper is to estimate and analyze Bouguer density using Nettleton method at Banten NPP Site. Methodology used in this paper is Nettleton method that applied in three different slices (A-B, A-C and A-D) with density assumption range between 1700 and 3300 kg/m"3. Nettleton method is based on minimum correlation between gravity anomaly and topography to determine density correction. The result shows that slice A-B which covers rough topography difference, Nettleton method failed. While using the other two slices, Nettleton method yield with a different density value, 2700 kg/m"3 for A-C and 2300 kg/m"3 for A-D. A-C provides the lowest correlation value which represents the Upper Banten tuff and Gede Mt. volcanic rocks in accordance with Quartenary rocks exist in the studied area. (author)

  7. 40 CFR 1400.8 - Access to off-site consequence analysis information by Federal government officials.

    2010-07-01

    ... 40 Protection of Environment 32 2010-07-01 2010-07-01 false Access to off-site consequence... MANAGEMENT PROGRAMS UNDER THE CLEAN AIR ACT SECTION 112(r)(7); DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION Access to Off-Site Consequence Analysis...

  8. Cleavage/alteration of interleukin-8 by matrix metalloproteinase-9 in the female lower genital tract.

    Zariffard, M Reza; Anastos, Kathryn; French, Audrey L; Munyazesa, Elisaphane; Cohen, Mardge; Landay, Alan L; Spear, Gregory T

    2015-01-01

    Interleukin-8 (IL-8, CXCL8) plays important roles in immune responses at mucosal sites including in the lower genital tract. Since several types of bacteria produce proteases that cleave IL-8 and many types of bacteria can be present in lower genital tract microbiota, we assessed genital fluids for IL-8 cleavage/alteration. Genital fluids collected by lavage from 200 women (23 HIV-seronegative and 177 HIV-seropositive) were tested for IL-8 cleavage/alteration by ELISA. IL-8 cleaving/altering activity was observed in fluids from both HIV-positive (28%) and HIV-negative women (35%). There was no clear relationship between the activity and the types of bacteria present in the lower genital tract as determined by high-throughput sequencing of the 16S rRNA gene. Protease inhibitors specific for matrix metalloproteinases (MMPs) reduced the activity and a multiplex assay that detects both inactive and active MMPs showed the presence of multiple MMPs, including MMP-1, -3, -7, -8, -9, -10 and -12 in genital secretions from many of the women. The IL-8-cleaving/altering activity significantly correlated with active MMP-9 as well as with cleavage of a substrate that is acted on by several active MMPs. These studies show that multiple MMPs are present in the genital tract of women and strongly suggest that MMP-9 in genital secretions can cleave IL-8 at this mucosal site. These studies suggest that MMP-mediated cleavage of IL-8 can modulate inflammatory responses in the lower genital tract.

  9. Mutational analysis of Kex2 recognition sites and a disulfide bond in tannase from Aspergillus oryzae.

    Koseki, Takuya; Otsuka, Motohiro; Mizuno, Toshiyuki; Shiono, Yoshihito

    2017-01-22

    Aspergillus oryzae tannase (AoTanA), which contains two Kex2 recognition sites at positions Arg311 and Arg316, consists of two subunits that are generated by the cleavage of tannase gene product by the Kex2 protease. Based on the crystal structure of feruloyl esterase from Aspergillus oryzae (AoFaeB), which has been classified as a member of the fungal tannase family, the catalytic triad residues of AoTanA are predicted to be Ser195, Asp455, and His501, with the serine and histidine residues brought together by a disulfide bond of the neighboring cysteines, Cys194 and Cys502. In this study, we investigated the functional role of the Kex2 recognition sites and disulfide bond between the neighboring cysteines in AoTanA. We constructed a double variant (R311A/R316A), a seven amino-acid deletion variant of region Lys310-Arg316 (ΔKR), and two single variants (C194A and C502A). While the R311A/R316A variant exhibited the two bands similar to wild type by SDS-PAGE after treatment with endoglycosidase H, the ΔKR variant exhibited only one band. R311A/R316A variation had no effect on tannase activity and stability. Meanwhile, the ΔKR variant exhibited higher activity compared to the wild-type. The activities of the C194A and C502A variants decreased considerably (<0.24% of the wild-type) toward methyl gallate. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. A Subset of Membrane-Altering Agents and γ-Secretase Modulators Provoke Nonsubstrate Cleavage by Rhomboid Proteases

    Siniša Urban

    2014-09-01

    Full Text Available Rhomboid proteases are integral membrane enzymes that regulate cell signaling, adhesion, and organelle homeostasis pathways, making substrate specificity a key feature of their function. Interestingly, we found that perturbing the membrane pharmacologically in living cells had little effect on substrate processing but induced inappropriate cleavage of nonsubstrates by rhomboid proteases. A subclass of drugs known to modulate γ-secretase activity acted on the membrane directly and induced nonsubstrate cleavage by rhomboid proteases but left true substrate cleavage sites unaltered. These observations highlight an active role for the membrane in guiding rhomboid selectivity and caution that membrane-targeted drugs should be evaluated for cross-activity against membrane-resident enzymes that are otherwise unrelated to the intended drug target. Furthermore, some γ-secretase-modulating activity or toxicity could partly result from global membrane effects.

  11. Biodiversity Analysis of Vegetation on the Nevada Test Site

    W. K. Ostler; D. J. Hansen

    2001-01-01

    The Nevada Test Site (NTS) located in south central Nevada encompasses approximately 3,561 square kilometers and straddles two major North American deserts, Mojave and Great Basin. Transitional areas between the two desert types have been created by gradients in elevation, precipitation, temperature, and soils. From 1996-1998, more than 1,500 ecological landform units were sampled at the NTS for numerous biotic and abiotic parameters. These data provide a basis for spatial evaluations of biodiversity over landscape scales at the NTS. Species diversity maps (species richness vs. species abundance) have been produced. Differences in ecosystem diversity at the ecoregion, alliance, association, and ecological landform unit levels are presented. Spatial distribution maps of species presence and abundance provide evidence of where transition zones occur and the resulting impact on biodiversity. The influences of abiotic factors (elevation, soil, precipitation) and anthropogenic disturbance on biodiversity are assessed

  12. Biodiversity analysis of vegetation on the Nevada Test Site

    Ostler, W. K.; Hansen, D. J.

    2000-01-01

    The Nevada Test Site (NTS), located in south-central Nevada, encompasses approximately 3,500 square kilometers and straddles two major North American deserts, Mojave and Great Basin. Transitional areas between the two desert types have been created by gradients in elevation, precipitation, temperature, and soils. From 1996 to 1998, more than 1,500 ecological landform units were sampled at the NTS for numerous biotic and abiotic parameters. The data provide a basis for spatial evaluations of biodiversity over landscape scales at the NTS. Biodiversity maps (species richness vs. species abundance) have been produced. Differences in biodiversity among ecoregions and vegetation alliances are presented. Spatial distribution maps of species' presence and abundance provide evidence of where transition zones occur and the resulting impact on biodiversity. The influences of abiotic factors, such as elevation, soil, and precipitation, on biodiversity are assessed

  13. Analysis for the siting of a repository in Argentina

    Palacios, E.; Ciallella, N.; Matar, J.; Perucca, J.C.

    1984-02-01

    The first step in the selection of suitable geological formation for disposal of highly radioactive waste in Argentina was to analyse all the country's known occurrences of granite rock, on the basis of bibliographic material. For each occurrence, a determination was made of its petrographic and structural characteristics, the dimensions and specifications of the rock bodies, the seismic and hydrogeological characteristics of the region, its mining and oil-bearing potential, and the demographic, agricultural, cattle-breeding and industrial features of the areas under study. In this way, it was possible to identify something like 200 granite occurrences. In the second stage the following elimination criteria having been applied: (a) bodies located within seismic zones or areas in which mining or oil-drilling operations are carried on; (b) petrographic characteristics of rock indicating significant alterations, such as an advanced state of erosion or decomposition; and (c) bodies located in areas whose hydrogeological characteristics are known to be unfavourable. As a further selection criterion, account was taken of certain unfavourable characteristics from the point of view of the construction and operation of the storage facility. Sites within zones of dense population, tourism or difficult access were considered to be unfavourable. As a result of this second stage, seven formations were identified. A third stage, based on a survey of the preselected granite bodies led to the identificaton of the massifs of La Esperanza and Chasico in Rio Negro province and the Calcatapul and Medio ranges in Chubut province as the most suitable sites for continuing the detailed studies

  14. Analysis of cavern stability at the West Hackberry SPR site.

    Ehgartner, Brian L.; Sobolik, Steven Ronald

    2009-05-01

    This report presents computational analyses that simulate the structural response of caverns at the Strategic Petroleum Reserve (SPR) West Hackberry site. The cavern field comprises 22 caverns. Five caverns (6, 7, 8, 9, 11) were acquired from industry and have unusual shapes and a history dating back to 1946. The other 17 caverns (101-117) were leached according to SPR standards in the mid-1980s and have tall cylindrical shapes. The history of the caverns and their shapes are simulated in a three-dimensional geomechanics model of the site that predicts deformations, strains, and stresses. Future leaching scenarios corresponding to oil drawdowns using fresh water are also simulated by increasing the volume of the caverns. Cavern pressures are varied in the model to capture operational practices in the field. The results of the finite element model are interpreted to provide information on the current and future status of subsidence, well integrity, and cavern stability. The most significant results in this report are relevant to Cavern 6. The cavern is shaped like a bowl with a large ceiling span and is in close proximity to Cavern 9. The analyses predict tensile stresses at the edge of the ceiling during repressuization of Cavern 6 following workover conditions. During a workover the cavern is at low pressure to service a well. The wellhead pressures are atmospheric. When the workover is complete, the cavern is repressurized. The resulting elastic stresses are sufficient to cause tension around the edge of the large ceiling span. With time, these stresses relax to a compressive state because of salt creep. However, the potential for salt fracture and propagation exists, particularly towards Cavern 9. With only 200 ft of salt between the caverns, the operational consequences must be examined if the two caverns become connected. A critical time may be during a workover of Cavern 9 in part because of the operational vulnerabilities, but also because dilatant damage is

  15. Analysis of cavern stability at the Bryan Mound SPR site.

    Ehgartner, Brian L.; Sobolik, Steven Ronald

    2009-04-01

    This report presents computational analyses that simulate the structural response of caverns at the Strategic Petroleum Reserve Bryan Mound site. The cavern field comprises 20 caverns. Five caverns (1, 2, 4, and 5; 3 was later plugged and abandoned) were acquired from industry and have unusual shapes and a history dating back to 1946. The other 16 caverns (101-116) were leached according to SPR standards in the mid-1980s and have tall cylindrical shapes. The history of the caverns and their shapes are simulated in a 3-D geomechanics model of the site that predicts deformations, strains, and stresses. Future leaching scenarios due to oil drawdowns using fresh water are also simulated by increasing the volume of the caverns. Cavern pressures are varied in the model to capture operational practices in the field. The results of the finite element model are interpreted to provide information on the current and future status of subsidence, well integrity, and cavern stability. The most significant result in this report is relevant to caverns 1, 2, and 5. The caverns have non-cylindrical shapes and have potential regions where the surrounding salt may be damaged during workover procedures. During a workover the normal cavern operating pressure is lowered to service a well. At this point the wellhead pressures are atmospheric. When the workover is complete, the cavern is repressurized. The resulting elastic stresses are sufficient to cause tension and large deviatoric stresses at several locations. With time, these stresses relax to a compressive state due to salt creep. However, the potential for salt damage and fracturing exists. The analyses predict tensile stresses at locations with sharp-edges in the wall geometry, or in the case of cavern 5, in the neck region between the upper and lower lobes of the cavern. The effects do not appear to be large-scale, however, so the only major impact is the potential for stress-induced salt falls in cavern 5, potentially leading to

  16. 40 CFR 60.1115 - What is a siting analysis?

    2010-07-01

    ... unit affects ambient air quality, visibility, soils, vegetation, and other relevant factors. The... Waste Combustion Units for Which Construction is Commenced After August 30, 1999 or for Which... environmental and social costs resulting from its location and construction. The analysis must also consider...

  17. Implementation of a combinatorial cleavage and deprotection scheme

    Nielsen, John; Rasmussen, Palle H.

    1996-01-01

    Phthalhydrazide libraries are synthesized in solution from substituted hydrazines and phthalimides in several different library formats including single compounds, indexed sub-libraries and a full library. When carried out during solid-phase synthesis, this combinatorial cleavage and deprotection...

  18. Text in social networking Web sites: A word frequency analysis of Live Spaces

    Thelwall, Mike

    2008-01-01

    Social networking sites are owned by a wide section of society and seem to dominate Web usage. Despite much research into this phenomenon, little systematic data is available. This article partially fills this gap with a pilot text analysis of one social networking site, Live Spaces. The text in 3,071 English language Live Spaces sites was monitored daily for six months and word frequency statistics calculated and compared with those from the British National Corpus. The results confirmed the...

  19. Mass spectrometry analysis of tank wastes at the Hanford Site

    Campbell, J.A.; Mong, G.M.; Clauss, S.A.

    1995-01-01

    Twenty-five of the 177 high-level waste storage tanks at the Hanford Site in southeastern Washington are being watched closely because of the possibility that flammable gas mixtures may be produced from the mixed wastes contained in the storage tanks. One tank in particular, Tank 241-SY-101 (Tank 101-SY), has exhibited episodic releases of flammable gas mixtures since its final filling in the early 1980s. It has been postulated that the organic compounds present in the waste may be precursors to the production of hydrogen. Mass spectrometry has proven to be an invaluable tool for the identification of organic components in wastes from Tank 101-SY and C-103. A suite of physical and chemical analyses has been performed in support of activities directed toward the resolution of an Unresolved Safety Question concerning the potential for a floating organic layer in Hanford Waste Tank 241-C-103 to sustain a pool fire. The aqueous layer underlying the floating organic material was also analyzed for organic components

  20. Hanford Site Composite Analysis Technical Approach Description: Groundwater

    Budge, T. J. [CH2M HILL Plateau Remediation Company, Richland, WA (United States)

    2017-10-02

    The groundwater facet of the revised CA is responsible for generating predicted contaminant concentration values over the entire analysis spatial and temporal domain. These estimates will be used as part of the groundwater pathway dose calculation facet to estimate dose for exposure scenarios. Based on the analysis of existing models and available information, the P2R Model was selected as the numerical simulator to provide these estimates over the 10,000-year temporal domain of the CA. The P2R Model will use inputs from initial plume distributions, updated for a start date of 1/1/2017, and inputs from the vadose zone facet, created by a tool under development as part of the ICF, to produce estimates of hydraulic head, transmissivity, and contaminant concentration over time. A recommendation of acquiring 12 computer processors and 2 TB of hard drive space is made to ensure that the work can be completed within the anticipated schedule of the revised CA.

  1. Cleavage events and sperm dynamics in chick intrauterine embryos.

    Hyung Chul Lee

    Full Text Available This study was undertaken to elucidate detailed event of early embryogenesis in chicken embryos using a noninvasive egg retrieval technique before oviposition. White Leghorn intrauterine eggs were retrieved from 95 cyclic hens aged up to 54-56 weeks and morphogenetic observation was made under both bright field and fluorescent image in a time course manner. Differing from mammals, asymmetric cleavage to yield preblastodermal cells was observed throughout early embryogenesis. The first two divisions occurred synchronously and four polarized preblastodermal cells resulted after cruciform cleavage. Then, asynchronous cleavage continued in a radial manner and overall cell size in the initial cleavage region was smaller than that in the distal area. Numerous sperms were visible, regardless of zygotic nuclei formation. Condensed sperm heads were present mainly in the perivitelline space and cytoplasm, and rarely in the yolk region, while decondensed sperm heads were only visible in the yolk. In conclusion, apparent differences in sperm dynamics and early cleavage events compared with mammalian embryos were detected in chick embryo development, which demonstrated polarized cleavage with penetrating supernumerary sperm into multiple regions.

  2. Neutron activation analysis to the profile surface sediments from several sites on the Havana Bay

    Diaz Riso, O.; Gelen, A.; Lopez, N.; Gonzalez, H.; Manso, M.V.; Graciano, A.M.; Nogueira, C.A.; Beltran, J.; Soto, J.

    2003-01-01

    Instrumental neutron activation analysis (INAA) technique was employed to analyze the surface sediments from several sites on the Havana Bay, Cuba. Measurements of heavy and trace elements in the sediments are reported. The results show that the concentration of the elements is site dependent. The data suggest that an anthropogenic input into the bay from domestic sewage and industries occurred

  3. Dynamics of bleomycin interaction with a strongly bound hairpin DNA substrate, and implications for cleavage of the bound DNA.

    Bozeman, Trevor C; Nanjunda, Rupesh; Tang, Chenhong; Liu, Yang; Segerman, Zachary J; Zaleski, Paul A; Wilson, W David; Hecht, Sidney M

    2012-10-31

    Recent studies involving DNAs bound strongly by bleomycins have documented that such DNAs are degraded by the antitumor antibiotic with characteristics different from those observed when studying the cleavage of randomly chosen DNAs in the presence of excess Fe·BLM. In the present study, surface plasmon resonance has been used to characterize the dynamics of BLM B(2) binding to a strongly bound hairpin DNA, to define the effects of Fe(3+), salt, and temperature on BLM-DNA interaction. One strong primary DNA binding site, and at least one much weaker site, were documented. In contrast, more than one strong cleavage site was found, an observation also made for two other hairpin DNAs. Evidence is presented for BLM equilibration between the stronger and weaker binding sites in a way that renders BLM unavailable to other, less strongly bound DNAs. Thus, enhanced binding to a given site does not necessarily result in increased DNA degradation at that site; i.e., for strongly bound DNAs, the facility of DNA cleavage must involve other parameters in addition to the intrinsic rate of C-4' H atom abstraction from DNA sugars.

  4. Single molecule TPM analysis of the catalytic pentad mutants of Cre and Flp site-specific recombinases: contributions of the pentad residues to the pre-chemical steps of recombination

    Fan, Hsiu-Fang; Cheng, Yong-Song; Ma, Chien-Hui; Jayaram, Makkuni

    2015-01-01

    Cre and Flp site-specific recombinase variants harboring point mutations at their conserved catalytic pentad positions were characterized using single molecule tethered particle motion (TPM) analysis. The findings reveal contributions of these amino acids to the pre-chemical steps of recombination. They suggest functional differences between positionally conserved residues in how they influence recombinase-target site association and formation of ‘non-productive’, ‘pre-synaptic’ and ‘synaptic’ complexes. The most striking difference between the two systems is noted for the single conserved lysine. The pentad residues in Cre enhance commitment to recombination by kinetically favoring the formation of pre-synaptic complexes. These residues in Flp serve a similar function by promoting Flp binding to target sites, reducing non-productive binding and/or enhancing the rate of assembly of synaptic complexes. Kinetic comparisons between Cre and Flp, and between their derivatives lacking the tyrosine nucleophile, are consistent with a stronger commitment to recombination in the Flp system. The effect of target site orientation (head-to-head or head-to-tail) on the TPM behavior of synapsed DNA molecules supports the selection of anti-parallel target site alignment prior to the chemical steps. The integrity of the synapse, whose establishment/stability is fostered by strand cleavage in the case of Flp but not Cre, appears to be compromised by the pentad mutations. PMID:25765648

  5. Hanford Site Composite Analysis Technical Approach Description: Integrated Computational Framework.

    Smith, K. J. [CH2M HILL Plateau Remediation Company, Richland, WA (United States)

    2017-09-14

    The U.S. Department of Energy (DOE) in DOE O 435.1 Chg. 1, Radioactive Waste Management, requires the preparation and maintenance of a composite analysis (CA). The primary purpose of the CA is to provide a reasonable expectation that the primary public dose limit is not likely to be exceeded by multiple source terms that may significantly interact with plumes originating at a low-level waste disposal facility. The CA is used to facilitate planning and land use decisions that help assure disposal facility authorization will not result in long-term compliance problems; or, to determine management alternatives, corrective actions, or assessment needs if potential problems are identified.

  6. Hanford Site Composite Analysis Technical Approach Description: Waste Form Release.

    Hardie, S. [CH2M HILL Plateau Remediation Company, Richland, WA (United States); Paris, B. [CH2M HILL Plateau Remediation Company, Richland, WA (United States); Apted, M. [CH2M HILL Plateau Remediation Company, Richland, WA (United States)

    2017-09-14

    The U.S. Department of Energy (DOE) in DOE O 435.1 Chg. 1, Radioactive Waste Management, requires the preparation and maintenance of a composite analysis (CA). The primary purpose of the CA is to provide a reasonable expectation that the primary public dose limit is not likely to be exceeded by multiple source terms that may significantly interact with plumes originating at a low-level waste disposal facility. The CA is used to facilitate planning and land use decisions that help assure disposal facility authorization will not result in long-term compliance problems; or, to determine management alternatives, corrective actions or assessment needs, if potential problems are identified.

  7. Hanford Site Composite Analysis Technical Approach Description: Atmospheric Transport Modeling.

    Sun, B. [CH2M HILL Plateau Remediation Company, Richland, WA (United States); Lehman, L. L. [CH2M HILL Plateau Remediation Company, Richland, WA (United States)

    2017-10-02

    The U.S. Department of Energy (DOE) in DOE O 435.1 Chg. 1, Radioactive Waste Management, requires the preparation and maintenance of a composite analysis (CA). The primary purpose of the CA is to provide a reasonable expectation that the primary public dose limit is not likely to be exceeded by multiple source terms that may significantly interact with plumes originating at a low-level waste disposal facility. The CA is used to facilitate planning and land use decisions that help assure disposal facility authorization will not result in long-term compliance problems; or, to determine management alternatives, corrective actions or assessment needs, if potential problems are identified.

  8. Use of safety analysis to site comfirmation procedure in case of hard rock repository

    Peltonen, E.K.

    1984-02-01

    The role of safety analysis in a confirmation procedure of a candidate disposal site of radioactive wastes is discussed. Items dealt with include principle reasons and practical goals of the use of safety analysis, methodology of safety analysis and assessment, as well as usefulness and adequacy of the present safety analysis. Safety analysis is a tool, which enables one to estimate quantitatively the possible radiological impacts from the disposal. The results can be compared with the criteria and the suitability conclusions drawn. Because of its systems analytical nature safety analysis is an effective method to reveal, what are the most important factors of the disposal system and the most critical site characteristics inside the lumped parameters often provided by the experimental site investigation methods. Furthermore it gives information on the accuracy needs of different site properties. This can be utilized to judge whether the quality and quantity of the measurements for the characterization are sufficient as well as to guide the further site investigations. A more practical discussion regarding the applicability of the use of safety analysis is presented by an example concerning the assessment of a Finnish candidate site for low- and intermediate-level radioactive waste repository. (author)

  9. On-Site Processing and Subsampling of Surface Soil Samples for the Analysis of Explosives

    Hewitt, Alan D

    2003-01-01

    The on-site implementation of a sampling and analysis plan for the determination of explosives residues exposed a large uncertainty in our ability to quickly obtain representative subsamples from either large (>500 g...

  10. SKI2 mediates degradation of RISC 5′-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis

    Branscheid, Anja; Marchais, Antonin; Schott, Gregory; Lange, Heike; Gagliardi, Dominique; Andersen, Stig Uggerhøj; Voinnet, Olivier; Brodersen, Peter

    2015-01-01

    Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20–24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5′, but not 3′-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5′ to the cleavage site, but several examples of 3′-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5′-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5′-cleavage fragments. PMID:26464441

  11. SKI2 mediates degradation of RISC 5'-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis.

    Branscheid, Anja; Marchais, Antonin; Schott, Gregory; Lange, Heike; Gagliardi, Dominique; Andersen, Stig Uggerhøj; Voinnet, Olivier; Brodersen, Peter

    2015-12-15

    Small regulatory RNAs are fundamental in eukaryotic and prokaryotic gene regulation. In plants, an important element of post-transcriptional control is effected by 20-24 nt microRNAs (miRNAs) and short interfering RNAs (siRNAs) bound to the ARGONAUTE1 (AGO1) protein in an RNA induced silencing complex (RISC). AGO1 may cleave target mRNAs with small RNA complementarity, but the fate of the resulting cleavage fragments remains incompletely understood. Here, we show that SKI2, SKI3 and SKI8, subunits of a cytoplasmic cofactor of the RNA exosome, are required for degradation of RISC 5', but not 3'-cleavage fragments in Arabidopsis. In the absence of SKI2 activity, many miRNA targets produce siRNAs via the RNA-dependent RNA polymerase 6 (RDR6) pathway. These siRNAs are low-abundant, and map close to the cleavage site. In most cases, siRNAs were produced 5' to the cleavage site, but several examples of 3'-spreading were also identified. These observations suggest that siRNAs do not simply derive from RDR6 action on stable 5'-cleavage fragments and hence that SKI2 has a direct role in limiting secondary siRNA production in addition to its function in mediating degradation of 5'-cleavage fragments. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations

    Zuo, Zhicheng; Liu, Jin

    2016-11-01

    The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has emerged as a versatile toolbox for genome editing. Despite recent advances in the mechanism studies on spCas9-sgRNA-mediated double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg2+ ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg2+ bound. The simulation results showed that binding of two Mg2+ ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we demonstrated with our simulations that Cas9-catalyzed DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.

  13. Synthesis, spectral, crystal structure, thermal behavior, antimicrobial and DNA cleavage potential of two octahedral cadmium complexes: A supramolecular structure

    Montazerozohori, M.; Musavi, S.A.; Masoudiasl, A.; Naghiha, A.; Dušek, Michal; Kučeráková, Monika

    2015-01-01

    Roč. 137, FEB (2015), s. 389-396 ISSN 1386-1425 R&D Projects: GA ČR(CZ) GA14-03276S Institutional support: RVO:68378271 Keywords : Schiff base * Cd(II) * DNA cleavage * TG/DTG analysis * X-ray structure analysis Subject RIV: BM - Solid Matter Physics ; Magnetism Impact factor: 2.653, year: 2015

  14. Hanford Site Composite Analysis Technical Approach Description: Vadose Zone

    Williams, M. D. [INTERA Inc., Austin, TX (United States); Nichols, W. E. [CH2M, Richland, WA (United States); Ali, A. [INTERA Inc., Austin, TX (United States); Allena, P. [INTERA Inc., Austin, TX (United States); Teague, G. [INTERA Inc., Austin, TX (United States); Hammond, T. B. [INTERA Inc., Austin, TX (United States)

    2017-10-30

    The U.S. Department of Energy (DOE) in DOE O 435.1 Chg. 1, Radioactive Waste Management, and DOE M 435.1 Chg 1, Radioactive Waste Management Manual, requires the preparation and maintenance of a composite analysis (CA). The primary purpose of the CA is to provide a reasonable expectation that the primary public dose limit is not likely to be exceeded by multiple source terms that may significantly interact with plumes originating at a low-level waste disposal facility. The CA is used to facilitate planning and land use decisions that help assure disposal facility authorization will not result in long-term compliance problems; or, to determine management alternatives, corrective actions, or assessment needs, if potential problems are identified.

  15. Inhibition of influenza virus infection and hemagglutinin cleavage by the protease inhibitor HAI-2

    Hamilton, Brian S.; Chung, Changik; Cyphers, Soreen Y.; Rinaldi, Vera D.; Marcano, Valerie C.; Whittaker, Gary R., E-mail: grw7@cornell.edu

    2014-07-25

    Highlights: • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza HA cleavage activation. • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza virus infection. • Comparative analysis of HAI-2 for vesicular stomatitis virus and human parainfluenza virus type-1. • Analysis of the activity of HAI-2 in a mouse model of influenza. - Abstract: Influenza virus remains a significant concern to public health, with the continued potential for a high fatality pandemic. Vaccination and antiviral therapeutics are effective measures to circumvent influenza virus infection, however, multiple strains have emerged that are resistant to the antiviral therapeutics currently on the market. With this considered, investigation of alternative antiviral therapeutics is being conducted. One such approach is to inhibit cleavage activation of the influenza virus hemagglutinin (HA), which is an essential step in the viral replication cycle that permits viral-endosome fusion. Therefore, targeting trypsin-like, host proteases responsible for HA cleavage in vivo may prove to be an effective therapeutic. Hepatocyte growth factor activator inhibitor 2 (HAI-2) is naturally expressed in the respiratory tract and is a potent inhibitor of trypsin-like serine proteases, some of which have been determined to cleave HA. In this study, we demonstrate that HAI-2 is an effective inhibitor of cleavage of HA from the human-adapted H1 and H3 subtypes. HAI-2 inhibited influenza virus H1N1 infection in cell culture, and HAI-2 administration showed protection in a mouse model of influenza. HAI-2 has the potential to be an effective, alternative antiviral therapeutic for influenza.

  16. Inhibition of influenza virus infection and hemagglutinin cleavage by the protease inhibitor HAI-2

    Hamilton, Brian S.; Chung, Changik; Cyphers, Soreen Y.; Rinaldi, Vera D.; Marcano, Valerie C.; Whittaker, Gary R.

    2014-01-01

    Highlights: • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza HA cleavage activation. • Biochemical and cell biological analysis of HAI-2 as an inhibitor of influenza virus infection. • Comparative analysis of HAI-2 for vesicular stomatitis virus and human parainfluenza virus type-1. • Analysis of the activity of HAI-2 in a mouse model of influenza. - Abstract: Influenza virus remains a significant concern to public health, with the continued potential for a high fatality pandemic. Vaccination and antiviral therapeutics are effective measures to circumvent influenza virus infection, however, multiple strains have emerged that are resistant to the antiviral therapeutics currently on the market. With this considered, investigation of alternative antiviral therapeutics is being conducted. One such approach is to inhibit cleavage activation of the influenza virus hemagglutinin (HA), which is an essential step in the viral replication cycle that permits viral-endosome fusion. Therefore, targeting trypsin-like, host proteases responsible for HA cleavage in vivo may prove to be an effective therapeutic. Hepatocyte growth factor activator inhibitor 2 (HAI-2) is naturally expressed in the respiratory tract and is a potent inhibitor of trypsin-like serine proteases, some of which have been determined to cleave HA. In this study, we demonstrate that HAI-2 is an effective inhibitor of cleavage of HA from the human-adapted H1 and H3 subtypes. HAI-2 inhibited influenza virus H1N1 infection in cell culture, and HAI-2 administration showed protection in a mouse model of influenza. HAI-2 has the potential to be an effective, alternative antiviral therapeutic for influenza

  17. Determinants of the VP1/2A junction cleavage by the 3C protease in foot-and-mouth disease virus infected cells

    Kristensen, Thea; Normann, Preben; Gullberg, Maria

    2017-01-01

    . Interestingly, in contrast to the serotype O virus results, no second site mutations occurred within the VP1 coding region of serotype A viruses with the blocked VP1/2A cleavage site. However, some of these viruses acquired changes in the 2C protein that is involved in enterovirus morphogenesis. These results...

  18. Environmental-pathways analysis for evaluation of a low-level waste disposal site

    Lee, D.W.; Ketelle, R.H.; Pin, F.G.; Hill, G.S.

    1983-01-01

    The suitability of a site for the shallow land burial of low-level waste is evaluated by an environmental-pathways analysis. The environmental-pathways analysis considers the probable paths for the transport of contamination to man and models the long-term transport of contamination to determine the resulting dose-to-man. The model of the long-term transport of contamination is developed for a proposed site using data obtained from a comprehensive laboratory and field investigation. The proposed site is located at the US Department of Energy Portsmouth Reservation, Piketon, Ohio and is planned to accept low-level radioactive waste generated by the enrichment of uranium. Laboratory studies were performed to characterize the waste and determine the wastes' leaching and retardation characteristics with site soils and groundwater. Comprehensive drilling, sampling and laboratory investigations were performed to provide the necessary information for interpreting the site's geology and hydrology. Field tests were performed to further quantify the site's hydrology. The pathway of greatest concern is the migration of contaminated groundwater and subsequent consumption by man. This pathway was modeled using a numerical simulation of the long-term transport of contamination. Conservative scenarios were developed for leachate generation and migration through the geohydrologic system. The dose-to-man determined from the pathways analysis formed the basis for evaluating site acceptability and providing recommendations for site design and development

  19. Environmental pathways analysis for evaluation of a low-level waste disposal site

    Lee, D.W.; Ketelle, R.H.; Pin, F.G.; Hill, G.S.

    1984-01-01

    The suitability of a site for the shallow land burial of low-level waste is evaluated by an environmental pathways analysis. The environmental pathways analysis considers the probable paths for the transport of contamination to man and models the long-term transport of contamination to determine the resulting dose to man. The model of the long-term transport of contamination is developed for a proposed site using data obtained from a comprehensive laboratory and field investigation. The proposed site is located at the US Department of Energy Portsmouth Reservation, Piketon, Ohio, and is planned to accept low-level radioactive waste generated by the enrichment of uranium. Laboratory studies were performed to characterize the waste and determine the wastes' leaching and retardation characteristics with site soils and groundwater. Comprehensive drilling, sampling and laboratory investigations were performed to provide the necessary information for interpreting the site's geology and hydrology. Field tests were performed to further quantify the site's hydrology. The pathway of greatest concern is the migration of contaminated groundwater and subsequent consumption by man. This pathway was modelled using a numerical simulation of the long-term transport of contamination. Conservative scenarios were developed for leachate generation and migration through the geohydrologic system. The dose to man determined from the pathways analysis formed the basis for evaluating site acceptability and providing recommendations for site design and development. (author)

  20. NPP Siting in Western Part of Java Island Indonesia: Regional Analysis Stage

    Sastratenaya, A.S.; Yuliastuti

    2011-01-01

    Full text of publication follows: Considering that Banten and West Java Provinces are dense regions of industry, therefore they require a large amount of electricity. Nuclear power plant is one option to be considered to anticipate the future electricity demand. To support the program, it is needed to look for some potential locations through NPP siting. The siting should meet the requirement of safety, safety aspects of the natural external events, human induced external events, public and environmental safety. Site selection is performed in several stages, where each stage has specific assessment criteria. Siting is commenced with pre-survey activity to obtain several interest areas, the activity covers a wide area but the used data is very limited and only apply general criteria. The following activities after pre survey are site survey consisting of (1) regional analysis, (2) site screening, and (3) comparison and ranking stages. The objective of regional analysis is to obtain potential sites in the study area of 150 km radius from each interest area by using both general and specific criteria. The potential sites then screened to obtain selected candidate sites by using more detailed secondary data as well as survey activities such as geophysical investigation, a few of drilling, etc., within the radius of 50 km from each potential site. All the selected candidate sites are then compared and ranked to obtain preferred candidate site. Site evaluation is the next step to evaluate all site-specific parameter to obtain design basis parameters and as the basis for preparing site permit document. This paper presents the methodology and result of regional analysis stage. The objective of the activity is to obtain potential sites in the north coast of West Java and Banten Provinces by considering fourteen study aspects which could be categorize into safety related aspects, non-safety related aspect and public education. However, this paper only considers the safety

  1. Systems Analysis, Scenario Construction and Consequence Analysis Definition for SITE-94

    Chapman, N A; Robinson, P [Intera Information Technologies Ltd (United Kingdom); Andersson, Johan; Wingefors, S [Swedish Nuclear Power Inspectorate, Stockholm (Sweden); Skagius, K; Wiborgh, M [Kemakta Konsult AB, Stockholm (Sweden); Wene, C O [Chalmers Inst. of Technology, Goeteborg (Sweden)

    1995-06-01

    SITE-94 is a performance assessment of a hypothetical repository at a real site. The main objective of the project is to determine how site specific data should be assimilated into the performance assessment process and to evaluate how uncertainties inherent in site characterization will influence performance assessment results. This report uses scenario definition work as a vehicle to introduce the systems approach to performance assessment which has been developed and tested in SITE-94 and which constitutes one of the main advances made during the project. The results of the application of the methodology are presented separately, in the SITE-94 Summary Report. 40 refs, 21 figs, 12 tabs.

  2. Systems Analysis, Scenario Construction and Consequence Analysis Definition for SITE-94

    Chapman, N.A.; Robinson, P.; Andersson, Johan; Wingefors, S.; Skagius, K.; Wiborgh, M.; Wene, C.O.

    1995-06-01

    SITE-94 is a performance assessment of a hypothetical repository at a real site. The main objective of the project is to determine how site specific data should be assimilated into the performance assessment process and to evaluate how uncertainties inherent in site characterization will influence performance assessment results. This report uses scenario definition work as a vehicle to introduce the systems approach to performance assessment which has been developed and tested in SITE-94 and which constitutes one of the main advances made during the project. The results of the application of the methodology are presented separately, in the SITE-94 Summary Report. 40 refs, 21 figs, 12 tabs

  3. Geographical information system (GIS) suitability analysis of radioactive waste repository site in Pahang, Malaysia

    Faizal Azrin Abd Razalim; Noraini Surip; Ahmad Hasnulhadi; Nazran Harun; Nurul Nadia Abd Malek; Roziah Che Musa

    2010-01-01

    The aim of this project is to identify a suitable site for radioactive waste repository in Pahang using remote sensing and geographical information system (GIS) technologies. There are ten parameters considered in the analysis, which divided into Selection Criteria and Exclusion Criteria. The Selection Criteria parameters consists of land use, rainfall, lineament, slope, groundwater potential and elevation while Exclusion Criteria parameters consist of urban, protected land and island. Furthermore, all parameters were integrated, given weight age and ranked for site selection evaluation in GIS environment. At the first place, about twelve sites have been identified as suitable sites for radioactive waste repository throughout the study area. These sites were further analysed by ground checking on the physical setting including geological, drainage, and population density in order to finalise three most suitable sites for radioactive waste repository. (author)

  4. White Oak Creek Embayment site characterization and contaminant screening analysis

    Blaylock, B.G.; Ford, C.J.; Frank, M.L.; Hoffman, F.O.; Hook, L.A.

    1993-01-01

    Analyses of sediment samples collected near the mouth of White Oak Creek during the summer of 1990 revealed 137 Cs concentrations [> 10 6 Bq/kg dry wt (> 10 4 pCi/g dry wt)] near the sediment surface. Available evidence indicates that these relatively high concentrations of 137 Cs now at the sediment surface were released from White Oak Dam in the mid-1950s and had accumulated at depositionalsites in the embayment. These accumulated sediments are being eroded and transported downstream primarily during winter low-water levels by flood events and by a combination of normal downstream flow and the water turbulence created by the release of water from Melton Hill Dam during hydropower generation cycles. This report provides a more thorough characterization of the extent of contamination in WOCE than was previously available. Environmental samples collected from WOCE were analyzed for organic, inorganic, and radiological contaminants in fish, water, and sediment. These results were used to conduct a human health effects screening analysis. Walkover radiation surveys conducted inside the fenced area surrounding the WOCE at summer-pool (741 ft MSL) and at winter-pool (733 ft MSL) level, indicated a maximum exposure rate of 3 mR h 1 1 m above the soil surface

  5. Development of probabilistic seismic hazard analysis for international sites, challenges and guidelines

    Fernandez Ares, Antonio, E-mail: antonio.fernandez@rizzoassoc.com [Paul C. Rizzo Associates, Inc., 500 Penn Center Boulevard, Penn Center East, Suite 100, Pittsburgh, PA 15235 (United States); Fatehi, Ali, E-mail: ali.fatehi@rizzoassoc.com [Paul C. Rizzo Associates, Inc., 500 Penn Center Boulevard, Penn Center East, Suite 100, Pittsburgh, PA 15235 (United States)

    2013-06-15

    Research highlights: ► Site-specific seismic hazard study and suggestions for overcoming those challenges that are inherent to the significant amounts of epistemic uncertainty for sites at remote locations. ► Main aspects of probabilistic seismic hazard analysis (PSHA). ► Regional and site geology in the context of a probabilistic seismic hazard analysis (PSHA), including state-of-the-art ground motion estimation methods, and geophysical conditions. ► Senior seismic hazard analysis (SSHAC) as a mean to incorporate the opinions and contributions of the informed scientific community. -- Abstract: This article provides guidance to conduct a site-specific seismic hazard study, giving suggestions for overcoming those challenges that are inherent to the significant amounts of epistemic uncertainty for sites at remote locations. The text follows the general process of a seismic hazard study, describing both the deterministic and probabilistic approaches. Key and controversial items are identified in the areas of recorded seismicity, seismic sources, magnitude, ground motion models, and local site effects. A case history corresponding to a seismic hazard study in the Middle East for a Greenfield site in a remote location is incorporated along the development of the recommendations. Other examples of analysis case histories throughout the World are presented as well.

  6. Diaphanous gene mutation affects spiral cleavage and chirality in snails

    Kuroda, Reiko; Fujikura, Kohei; Abe, Masanori; Hosoiri, Yuji; Asakawa, Shuichi; Shimizu, Miho; Umeda, Shin; Ichikawa, Futaba; Takahashi, Hiromi

    2016-01-01

    L-R (left and right) symmetry breaking during embryogenesis and the establishment of asymmetric body plan are key issues in developmental biology, but the onset including the handedness-determining gene locus still remains unknown. Using pure dextral (DD) and sinistral (dd) strains of the pond snail Lymnaea stagnalis as well as its F2 through to F10 backcrossed lines, the single handedness-determining-gene locus was mapped by genetic linkage analysis, BAC cloning and chromosome walking. We have identified the actin-related diaphanous gene Lsdia1 as the strongest candidate. Although the cDNA and derived amino acid sequences of the tandemly duplicated Lsdia1 and Lsdia2 genes are very similar, we could discriminate the two genes/proteins in our molecular biology experiments. The Lsdia1 gene of the sinistral strain carries a frameshift mutation that abrogates full-length LsDia1 protein expression. In the dextral strain, it is already translated prior to oviposition. Expression of Lsdia1 (only in the dextral strain) and Lsdia2 (in both chirality) decreases after the 1-cell stage, with no asymmetric localization throughout. The evolutionary relationships among body handedness, SD/SI (spiral deformation/spindle inclination) at the third cleavage, and expression of diaphanous proteins are discussed in comparison with three other pond snails (L. peregra, Physa acuta and Indoplanorbis exustus). PMID:27708420

  7. Enzymatic carotenoid cleavage in star fruit (Averrhoa carambola).

    Fleischmann, Peter; Watanabe, Naoharu; Winterhalter, Peter

    2003-05-01

    This paper presents the first description of an enzyme fraction exhibiting carotenoid cleavage activity isolated from fruit skin of Averrhoa carambola. Partial purification of the enzyme could be achieved by acetone precipitation, ultrafiltration (300 kDa, 50 kDa), isoelectric focusing (pH 3-10) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (7.5%). In this way, an enzymatically active protein fraction was obtained, consisting of four proteins in the molecular weight range of between 12 and 90 kDa. Using beta-carotene as substrate, the enzyme activity was detected spectrophotometrically at 505 nm. The main reaction product, detected by GC analysis, was beta-ionone. This proves that the isolated enzymes are closely related to aroma metabolism and release of star fruit. The time constant of the reaction was 16.6 min, the Michaelis Constant K(m)=3.6 micromol 1(-1) and the maximum velocity V(max)=10.5 x 10(-3) micromol l(-1) s(-1) mg((Protein))(-1). The optimum temperature was 45 degrees C.

  8. Analysis and evaluation of the Gorleben site characteristics. Report on the working package 2. Preliminary safety analysis Gorleben

    Kukla, Peter; Pechnig, Renate; Urai, Janos

    2011-10-01

    The report on analysis and evaluation of the Gorleben site characteristics covers the following chapters: Site characteristics on the hydrology of the cover rock: hydrology and hydraulic properties, geothermal studies, ground water properties and motion, model calculations. Site characteristics on geology of the cover and adjoining rock: borehole explorations, geologic development, 3D geometry of the salt dome Gorleben, geologic development of the salt structure Gorleben. Site characteristics on the saliniferous formation: geophysical exploration, modeling of the internal structure of the salt dome Gorleben, fluids in the salt dome Gorleben. Geotechnical studies at the site: geothermal studies, hydraulic data on the saliniferous formation, thermal model calculations, geomechanical in-situ measurements, mechanical and transport properties, thermo-mechanical modeling. Summarized evaluation of the site characteristics and further research requirement.

  9. Cell-based land use screening procedure for regional siting analysis

    Jalbert, J.S.; Dobson, J.E.

    1976-01-01

    An energy facility site-screening methodology which permits the land resource planner to identify candidate siting areas was developed. Through the use of spatial analysis procedures and computer graphics, a selection of candidate areas is obtained. Specific sites then may be selected from among candidate areas for environmental impact analysis. The computerized methodology utilizes a cell-based geographic information system for specifying the suitability of candidate areas for an energy facility. The criteria to be considered may be specified by the user and weighted in terms of importance. Three primary computer programs have been developed. These programs produce thematic maps, proximity calculations, and suitability calculations. Programs are written so as to be transferrable to regional planning or regulatory agencies to assist in rational and comprehensive power plant site identification and analysis

  10. Evidence of Alternative Cystatin C Signal Sequence Cleavage Which Is Influenced by the A25T Polymorphism.

    Annie Nguyen

    Full Text Available Cystatin C (Cys C is a small, potent, cysteine protease inhibitor. An Ala25Thr (A25T polymorphism in Cys C has been associated with both macular degeneration and late-onset Alzheimer's disease. Previously, studies have suggested that this polymorphism may compromise the secretion of Cys C. Interestingly, we found that untagged A25T, A25T tagged C-terminally with FLAG, or A25T FLAG followed by green fluorescent protein (GFP, were all secreted as efficiently from immortalized human cells as their wild-type (WT counterparts (e.g., 112%, 100%, and 88% of WT levels from HEK-293T cells, respectively. Supporting these observations, WT and A25T Cys C variants also showed similar intracellular steady state levels. Furthermore, A25T Cys C did not activate the unfolded protein response and followed the same canonical endoplasmic reticulum (ER-Golgi trafficking pathway as WT Cys C. WT Cys C has been shown to undergo signal sequence cleavage between residues Gly26 and Ser27. While the A25T polymorphism did not affect Cys C secretion, we hypothesized that it may alter where the Cys C signal sequence is preferentially cleaved. Under normal conditions, WT and A25T Cys C have the same signal sequence cleavage site after Gly26 (referred to as 'site 2' cleavage. However, in particular circumstances when the residues around site 2 are modified (such as by the presence of an N-terminal FLAG tag immediately after Gly26, or by a Gly26Lys (G26K mutation, A25T has a significantly higher likelihood than WT Cys C of alternative signal sequence cleavage after Ala20 ('site 1' or even earlier in the Cys C sequence. Overall, our results indicate that the A25T polymorphism does not cause a significant reduction in Cys C secretion, but instead predisposes the protein to be cleaved at an alternative signal sequence cleavage site if site 2 is hindered. Additional N-terminal amino acids resulting from alternative signal sequence cleavage may, in turn, affect the protease

  11. An approach to siting nuclear power plants: the relevance of earthquakes, faults and decision analysis

    Nair, K.; Brogan, G.E.; Cluff, L.S.; Idriss, I.M.; Mao, K.T.

    1975-01-01

    The regional approach to nuclear power plant siting described in this paper identifies candidate sites within the region and ranks these sites by using decision-analysis concepts. The approach uses exclusionary criteria to eliminate areas from consideration and to identify those areas which are most likely to contain candidate sites. These areas are then examined in greater detail to identify candidate sites, and the number of sites under consideration is reduced to a reasonably manageable number, approximately 15. These sites are then ranked using concepts of decision analysis. The exclusionary criteria applied relate primarily to regulatory-agency safety requirements and essential functional requirements. Examples of such criteria include proximity to population centres, presence of active faults, and the availability of cooling water. In many areas of the world, the presence of active faults and potential negative effects of earthquakes are dominant exclusionary criteria. To apply the 'active fault' criterion the region must be studied to locate and assess the activity of all potentially active faults. This requires complementary geologic (including geomorphic), historical, seismological, geodetic and geophysical investigations of the entire region. Site response studies or empirical attenuation correlations can be used to determine the relevant parameters of anticipated shaking from postulated earthquakes, and analytical testing and evaluation can be used to assess the potential extent of ground failure during an earthquake. After candidate sites are identified, an approach based on decision analysis is used to rank them. This approach uses the preferences and judgements of consumers, utility companies, the government, and other groups concerned with siting and licensing issues in the ranking process. Both subjective and objective factors are processed in a logical manner, as are the monetary and non-monetary factors and achievement of competing environmental

  12. 40 CFR 1400.3 - Public access to paper copies of off-site consequence analysis information.

    2010-07-01

    ...-site consequence analysis information. 1400.3 Section 1400.3 Protection of Environment ENVIRONMENTAL... PROGRAMS UNDER THE CLEAN AIR ACT SECTION 112(r)(7); DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION DISTRIBUTION OF OFF-SITE CONSEQUENCE ANALYSIS INFORMATION Public Access § 1400.3 Public access to...

  13. URBAN RAIN GAUGE SITING SELECTION BASED ON GIS-MULTICRITERIA ANALYSIS

    Y. Fu

    2016-06-01

    Full Text Available With the increasingly rapid growth of urbanization and climate change, urban rainfall monitoring as well as urban waterlogging has widely been paid attention. In the light of conventional siting selection methods do not take into consideration of geographic surroundings and spatial-temporal scale for the urban rain gauge site selection, this paper primarily aims at finding the appropriate siting selection rules and methods for rain gauge in urban area. Additionally, for optimization gauge location, a spatial decision support system (DSS aided by geographical information system (GIS has been developed. In terms of a series of criteria, the rain gauge optimal site-search problem can be addressed by a multicriteria decision analysis (MCDA. A series of spatial analytical techniques are required for MCDA to identify the prospective sites. With the platform of GIS, using spatial kernel density analysis can reflect the population density; GIS buffer analysis is used to optimize the location with the rain gauge signal transmission character. Experiment results show that the rules and the proposed method are proper for the rain gauge site selection in urban areas, which is significant for the siting selection of urban hydrological facilities and infrastructure, such as water gauge.

  14. Sentiment Analysis of Web Sites Related to Vaginal Mesh Use in Pelvic Reconstructive Surgery.

    Hobson, Deslyn T G; Meriwether, Kate V; Francis, Sean L; Kinman, Casey L; Stewart, J Ryan

    2018-05-02

    The purpose of this study was to utilize sentiment analysis to describe online opinions toward vaginal mesh. We hypothesized that sentiment in legal Web sites would be more negative than that in medical and reference Web sites. We generated a list of relevant key words related to vaginal mesh and searched Web sites using the Google search engine. Each unique uniform resource locator (URL) was sorted into 1 of 6 categories: "medical", "legal", "news/media", "patient generated", "reference", or "unrelated". Sentiment of relevant Web sites, the primary outcome, was scored on a scale of -1 to +1, and mean sentiment was compared across all categories using 1-way analysis of variance. Tukey test evaluated differences between category pairs. Google searches of 464 unique key words resulted in 11,405 URLs. Sentiment analysis was performed on 8029 relevant URLs (3472 legal, 1625 "medical", 1774 "reference", 666 "news media", 492 "patient generated"). The mean sentiment for all relevant Web sites was +0.01 ± 0.16; analysis of variance revealed significant differences between categories (P Web sites categorized as "legal" and "news/media" had a slightly negative mean sentiment, whereas those categorized as "medical," "reference," and "patient generated" had slightly positive mean sentiments. Tukey test showed differences between all category pairs except the "medical" versus "reference" in comparison with the largest mean difference (-0.13) seen in the "legal" versus "reference" comparison. Web sites related to vaginal mesh have an overall mean neutral sentiment, and Web sites categorized as "medical," "reference," and "patient generated" have significantly higher sentiment scores than related Web sites in "legal" and "news/media" categories.

  15. Replacement tunnelled dialysis catheters for haemodialysis access: Same site, new site, or exchange — A multivariate analysis and risk score

    Tapping, C.R.; Scott, P.M.; Lakshminarayan, R.; Ettles, D.F.; Robinson, G.J.

    2012-01-01

    Aim: To identify variables related to complications following tunnelled dialysis catheter (TDC) replacement and stratifying the risk to reduce morbidity in patients with end-stage renal disease. Materials and methods: One hundred and forty TDCs (Split Cath, medCOMP) were replaced in 140 patients over a 5 year period. Multiple variables were retrospectively collected and analysed to stratify the risk and to predict patients who were more likely to suffer from complications. Multivariate regression analysis was used to identify variables predictive of complications. Results: There were six immediate complications, 42 early complications, and 37 late complications. Multivariate analysis revealed that variables significantly associated to complications were: female sex (p = 0.003; OR 2.9); previous TDC in the same anatomical position in the past (p = 0.014; OR 4.1); catheter exchange (p = 0.038; OR 3.8); haemoglobin 15 s (p = 0.002; OR 4.1); and C-reactive protein >50 mg/l (p = 0.007; OR 4.6). A high-risk score, which used the values from the multivariate analysis, predicted 100% of the immediate complications, 95% of the early complications, and 68% of the late complications. Conclusion: Patients can now be scored prior to TDC replacement. A patient with a high-risk score can be optimized to reduce the chance of complications. Further prospective studies to confirm that rotating the site of TDC reduces complications are warranted as this has implications for current guidelines.

  16. Secondary isotope effects on alpha-cleavage reactions

    Ingemann, S.; Hammerum, S.

    1980-01-01

    Kinetic deuterium isotope effects on mass spectral reactions have in several instances been utilized to provide structural information and to answer mechanistic questions. Typically, the influence of the deuterium label on the rate of one of a number of competing reactions has been studied. Secondary isotope effects have usually been assumed to be relatively insignificant in comparison with the observed kinetic effects, even though various workers have shown that secondary isotope effects may indeed exert a considerable influence on the rates of competing simple cleavages. Recent studies have provided quantitative data to show that the mere presence of deuterium atoms up to six bonds away may influence the rate of a simple cleavage reaction. In relation to an investigation of rearrangements accompanying simple cleavage reactions, a semi-quantitative measure was needed of the variation of the secondary isotope effect with the number of bonds between the deuterium label and the point of rupture. The influence has therefore been examined of the presence of remote deuterium atoms on a typical simple cleavage reaction, the α-cleavage of aliphatic amines. As a model compound, N-methyldipentylamine was chosen, systematically labelled with deuterium. (author)

  17. Risk analysis of NPP in multi-unit site for configuration of AAC power source

    Kim, Myung Ki

    2000-01-01

    Because of the difficulties in finding new sites for nuclear power plants, more units are being added to the existing sites. In these multi-unit sites, appropriate countermeasures should be established to cope with the potential station blackout (SBO) accident. Currently, installation of additional diesel generator (DG) is considered to ensure an alternative AC power source, but it has not been decided yet how many DGs should be installed in a multi-unit site. In this paper, risk informed decision making method, which evaluates reliability of electrical system, core damage frequency, and site average core damage frequency, is introduced to draw up the suitable number of DG in multi-unit site. The analysis results show that installing two DGs lowered the site average core damage frequency by 1.4% compared to one DG in six unit site. In the light of risk-informed decisions in regulatory guide 1.174, there is no difference of safety between two alternatives. It is concluded that one emergency diesel generator sufficiently guarantees safety against station blackout of nuclear power plants in multi-unit site. (author)

  18. A method for the automated detection phishing websites through both site characteristics and image analysis

    White, Joshua S.; Matthews, Jeanna N.; Stacy, John L.

    2012-06-01

    Phishing website analysis is largely still a time-consuming manual process of discovering potential phishing sites, verifying if suspicious sites truly are malicious spoofs and if so, distributing their URLs to the appropriate blacklisting services. Attackers increasingly use sophisticated systems for bringing phishing sites up and down rapidly at new locations, making automated response essential. In this paper, we present a method for rapid, automated detection and analysis of phishing websites. Our method relies on near real-time gathering and analysis of URLs posted on social media sites. We fetch the pages pointed to by each URL and characterize each page with a set of easily computed values such as number of images and links. We also capture a screen-shot of the rendered page image, compute a hash of the image and use the Hamming distance between these image hashes as a form of visual comparison. We provide initial results demonstrate the feasibility of our techniques by comparing legitimate sites to known fraudulent versions from Phishtank.com, by actively introducing a series of minor changes to a phishing toolkit captured in a local honeypot and by performing some initial analysis on a set of over 2.8 million URLs posted to Twitter over a 4 days in August 2011. We discuss the issues encountered during our testing such as resolvability and legitimacy of URL's posted on Twitter, the data sets used, the characteristics of the phishing sites we discovered, and our plans for future work.

  19. Examination of a Social-Networking Site Activities Scale (SNSAS) Using Rasch Analysis

    Alhaythami, Hassan; Karpinski, Aryn; Kirschner, Paul; Bolden, Edward

    2017-01-01

    This study examined the psychometric properties of a social-networking site (SNS) activities scale (SNSAS) using Rasch Analysis. Items were also examined with Rasch Principal Components Analysis (PCA) and Differential Item Functioning (DIF) across groups of university students (i.e., males and females from the United States [US] and Europe; N =…

  20. High resolution or optimum resolution? Spatial analysis of the Federmesser site at Andernach, Germany

    Stapert, D; Street, M

    1997-01-01

    This paper discusses spatial analysis at site level. It is suggested that spatial analysis has to proceed in several levels, from global to more detailed questions, and that optimum resolution should be established when applying any quantitative methods in this field. As an example, the ring and

  1. Sampling and Analysis Instruction for the 120-F-1 Glass Dump Site

    Brown, T.M.

    1998-01-01

    This sampling and analysis instruction has been prepared to clearly define the sampling and analysis activities to be performed to develop the basis for surveillance and maintenance of the 120-F-1 Glass Dumpsite. The purpose of this investigation is to augment historical information and obtain data to establish a technical basis for surveillance and maintenance at the site

  2. Time Spent on Social Network Sites and Psychological Well-Being: A Meta-Analysis.

    Huang, Chiungjung

    2017-06-01

    This meta-analysis examines the relationship between time spent on social networking sites and psychological well-being factors, namely self-esteem, life satisfaction, loneliness, and depression. Sixty-one studies consisting of 67 independent samples involving 19,652 participants were identified. The mean correlation between time spent on social networking sites and psychological well-being was low at r = -0.07. The correlations between time spent on social networking sites and positive indicators (self-esteem and life satisfaction) were close to 0, whereas those between time spent on social networking sites and negative indicators (depression and loneliness) were weak. The effects of publication outlet, site on which users spent time, scale of time spent, and participant age and gender were not significant. As most included studies used student samples, future research should be conducted to examine this relationship for adults.

  3. An economic analysis of a monitored retrievable storage site for Tennessee. Final report and appendices

    Fox, W.F.; Mayo, J.W.; Hansen, L.T.; Quindry, K.E.

    1985-12-17

    The United States Department of Energy is charged with the task of identifying potential sites for a Monitored Retrievable Storage (MRS) Facility and reporting the results of its analysis to Congress by January 1986. DOE chose three finalist sites from 11 sites DOE analysts evaluated earlier. All three are in Tennessee, including two in Oak Ridge and one in Trousdale/Smith Counties. This paper is a summary of research undertaken on the economic effects of establishing the MRS facility in Tennessee. All three locations were considered in the analysis, but on some occasions attention is focused on the site preferred by DOE. The research was undertaken by the Center for Business and Economic Research (CBER), College of Business Administration, the University of Tennessee, Knoxville, under contract with the Tennessee Department of Economic and Community Development.

  4. An economic analysis of a monitored retrievable storage site for Tennessee

    Fox, W.F.; Mayo, J.W.; Hansen, L.T.; Quindry, K.E.

    1985-12-17

    The United States Department of Energy is charged with the task of identifying potential sites for a Monitored Retrievable Storage (MRS) Facility and reporting the results of its analysis to Congress by January 1986. DOE chose three finalist sites from 11 sites DOE analysts evaluated earlier. All three are in Tennessee, including two in Oak Ridge and one in Trousdale/Smith Counties. This paper is a summary of research undertaken on the economic effects of establishing the MRS facility in Tennessee. All three locations were considered in the analysis, but on some occasions attention is focused on the site preferred by DOE. The research was undertaken by the Center for Business and Economic Research (CBER), College of Business Administration, the University of Tennessee, Knoxville, under contract with the Tennessee Department of Economic and Community Development.

  5. Large-scale analysis of phosphorylation site occupancy in eukaryotic proteins

    Rao, R Shyama Prasad; Møller, Ian Max

    2012-01-01

    in proteins is currently lacking. We have therefore analyzed the occurrence and occupancy of phosphorylated sites (~ 100,281) in a large set of eukaryotic proteins (~ 22,995). Phosphorylation probability was found to be much higher in both the  termini of protein sequences and this is much pronounced...... maximum randomness. An analysis of phosphorylation motifs indicated that just 40 motifs and a much lower number of associated kinases might account for nearly 50% of the known phosphorylations in eukaryotic proteins. Our results provide a broad picture of the phosphorylation sites in eukaryotic proteins.......Many recent high throughput technologies have enabled large-scale discoveries of new phosphorylation sites and phosphoproteins. Although they have provided a number of insights into protein phosphorylation and the related processes, an inclusive analysis on the nature of phosphorylated sites...

  6. Application of instrumental neutron activation analysis to study ceramic fragments from Damascus Castle site, Syria

    Bakraji, E.H.

    2005-01-01

    Thirty-three archaeological ceramic fragment samples from Damascus Castle archaeological site, Damascus city, Syria, were analyzed using instrumental neutron activation analysis (INAA). 36 elements were determined. These elemental concentrations have been processed using two multivariate statistical methods, cluster and factor analysis in order to determine similarities and correlation between the various samples. Factor analysis confirms that 84.8% of the ceramics samples classified by cluster analysis are correctly classified by cluster analysis. The results provided persuasive evidence that Castle pottery used at least four different clay sources. Moreover, by means of systematic local analysis it will be clear whether these sources are local or not. (author)

  7. A new cultural cleavage in post-modern society

    Jan-Erik Lane

    2007-09-01

    Full Text Available The attitudes towards gender and homosexuality tend to be linked at the micro level (individuals, which explains the political saliency of this newly emerging cleavage. At the macro level (country, the main finding is that the value orientations towards gender and homosexuality are strongly embedded in the basic cultural or civilisation differences among countries. As developing countries modernise and enter post-modernity, they will also experience the gender cleavage, especially when they adhere to an individualistic culture. Cultural cleavages in the post-modern society, whether in rich or developing countries, can only be properly researched by the survey method. It opens up a large area for both micro and macro analyses in the social sciences.

  8. Computer analysis of protein functional sites projection on exon structure of genes in Metazoa.

    Medvedeva, Irina V; Demenkov, Pavel S; Ivanisenko, Vladimir A

    2015-01-01

    Study of the relationship between the structural and functional organization of proteins and their coding genes is necessary for an understanding of the evolution of molecular systems and can provide new knowledge for many applications for designing proteins with improved medical and biological properties. It is well known that the functional properties of proteins are determined by their functional sites. Functional sites are usually represented by a small number of amino acid residues that are distantly located from each other in the amino acid sequence. They are highly conserved within their functional group and vary significantly in structure between such groups. According to this facts analysis of the general properties of the structural organization of the functional sites at the protein level and, at the level of exon-intron structure of the coding gene is still an actual problem. One approach to this analysis is the projection of amino acid residue positions of the functional sites along with the exon boundaries to the gene structure. In this paper, we examined the discontinuity of the functional sites in the exon-intron structure of genes and the distribution of lengths and phases of the functional site encoding exons in vertebrate genes. We have shown that the DNA fragments coding the functional sites were in the same exons, or in close exons. The observed tendency to cluster the exons that code functional sites which could be considered as the unit of protein evolution. We studied the characteristics of the structure of the exon boundaries that code, and do not code, functional sites in 11 Metazoa species. This is accompanied by a reduced frequency of intercodon gaps (phase 0) in exons encoding the amino acid residue functional site, which may be evidence of the existence of evolutionary limitations to the exon shuffling. These results characterize the features of the coding exon-intron structure that affect the functionality of the encoded protein and

  9. New Insight into the Cleavage Reaction of Nostoc sp. Strain PCC 7120 Carotenoid Cleavage Dioxygenase in Natural and Nonnatural Carotenoids

    Heo, Jinsol; Kim, Se Hyeuk

    2013-01-01

    Carotenoid cleavage dioxygenases (CCDs) are enzymes that catalyze the oxidative cleavage of carotenoids at a specific double bond to generate apocarotenoids. In this study, we investigated the activity and substrate preferences of NSC3, a CCD of Nostoc sp. strain PCC 7120, in vivo and in vitro using natural and nonnatural carotenoid structures. NSC3 cleaved β-apo-8′-carotenal at 3 positions, C-13C-14, C-15C-15′, and C-13′C-14′, revealing a unique cleavage pattern. NSC3 cleaves the natural structure of carotenoids 4,4′-diaponeurosporene, 4,4′-diaponeurosporen-4′-al, 4,4′-diaponeurosporen-4′-oic acid, 4,4′-diapotorulene, and 4,4′-diapotorulen-4′-al to generate novel cleavage products (apo-14′-diaponeurosporenal, apo-13′-diaponeurosporenal, apo-10′-diaponeurosporenal, apo-14′-diapotorulenal, and apo-10′-diapotorulenal, respectively). The study of carotenoids with natural or nonnatural structures produced by using synthetic modules could provide information valuable for understanding the cleavage reactions or substrate preferences of other CCDs in vivo and in vitro. PMID:23524669

  10. Identification of Rbd2 as a candidate protease for sterol regulatory element binding protein (SREBP) cleavage in fission yeast

    Kim, Jinsil; Ha, Hye-Jeong [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of); Kim, Sujin [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of); Department of Functional Genomics, University of Science and Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 34113 (Korea, Republic of); Choi, Ah-Reum [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of); Lee, Sook-Jeong [Department of New Drug Discovery and Development, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134 (Korea, Republic of); Hoe, Kwang-Lae, E-mail: kwanghoe@cnu.ac.kr [Department of New Drug Discovery and Development, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134 (Korea, Republic of); Kim, Dong-Uk, E-mail: kimdongu@kribb.re.kr [Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141 (Korea, Republic of)

    2015-12-25

    Lipid homeostasis in mammalian cells is regulated by sterol regulatory element-binding protein (SREBP) transcription factors that are activated through sequential cleavage by Golgi Site-1 and Site-2 proteases. Fission yeast SREBP, Sre1, engages a different mechanism involving the Golgi Dsc E3 ligase complex, but it is not clearly understood exactly how Sre1 is proteolytically cleaved and activated. In this study, we screened the Schizosaccharomyces pombe non-essential haploid deletion collection to identify missing components of the Sre1 cleavage machinery. Our screen identified an additional component of the SREBP pathway required for Sre1 proteolysis named rhomboid protein 2 (Rbd2). We show that an rbd2 deletion mutant fails to grow under hypoxic and hypoxia-mimetic conditions due to lack of Sre1 activity and that this growth phenotype is rescued by Sre1N, a cleaved active form of Sre1. We found that the growth inhibition phenotype under low oxygen conditions is specific to the strain with deletion of rbd2, not any other fission yeast rhomboid-encoding genes. Our study also identified conserved residues of Rbd2 that are required for Sre1 proteolytic cleavage. All together, our results suggest that Rbd2 is a functional SREBP protease with conserved residues required for Sre1 cleavage and provide an important piece of the puzzle to understand the mechanisms for Sre1 activation and the regulation of various biological and pathological processes involving SREBPs. - Highlights: • An rbd2-deleted yeast strain shows defects in growth in response to low oxygen levels. • rbd2-deficient cells fail to generate cleaved Sre1 (Sre1N) under hypoxic conditions. • Expression of Sre1N rescues the rbd2 deletion mutant growth phenotype. • Rbd2 contains conserved residues potentially critical for catalytic activity. • Mutation of the conserved Rbd2 catalytic residues leads to defects in Sre1 cleavage.

  11. GIS and Multicriteria Decision Analysis for Landfill Site Selection in AL-HashimyahQadaa

    Alanbari, Mohammad Ali; Al-Ansari, Nadhir; Jasim, Hadeel Kareem

    2014-01-01

    Waste management system is not well regulated in Iraq. Despite the fact that there are various techniques used for solid waste disposal, landfill is the most common mode for the disposal of solid waste in Iraq, landfill site selection criteria is quite complex process and it depends on several regulation and factors. In this study landfill site selection is performed by using Multicriteria Decision Analysis (MCDA) and Geographic Information System (GIS) for the Al-Hashimyahqadaa. Existing lan...

  12. Approaches to the process of measuring and analysis of web site visits

    Stankić Rade M.

    2003-01-01

    Full Text Available How to learn more about your Web-site's visitors by using Web-traffic statistics? One of the principal methods used by online advertisers, online communities and online businesses to keep track of their visitors' behavior is to employ tracking devices. Data gathered through log-file analysis customer registration, cookies and other tracking devices can be used to personalize each visitor's experience, find trends in customer use and measure the effectiveness of a Web site over time.

  13. Analysis of the portfolio of sites to characterize for selecting a nuclear repository

    Keeney, R.L.

    1987-01-01

    The US Department of Energy has selected three sites, from five nominated, to characterize for a nuclear repository to permanently dispose of nuclear waste. This decision was made without the benefit of an analysis of this portfolio problem. This paper analyzes different portfolios of three sites for simultaneous characterization and strategies for sequential characterization. Characterization of each site, which involves significant subsurface excavation, is now estimated to cost $1 billion. Mainly because of the high characterization costs, sequential characterization strategies are identified which are the equivalent of $1.7-2.0 billion less expensive than the selected DOE simultaneous characterization of the three sites. If three sites are simultaneously characterized, one portfolio is estimated to be the equivalent of $100-400 million better than the selected DOE portfolio. Because of these potential savings and several other complicating factors that may influence the relative desirability of characterization strategies, a thorough analysis of characterization strategies that addresses the likelihood of finding disqualifying conditions during site characterization, uncertainties, and dependencies in forecast site repository costs, preclosure and postclosure health and safety impacts, potential delays of both sequential and simultaneous characterization strategies, and the environmental, socioeconomic, and health and safety impacts of characterization activities is recommended

  14. A preliminary analysis of the risk of transporting nuclear waste to potential candidate commercial repository sites

    Madsen, M.M.

    1984-01-01

    In accordance with the provisions of the Nuclear Waste Policy Act of 1982, environmental assessments for potential candidate sites are required to provide a basis for selection of the first site for disposal of commercial radioactive waste in deep geologic repositories. A preliminary analysis of the impacts of transportation for each of the five potential sites will be described. Transportation was assumed to be entirely by truck or entirely by rail in order to obtain bounding impacts. This paper presents both radiological and nonradiological risks for the once-through fuel cycle

  15. Uncertainty Analysis Framework - Hanford Site-Wide Groundwater Flow and Transport Model

    Cole, Charles R.; Bergeron, Marcel P.; Murray, Christopher J.; Thorne, Paul D.; Wurstner, Signe K.; Rogers, Phillip M.

    2001-11-09

    Pacific Northwest National Laboratory (PNNL) embarked on a new initiative to strengthen the technical defensibility of the predictions being made with a site-wide groundwater flow and transport model at the U.S. Department of Energy Hanford Site in southeastern Washington State. In FY 2000, the focus of the initiative was on the characterization of major uncertainties in the current conceptual model that would affect model predictions. The long-term goals of the initiative are the development and implementation of an uncertainty estimation methodology in future assessments and analyses using the site-wide model. This report focuses on the development and implementation of an uncertainty analysis framework.

  16. Sensitive and fast mutation detection by solid phase chemical cleavage

    Hansen, Lise Lotte; Justesen, Just; Kruse, Torben A

    1996-01-01

    We have developed a solid phase chemical cleavage method (SpCCM) for screening large DNA fragments for mutations. All reactions can be carried out in microtiterwells from the first amplification of the patient (or test) DNA through the search for mutations. The reaction time is significantly...... reduced compared to the conventional chemical cleavage method (CCM), and even by using a uniformly labelled probe, the exact position and nature of the mutation can be revealed. The SpCCM is suitable for automatization using a workstation to carry out the reactions and a fluorescent detection-based DNA...

  17. Proteolytic cleavage orchestrates cofactor insertion and protein assembly in [NiFe]-hydrogenase biosynthesis.

    Senger, Moritz; Stripp, Sven T; Soboh, Basem

    2017-07-14

    Metalloenzymes catalyze complex and essential processes, such as photosynthesis, respiration, and nitrogen fixation. For example, bacteria and archaea use [NiFe]-hydrogenases to catalyze the uptake and release of molecular hydrogen (H 2 ). [NiFe]-hydrogenases are redox enzymes composed of a large subunit that harbors a NiFe(CN) 2 CO metallo-center and a small subunit with three iron-sulfur clusters. The large subunit is synthesized with a C-terminal extension, cleaved off by a specific endopeptidase during maturation. The exact role of the C-terminal extension has remained elusive; however, cleavage takes place exclusively after assembly of the [NiFe]-cofactor and before large and small subunits form the catalytically active heterodimer. To unravel the functional role of the C-terminal extension, we used an enzymatic in vitro maturation assay that allows synthesizing functional [NiFe]-hydrogenase-2 of Escherichia coli from purified components. The maturation process included formation and insertion of the NiFe(CN) 2 CO cofactor into the large subunit, endoproteolytic cleavage of the C-terminal extension, and dimerization with the small subunit. Biochemical and spectroscopic analysis indicated that the C-terminal extension of the large subunit is essential for recognition by the maturation machinery. Only upon completion of cofactor insertion was removal of the C-terminal extension observed. Our results indicate that endoproteolytic cleavage is a central checkpoint in the maturation process. Here, cleavage temporally orchestrates cofactor insertion and protein assembly and ensures that only cofactor-containing protein can continue along the assembly line toward functional [NiFe]-hydrogenase. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Peptidomics of Peptic Digest of Selected Potato Tuber Proteins: Post-Translational Modifications and Limited Cleavage Specificity.

    C K Rajendran, Subin R; Mason, Beth; Udenigwe, Chibuike C

    2016-03-23

    Bioinformatic tools are useful in predicting bioactive peptides from food proteins. This study was focused on using bioinformatics and peptidomics to evaluate the specificity of peptide release and post-translational modifications (PTMs) in a peptic digest of potato protein isolate. Peptides in the protein hydrolysate were identified by LC-MS/MS and subsequently aligned to their parent potato tuber proteins. Five major proteins were selected for further analysis, namely, lipoxygenase, α-1,4-glucan phosphorylase, annexin, patatin, and polyubiquitin, based on protein coverage, abundance, confidence levels, and function. Comparison of the in silico peptide profile generated with ExPASy PeptideCutter and experimental peptidomics data revealed several differences. The experimental peptic cleavage sites were found to vary in number and specificity from PeptideCutter predictions. Average peptide chain length was also found to be higher than predicted with hexapeptides as the smallest detected peptides. Moreover, PTMs, particularly Met oxidation and Glu/Asp deamidation, were observed in some peptides, and these were unaccounted for during in silico analysis. PTMs can be formed during aging of potato tubers, or as a result of processing conditions during protein isolation and hydrolysis. The findings provide insights on the limitations of current bioinformatics tools for predicting bioactive peptide release from proteins, and on the existence of structural modifications that can alter the peptide bioactivity and functionality.

  19. Site-specific analysis of radiological and physical parameters for cobbly soils at the Gunnison, Colorado, processing site

    1993-10-01

    The remedial action at the Gunnison, Colorado, processing site is being performed under the Uranium Mill Tailings Radiation Control Act (UMTRCA) of 1978 [Public Law (PL) 95-6041]. Under UMTRCA, the US Environmental Protection Agency (EPA) is charged with the responsibility of developing appropriate and applicable standards for the cleanup of radiologically contaminated land and buildings at 24 designated sites, including the Gunnison, Colorado, inactive processing site. The remedial action at the processing site will be conducted to remove the tailings and contaminated materials to meet the EPA bulk soil cleanup standards for surface and subsurface soils. The site areas disturbed by remedial action excavation will be either contoured or backfilled with radiologically uncontaminated soil and contoured to restore the site. The final contours will produce a final surface grade that will create positive drainage from the site

  20. Environmental analysis of the operation of Oak Ridge National Laboratory (X-10 site)

    Boyle, J.W.; Blumberg, R.; Cotter, S.J.

    1982-11-01

    An environmental analysis of the operation of the Oak Ridge National Laboratory (ORNL) facilities in Bethel Valley and Melton Valley was conducted to present to the public information concerning the extent to which recognizable effects, or potential effects, on the environment may occur. The analysis addresses current operations of the ORNL X-10 site and completed operations that may continue to have residual effects. Solid wastes from ORNL operations at the Y-12 site which are transported to the X-10 site for burial (e.g., Biology Division animal wastes) are included as part of X-10 site operation. Socioeconomic effects are associated primarily with the communities where employees live and with the Knoxville Bureau of Economic Analysis economic area as a whole. Therefore, ORNL employees at both Y-12 and X-10 sites are included in the ORNL socioeconomic impact analysis. An extensive base of environmental data was accumulated for this report. Over 80 reports related to ORNL facilities and/or operations are cited as well as many open-literature citations. Environmental effects of the operation of ORNL result from operational discharges from the onsite facilities; construction and/or modification of facilities, transportation to and from the site of persons, goods and services; socioeconomic impacts to the local, regional, and general population; and accidental discharges if they should occur. Operational discharges to the environnment are constrained by federal, state, and local regulations and by criteria established by the US Department of Energy to minimize adverse impacts. It is the purpose of this document to evaluate the operation of the ORNL insofar as impacts beyond the site boundary may occur or have the potential for occurrence.

  1. Environmental analysis of closure options for waste sites at the Savannah River Plant

    Gordon, D.E.; King, C.M.; Looney, B.B.; Stephenson, D.E.; Johnson, W.F.

    1987-01-01

    Previously acceptable waste management practices (e.g., the use of unlined seepage basins) for discarding of wastes from nuclear materials production has resulted in occasional cases of groundwater contamination beneath some disposal sites, mainly in water-table aquifers. Groundwater contaminants include volatile organic compounds, heavy metals, radionuclides, and other chemicals. The closure of active and inactive waste sites that have received hazardous and/or low-level radioactive materials at the Savannah River Plant (SRP) is planned as part of an overall program to protect groundwater quality. DOE developed and submitted to Congress a groundwater protection plan for SRP. This initial plan and subsequent revisions provide the basis for closure of SRP waste sites to comply with applicable groundwater protection requirements. An environmental analysis of the closure options for the criteria waste sites that have received hazardous and/or low-level radioactive wastes was conducted to provide technical support. The several parts of this environmental analysis include description of geohydrologic conditions; determination of waste inventories; definition of closure options; modeling of environmental pathways; assessment of risk; and analysis of project costs. Each of these components of the overall analysis is described in turn in the following paragraphs. Production operations at SRP have generated a variety of solid, hazardous, and low-level radioactive waste materials. Several locations onplant have been used as waste disposal sites for solid and liquid wastes. Seventy-six individual waste sites at 45 distinct geographical locations on SRP have received hazardous, low-level radioactive, or mixed wastes. These waste sites can be categorized into 26 groupings according to the function of the waste disposed. 15 refs., 6 figs., 5 tabs

  2. Environmental analysis of the operation of Oak Ridge National Laboratory (X-10 site)

    Boyle, J.W.; Blumberg, R.; Cotter, S.J.

    1982-11-01

    An environmental analysis of the operation of the Oak Ridge National Laboratory (ORNL) facilities in Bethel Valley and Melton Valley was conducted to present to the public information concerning the extent to which recognizable effects, or potential effects, on the environment may occur. The analysis addresses current operations of the ORNL X-10 site and completed operations that may continue to have residual effects. Solid wastes from ORNL operations at the Y-12 site which are transported to the X-10 site for burial (e.g., Biology Division animal wastes) are included as part of X-10 site operation. Socioeconomic effects are associated primarily with the communities where employees live and with the Knoxville Bureau of Economic Analysis economic area as a whole. Therefore, ORNL employees at both Y-12 and X-10 sites are included in the ORNL socioeconomic impact analysis. An extensive base of environmental data was accumulated for this report. Over 80 reports related to ORNL facilities and/or operations are cited as well as many open-literature citations. Environmental effects of the operation of ORNL result from operational discharges from the onsite facilities; construction and/or modification of facilities, transportation to and from the site of persons, goods and services; socioeconomic impacts to the local, regional, and general population; and accidental discharges if they should occur. Operational discharges to the environnment are constrained by federal, state, and local regulations and by criteria established by the US Department of Energy to minimize adverse impacts. It is the purpose of this document to evaluate the operation of the ORNL insofar as impacts beyond the site boundary may occur or have the potential for occurrence

  3. Special Analysis of the Area 3 Radioactive Waste Management Site at the Nevada National Security Site, Nye County, Nevada

    National Security Technologies, LLC, Environmental Management

    2012-09-30

    This report describes the methods and results of a special analysis (SA) of the Area 3 Radioactive Waste Management Site (RWMS) on the Nevada National Security Site (NNSS). The purpose of the SA is to determine if the approved performance assessment (PA) and composite analysis (CA) (Shott et al., 2001) remain valid. The Area 3 RWMS PA and CA were prepared as a single document and received conditional approval on October 6, 1999. A conditional Disposal Authorization Statement (DAS) for the Area 3 RWMS was issued on October 20, 1999. Since preparation of the approved PA and CA, new information and additional environmental monitoring data have been used to update the PA and CA. At the same time, continual advancements in computer processors and software have allowed improvement to the PA and CA models. Annual reviews of the PA and CA required by U.S. Department of Energy (DOE) Order DOE O 435.1 have documented multiple changes occurring since preparation of the PA and CA. Potentially important changes include: Development of a new and improved baseline PA and CA model implemented in the probabilistic GoldSim simulation platform. A significant increase in the waste inventory disposed at the site. Revision and updating of model parameters based on additional years of site monitoring data and new research and development results. Although changes have occurred, many important PA/CA issues remain unchanged, including the site conceptual model, important features, events, and processes, and the points of compliance. The SA is performed to document the current status of the PA/CA model and to quantitatively assess the impact of cumulative changes on the PA and CA results. The results of the SA are used to assess the validity of the approved PA/CA and make a determination if revision of the PA or CA is necessary. The SA was performed using the Area 3 RWMS, version 2.102, GoldSim model, the current baseline PA/CA model. Comparison of the maximum SA results with the PA

  4. Food and Beverage Brands that Market to Children and Adolescents on the Internet: A Content Analysis of Branded Web Sites

    Henry, Anna E.; Story, Mary

    2009-01-01

    Objective: To identify food and beverage brand Web sites featuring designated children's areas, assess marketing techniques present on those industry Web sites, and determine nutritional quality of branded food items marketed to children. Design: Systematic content analysis of food and beverage brand Web sites and nutrient analysis of food and…

  5. The Conformational Dynamics of Cas9 Governing DNA Cleavage Are Revealed by Single-Molecule FRET.

    Yang, Mengyi; Peng, Sijia; Sun, Ruirui; Lin, Jingdi; Wang, Nan; Chen, Chunlai

    2018-01-09

    Off-target binding and cleavage by Cas9 pose major challenges in its application. How the conformational dynamics of Cas9 govern its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single-molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms spontaneously transits among three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We also uncovered surprising long-range allosteric communication between the HNH domain and the RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated that a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the PAM-distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  6. Impaired Cleavage of Preproinsulin Signal Peptide Linked to Autosomal-Dominant Diabetes

    Liu, Ming; Lara-Lemus, Roberto; Shan, Shu-ou; Wright, Jordan; Haataja, Leena; Barbetti, Fabrizio; Guo, Huan; Larkin, Dennis; Arvan, Peter

    2012-01-01

    Recently, missense mutations upstream of preproinsulin’s signal peptide (SP) cleavage site were reported to cause mutant INS gene-induced diabetes of youth (MIDY). Our objective was to understand the molecular pathogenesis using metabolic labeling and assays of proinsulin export and insulin and C-peptide production to examine the earliest events of insulin biosynthesis, highlighting molecular mechanisms underlying β-cell failure plus a novel strategy that might ameliorate the MIDY syndrome. We find that whereas preproinsulin-A(SP23)S is efficiently cleaved, producing authentic proinsulin and insulin, preproinsulin-A(SP24)D is inefficiently cleaved at an improper site, producing two subpopulations of molecules. Both show impaired oxidative folding and are retained in the endoplasmic reticulum (ER). Preproinsulin-A(SP24)D also blocks ER exit of coexpressed wild-type proinsulin, accounting for its dominant-negative behavior. Upon increased expression of ER–oxidoreductin-1, preproinsulin-A(SP24)D remains blocked but oxidative folding of wild-type proinsulin improves, accelerating its ER export and increasing wild-type insulin production. We conclude that the efficiency of SP cleavage is linked to the oxidation of (pre)proinsulin. In turn, impaired (pre)proinsulin oxidation affects ER export of the mutant as well as that of coexpressed wild-type proinsulin. Improving oxidative folding of wild-type proinsulin may provide a feasible way to rescue insulin production in patients with MIDY. PMID:22357960

  7. TRAIL-induced cleavage and inactivation of SPAK sensitizes cells to apoptosis

    Polek, Tara C.; Talpaz, Moshe; Spivak-Kroizman, Taly R.

    2006-01-01

    Ste20-related proline-alanine-rich kinase (SPAK) has been linked to various cellular processes, including proliferation, differentiation, and ion transport regulation. Recently, we showed that SPAK mediates signaling by the TNF receptor, RELT. The presence of a caspase cleavage site in SPAK prompted us to study its involvement in apoptotic signaling induced by another TNF member, TRAIL. We show that TRAIL stimulated caspase 3-like proteases that cleaved SPAK at two distinct sites. Cleavage had little effect on the activity of SPAK but removed its substrate-binding domain. In addition, TRAIL reduced the activity of SPAK in HeLa cells in a caspase-independent manner. Thus, TRAIL inhibited SPAK by two mechanisms: activation of caspases, which removed its substrate-binding domain, and caspase-independent down-regulation of SPAK activity. Furthermore, reducing the amount of SPAK by siRNA increased the sensitivity of HeLa cells to TRAIL-induced apoptosis. Thus, TRAIL down-regulation of SPAK is an important event that enhances its apoptotic effects

  8. Artificial neural network for on-site quantitative analysis of soils using laser induced breakdown spectroscopy

    El Haddad, J. [Univ. Bordeaux, LOMA, UMR 5798, F-33400 Talence (France); CNRS, LOMA, UMR 5798, F-33400 Talence (France); Villot-Kadri, M.; Ismaël, A.; Gallou, G. [IVEA Solution, Centre Scientifique d' Orsay, Bât 503, 91400 Orsay (France); Michel, K.; Bruyère, D.; Laperche, V. [BRGM, Service Métrologie, Monitoring et Analyse, 3 avenue Claude Guillemin, B.P 36009, 45060 Orléans Cedex (France); Canioni, L. [Univ. Bordeaux, LOMA, UMR 5798, F-33400 Talence (France); CNRS, LOMA, UMR 5798, F-33400 Talence (France); Bousquet, B., E-mail: bruno.bousquet@u-bordeaux1.fr [Univ. Bordeaux, LOMA, UMR 5798, F-33400 Talence (France); CNRS, LOMA, UMR 5798, F-33400 Talence (France)

    2013-01-01

    Nowadays, due to environmental concerns, fast on-site quantitative analyses of soils are required. Laser induced breakdown spectroscopy is a serious candidate to address this challenge and is especially well suited for multi-elemental analysis of heavy metals. However, saturation and matrix effects prevent from a simple treatment of the LIBS data, namely through a regular calibration curve. This paper details the limits of this approach and consequently emphasizes the advantage of using artificial neural networks well suited for non-linear and multi-variate calibration. This advanced method of data analysis is evaluated in the case of real soil samples and on-site LIBS measurements. The selection of the LIBS data as input data of the network is particularly detailed and finally, resulting errors of prediction lower than 20% for aluminum, calcium, copper and iron demonstrate the good efficiency of the artificial neural networks for on-site quantitative LIBS of soils. - Highlights: ► We perform on-site quantitative LIBS analysis of soil samples. ► We demonstrate that univariate analysis is not convenient. ► We exploit artificial neural networks for LIBS analysis. ► Spectral lines other than the ones from the analyte must be introduced.

  9. Artificial neural network for on-site quantitative analysis of soils using laser induced breakdown spectroscopy

    El Haddad, J.; Villot-Kadri, M.; Ismaël, A.; Gallou, G.; Michel, K.; Bruyère, D.; Laperche, V.; Canioni, L.; Bousquet, B.

    2013-01-01

    Nowadays, due to environmental concerns, fast on-site quantitative analyses of soils are required. Laser induced breakdown spectroscopy is a serious candidate to address this challenge and is especially well suited for multi-elemental analysis of heavy metals. However, saturation and matrix effects prevent from a simple treatment of the LIBS data, namely through a regular calibration curve. This paper details the limits of this approach and consequently emphasizes the advantage of using artificial neural networks well suited for non-linear and multi-variate calibration. This advanced method of data analysis is evaluated in the case of real soil samples and on-site LIBS measurements. The selection of the LIBS data as input data of the network is particularly detailed and finally, resulting errors of prediction lower than 20% for aluminum, calcium, copper and iron demonstrate the good efficiency of the artificial neural networks for on-site quantitative LIBS of soils. - Highlights: ► We perform on-site quantitative LIBS analysis of soil samples. ► We demonstrate that univariate analysis is not convenient. ► We exploit artificial neural networks for LIBS analysis. ► Spectral lines other than the ones from the analyte must be introduced

  10. Integrative Analysis of CRISPR/Cas9 Target Sites in the Human HBB Gene

    Yumei Luo

    2015-01-01

    Full Text Available Recently, the clustered regularly interspaced short palindromic repeats (CRISPR system has emerged as a powerful customizable artificial nuclease to facilitate precise genetic correction for tissue regeneration and isogenic disease modeling. However, previous studies reported substantial off-target activities of CRISPR system in human cells, and the enormous putative off-target sites are labor-intensive to be validated experimentally, thus motivating bioinformatics methods for rational design of CRISPR system and prediction of its potential off-target effects. Here, we describe an integrative analytical process to identify specific CRISPR target sites in the human β-globin gene (HBB and predict their off-target effects. Our method includes off-target analysis in both coding and noncoding regions, which was neglected by previous studies. It was found that the CRISPR target sites in the introns have fewer off-target sites in the coding regions than those in the exons. Remarkably, target sites containing certain transcriptional factor motif have enriched binding sites of relevant transcriptional factor in their off-target sets. We also found that the intron sites have fewer SNPs, which leads to less variation of CRISPR efficiency in different individuals during clinical applications. Our studies provide a standard analytical procedure to select specific CRISPR targets for genetic correction.

  11. Supporting documents for LLL area 27 (410 area) safety analysis reports, Nevada Test Site

    Odell, B. N. [comp.

    1977-02-01

    The following appendices are common to the LLL Safety Analysis Reports Nevada Test Site and are included here as supporting documents to those reports: Environmental Monitoring Report for the Nevada Test Site and Other Test Areas Used for Underground Nuclear Detonations, U. S. Environmental Protection Agency, Las Vegas, Rept. EMSL-LV-539-4 (1976); Selected Census Information Around the Nevada Test Site, U. S. Environmental Protection Agency, Las Vegas, Rept. NERC-LV-539-8 (1973); W. J. Hannon and H. L. McKague, An Examination of the Geology and Seismology Associated with Area 410 at the Nevada Test Site, Lawrence Livermore Laboratory, Livermore, Rept. UCRL-51830 (1975); K. R. Peterson, Diffusion Climatology for Hypothetical Accidents in Area 410 of the Nevada Test Site, Lawrence Livermore Laboratory, Livermore, Rept. UCRL-52074 (1976); J. R. McDonald, J. E. Minor, and K. C. Mehta, Development of a Design Basis Tornado and Structural Design Criteria for the Nevada Test Site, Nevada, Lawrence Livermore Laboratory, Livermore, Rept. UCRL-13668 (1975); A. E. Stevenson, Impact Tests of Wind-Borne Wooden Missiles, Sandia Laboratories, Tonopah, Rept. SAND 76-0407 (1976); and Hydrology of the 410 Area (Area 27) at the Nevada Test Site.

  12. Site-specific analysis of radiological and physical parameters for cobbly soils at the Gunnison, Colorado, processing site

    1994-01-01

    The remedial action at the Gunnison, Colorado, processing site is being performed under the Uranium Mill Tailings Radiation Control Act (UMTRCA) of 1978. Under UMTRCA, the US Environmental Protection Agency (EPA) is charged with the responsibility of developing appropriate and applicable standards for the cleanup of radiologically contaminated land and buildings at 24 designated sites, including the Gunnison, Colorado, inactive processing site. Section 108 of Public Law 95-604 states that the US Department of Energy (DOE) shall ''select and perform remedial actions at the designated processing sites and disposal sites in accordance with the general standards'' prescribed by the EPA. Regulations governing the required remedial action at inactive uranium processing sites were promulgated by the EPA in 1983 and are contained in 40 CFR Part 192 (1993), Health and Environmental Protection Standards for Uranium and Thorium Mill Tailings. This document describes the radiological and physical parameters for the remedial action of the soil

  13. Site Classification using Multichannel Channel Analysis of Surface Wave (MASW) method on Soft and Hard Ground

    Ashraf, M. A. M.; Kumar, N. S.; Yusoh, R.; Hazreek, Z. A. M.; Aziman, M.

    2018-04-01

    Site classification utilizing average shear wave velocity (Vs(30) up to 30 meters depth is a typical parameter. Numerous geophysical methods have been proposed for estimation of shear wave velocity by utilizing assortment of testing configuration, processing method, and inversion algorithm. Multichannel Analysis of Surface Wave (MASW) method is been rehearsed by numerous specialist and professional to geotechnical engineering for local site characterization and classification. This study aims to determine the site classification on soft and hard ground using MASW method. The subsurface classification was made utilizing National Earthquake Hazards Reduction Program (NERHP) and international Building Code (IBC) classification. Two sites are chosen to acquire the shear wave velocity which is in the state of Pulau Pinang for soft soil and Perlis for hard rock. Results recommend that MASW technique can be utilized to spatially calculate the distribution of shear wave velocity (Vs(30)) in soil and rock to characterize areas.

  14. Guidance document for preparing water sampling and analysis plans for UMTRA Project sites. Revision 1

    1995-09-01

    A water sampling and analysis plan (WSAP) is prepared for each Uranium Mill Tailings Remedial Action (UMTRA) Project site to provide the rationale for routine ground water sampling at disposal sites and former processing sites. The WSAP identifies and justifies the sampling locations, analytical parameters, detection limits, and sampling frequency for the routine ground water monitoring stations at each site. This guidance document has been prepared by the Technical Assistance Contractor (TAC) for the US Department of Energy (DOE). Its purpose is to provide a consistent technical approach for sampling and monitoring activities performed under the WSAP and to provide a consistent format for the WSAP documents. It is designed for use by the TAC in preparing WSAPs and by the DOE, US Nuclear Regulatory Commission, state and tribal agencies, other regulatory agencies, and the public in evaluating the content of WSAPS

  15. Maintenance Plan for the Composite Analysis of the Hanford Site, Southeast Washington.

    Lehman, L. L. [Hanford Site (HNF), Richland, WA (United States); Nichols, W. E. [Hanford Site (HNF), Richland, WA (United States)

    2018-02-08

    The U.S. Department of Energy (DOE) manuals for radioactive waste management, DOE M 435.1-1 Chg 21 and DOE-STD-5002-2017, require that the Hanford Site maintain site performance assessments and composite analyses (CAs). This document describes the plan for maintaining the CA that supports waste disposal and remedial actions for the Hanford Site. An initial CA of the site was issued in 1998, conditionally approved in 1999, received further analysis to satisfy conditions in an addendum in 2001, and was approved in 2002. This document meets the maintenance plan requirements described in DOE M 435.1-1 Chg 2 and DOE-STD-5002-2017 and implements the requirements of the disposal authorization related to the CA for the U.S. Department of Energy, Richland Operations Office, the responsible field office, and its contractors.

  16. DNA Cleavage Activity of Diazonium Salts: Chemical Nucleases

    KIZIL, Murat

    2014-01-01

    4-Fenoldiazonium tetrafluoroborate and 4-benzoicaciddiazonium tetrafluoroborate was prepared and was shown to be an effective DNA cleavage agent in the presence of the 1-electron donor copper(II) chloride. Its mechanism involves the generation of the aryl radical cleaving DNA by hydrogen atom abstraction from deoxyribose sugar.

  17. Fe-Catalyzed Oxidative Cleavage of Unsaturated Fatty Acids

    Spannring, P.

    2013-01-01

    The oxidative cleavage of unsaturated fatty acids into aldehydes or carboxylic acids gives access to valuable products. The products can be used as chemical building blocks, as emulsifiers or in the paint or polymer industry. Ozonolysis is applied industrially to cleave the fatty acid oleic acid

  18. Kinetics of phycocyanobilin cleavage from C-phycocyanin by methanolysis

    Malwade, Chandrakant Ramkrishna; Roda Serrat, Maria Cinta; Christensen, Knud Villy

    2016-01-01

    Phycocyanobilin (PCB) is an important linear tetrapyrrolic molecule for food as well as pharmaceutical industry. It is obtained from blue-green algae, where it is attached covalently to phycobiliproteins (C-PC and APC) present in the light harvesting complexes. In this work, cleavage of PCB from...

  19. Y-12 site-specific earthquake response analysis and soil liquefaction assessment

    Ahmed, S.B.; Hunt, R.J.; Manrod, W.E. III.

    1995-01-01

    A site-specific earthquake response analysis and soil liquefaction assessment were performed for the Oak Ridge Y-12 Plant. The main purpose of these studies was to use the results of the analyses for evaluating the safety of the performance category -1, -2, and -3 facilities against the natural phenomena seismic hazards. Earthquake response was determined for seven (7), one dimensional soil columns (Fig. 12) using two horizontal components of the PC-3 design basis 2000-year seismic event. The computer program SHAKE 91 (Ref. 7) was used to calculate the absolute response accelerations on top of ground (soil/weathered shale) and rock outcrop. The SHAKE program has been validated for horizontal response calculations at periods less than 2.0 second at several sites and consequently is widely accepted in the geotechnical earthquake engineering area for site response analysis

  20. Addendum 1 Composite Analysis for the Area 5 Radioactive Waste Management Site at the Nevada Test Site, Nye County, Nevada

    Vefa Yucel

    2001-01-01

    A disposal authorization statement (DAS) was issued by the U.S. Department of Energy/Headquarters (DOE/HQ) on December 5, 2000, authorizing the DOE's National Nuclear Security Administration Nevada Operations Office to continue the operation of the Area 5 Radioactive Waste Management Site (RWMS) at the Nevada Test Site for the disposal of low-level waste and mixed low-level waste. Prior to the issuance of the DAS, the Low-Level Waste Disposal Facility Federal Review Group (LFRG) had conducted reviews of the performance assessment (PA) and the composite analysis (CA) for the Area 5 RWMS, in accordance with the requirements of the DOE Radioactive Waste Management Order DOE O 435.1. A brief history of the reviews is as follows. (The reviews were conducted by independent review teams chartered by the LFRG; the review findings and recommendations were issued in review team reports to the LFRG.) The LFRG accepted the initial PA, with conditions, on August 30, 1996. Revision 2.1 to the PA was issued in January 1998, implementing the conditions of acceptance of the 1996 PA. The LFRG reviewed Revision 2.1 as part of the Area 5 RWMS CA review during 2000, and found it acceptable. The CA and the Supplemental Information provided in response to issues identified during the initial review of the CA were accepted by the LFRG. The Supplemental Information (including the responses to four key issues) is included in the Review Team Report to the LFRG, which recommends that it be incorporated into the CA and issued to all known holders of the CA. The Area 5 RWMS DAS requires that the Supplemental Information generated during the DOE/HQ review of the CA be incorporated into the CA within one year of the date of issuance of the DAS. This report, the first addendum to the Area 5 CA, is prepared to fulfill that requirement. The Supplemental Information includes the following: Issues Identified in the Review Team Report; Crosswalk Presentation; and Maintaining Doses As Low As Reasonably

  1. Water-Level Data Analysis for the Saturated Zone Site-Scale Flow and Transport Model

    Tucci, P.

    2001-01-01

    This Analysis/Model Report (AMR) documents an updated analysis of water-level data performed to provide the saturated-zone, site-scale flow and transport model (CRWMS M and O 2000) with the configuration of the potentiometric surface, target water-level data, and hydraulic gradients for model calibration. The previous analysis was presented in ANL-NBS-HS-000034, Rev 00 ICN 01, Water-Level Data Analysis for the Saturated Zone Site-Scale Flow and Transport Model (USGS 2001). This analysis is designed to use updated water-level data as the basis for estimating water-level altitudes and the potentiometric surface in the SZ site-scale flow and transport model domain. The objectives of this revision are to develop computer files containing (1) water-level data within the model area (DTN: GS010908312332.002), (2) a table of known vertical head differences (DTN: GS0109083 12332.003), and (3) a potentiometric-surface map (DTN: GS010608312332.001) using an alternate concept from that presented in ANL-NBS-HS-000034, Rev 00 ICN 01 for the area north of Yucca Mountain. The updated water-level data include data obtained from the Nye County Early Warning Drilling Program (EWDP) and data from borehole USW WT-24. In addition to being utilized by the SZ site-scale flow and transport model, the water-level data and potentiometric-surface map contained within this report will be available to other government agencies and water users for ground-water management purposes. The potentiometric surface defines an upper boundary of the site-scale flow model, as well as provides information useful to estimation of the magnitude and direction of lateral ground-water flow within the flow system. Therefore, the analysis documented in this revision is important to SZ flow and transport calculations in support of total system performance assessment

  2. Analysis of biomolecular solvation sites by 3D-RISM theory.

    Sindhikara, Daniel J; Hirata, Fumio

    2013-06-06

    We derive, implement, and apply equilibrium solvation site analysis for biomolecules. Our method utilizes 3D-RISM calculations to quickly obtain equilibrium solvent distributions without either necessity of simulation or limits of solvent sampling. Our analysis of these distributions extracts highest likelihood poses of solvent as well as localized entropies, enthalpies, and solvation free energies. We demonstrate our method on a structure of HIV-1 protease where excellent structural and thermodynamic data are available for comparison. Our results, obtained within minutes, show systematic agreement with available experimental data. Further, our results are in good agreement with established simulation-based solvent analysis methods. This method can be used not only for visual analysis of active site solvation but also for virtual screening methods and experimental refinement.

  3. Groundwater flow analysis on local scale. Setting boundary conditions for groundwater flow analysis on site scale model in step 1

    Ohyama, Takuya; Saegusa, Hiromitsu; Onoe, Hironori

    2005-05-01

    Japan Nuclear Cycle Development Institute has been conducting a wide range of geoscientific research in order to build a foundation for multidisciplinary studies of the deep geological environment as a basis of research and development for geological disposal of nuclear wastes. Ongoing geoscientific research programs include the Regional Hydrogeological Study (RHS) project and Mizunami Underground Research Laboratory (MIU) project in the Tono region, Gifu Prefecture. The main goal of these projects is to establish comprehensive techniques for investigation, analysis, and assessment of the deep geological environment at several spatial scales. The RHS project is a local scale study for understanding the groundwater flow system from the recharge area to the discharge area. The surface-based Investigation Phase of the MIU project is a site scale study for understanding the groundwater flow system immediately surrounding the MIU construction site. The MIU project is being conducted using a multiphase, iterative approach. In this study, the hydrogeological modeling and groundwater flow analysis of the local scale were carried out in order to set boundary conditions of the site scale model based on the data obtained from surface-based investigations in Step 1 in site scale of the MIU project. As a result of the study, head distribution to set boundary conditions for groundwater flow analysis on the site scale model could be obtained. (author)

  4. Bias and Bias Correction in Multi-Site Instrumental Variables Analysis of Heterogeneous Mediator Effects

    Reardon, Sean F.; Unlu, Faith; Zhu, Pei; Bloom, Howard

    2013-01-01

    We explore the use of instrumental variables (IV) analysis with a multi-site randomized trial to estimate the effect of a mediating variable on an outcome in cases where it can be assumed that the observed mediator is the only mechanism linking treatment assignment to outcomes, as assumption known in the instrumental variables literature as the…

  5. A Content Analysis of U.S. Botanical and Horticultural Library Web Sites.

    Meyers, Michele M.

    The purpose of this study was to provide an introductory analysis of the content of the Web sites of botanical and horticultural libraries in the United States to determine what types of resources and information is included, whether or not information is organized and accessible, and to what extent botanical and horticultural libraries are using…

  6. Chemical Analysis of the Moon at the Surveyor VII Landing Site: Preliminary Results.

    Turkevich, A L; Franzgrote, E J; Patterson, J H

    1968-10-04

    The alpha-scattering experiment aboard Surveyor VII has provided a chemical analysis of the moon in the area of the crater Tycho. The preliminary results indicate a chemical composition similar to that already found at two mare sites, but with a lower concentration of elements of the iron group (titanium through copper).

  7. Security Analysis of the Electronic Management System for a Total Site Utility System

    Manso Cortes, Oscar

    2016-01-01

    This paper presents the Security Analysis of the Electronic Management System (EMS) of a Total Site Utility System as proposed under the scope of the Efenis project. The Efenis project has been funded by the European Commission via the seventh framework programme (EC FP7) with the aim to improve ...

  8. Identification of the provenience of Majolica from sites in the Caribbean using neutron activation analysis

    Olin, J.S.; Sayre, E.V.

    1975-01-01

    Tin-enamelled earthenware pottery from five early Spanish Colonial sites in the Dominican Republic and Venezuela were sampled and analyzed by neutron activation analysis in an attempt to determine whether these sherds had a common source. The tentative conclusion was that although several sources were indicated for the specimens analyzed the overall similarity in composition indicated that these sources were probably closely related

  9. 40 CFR 60.1130 - How do I make my siting analysis available to the public?

    2010-07-01

    ... separation plan. Include the normal business hours of each library. (5) An agenda of the topics that will be...) Distribute your siting analysis and revised materials separation plan to the main public libraries in the... public meeting. (2) The time of the public meeting. (3) The location of the public meeting. (4) The...

  10. Seismic hazard analysis for the NTS spent reactor fuel test site

    Campbell, K.W.

    1980-01-01

    An experiment is being directed at the Nevada Test Site to test the feasibility for storage of spent fuel from nuclear reactors in geologic media. As part of this project, an analysis of the earthquake hazard was prepared. This report presents the results of this seismic hazard assessment. Two distinct components of the seismic hazard were addressed: vibratory ground motion and surface displacement

  11. Social Capital, Self-Esteem, and Use of Online Social Network Sites: A Longitudinal Analysis

    Steinfield, Charles; Ellison, Nicole B.; Lampe, Cliff

    2008-01-01

    A longitudinal analysis of panel data from users of a popular online social network site, Facebook, investigated the relationship between intensity of Facebook use, measures of psychological well-being, and bridging social capital. Two surveys conducted a year apart at a large U.S. university, complemented with in-depth interviews with 18 Facebook…

  12. Radiological analysis of materials sampled on the old nuclear test site of In Ekker (Algeria)

    Chareyron, Bruno

    2010-01-01

    After having recalled the context of the French nuclear test campaign in Algeria between 1961 and 1966, this document reports and comments radiological measurements performed on the site of In Ekker, and also results of analysis performed in laboratory (contamination by cesium 137, americium 241, plutonium); recommendations are given

  13. Artificially regenerating longleaf pine on wet sites: preliminary analysis of effects of site preparation treatments on early survival and growth

    Benjamin O. Knapp; G. Geoff Wang; Joan L. Walker

    2010-01-01

    Our study, conducted over two years on poorly drained, sandy sites in Onslow County, NC, compared the effects of eight common site preparation treatments on early survival and growth of planted longleaf pine seedlings. Through two growing seasons, we found survival to be similar across all treatments (p = 0.8806), but root collar diameter was greatest with combinations...

  14. Cleavage of influenza RNA by using a human PUF-based artificial RNA-binding protein–staphylococcal nuclease hybrid

    Mori, Tomoaki; Nakamura, Kento; Masaoka, Keisuke; Fujita, Yusuke; Morisada, Ryosuke; Mori, Koichi; Tobimatsu, Takamasa; Sera, Takashi

    2016-01-01

    Various viruses infect animals and humans and cause a variety of diseases, including cancer. However, effective methodologies to prevent virus infection have not yet been established. Therefore, development of technologies to inactivate viruses is highly desired. We have already demonstrated that cleavage of a DNA virus genome was effective to prevent its replication. Here, we expanded this methodology to RNA viruses. In the present study, we used staphylococcal nuclease (SNase) instead of the PIN domain (PilT N-terminus) of human SMG6 as an RNA-cleavage domain and fused the SNase to a human Pumilio/fem-3 binding factor (PUF)-based artificial RNA-binding protein to construct an artificial RNA restriction enzyme with enhanced RNA-cleavage rates for influenzavirus. The resulting SNase-fusion nuclease cleaved influenza RNA at rates 120-fold greater than the corresponding PIN-fusion nuclease. The cleaving ability of the PIN-fusion nuclease was not improved even though the linker moiety between the PUF and RNA-cleavage domain was changed. Gel shift assays revealed that the RNA-binding properties of the PUF derivative used was not as good as wild type PUF. Improvement of the binding properties or the design method will allow the SNase-fusion nuclease to cleave an RNA target in mammalian animal cells and/or organisms. - Highlights: • A novel RNA restriction enzyme using SNase was developed tor cleave viral RNA. • Our enzyme cleaved influenza RNA with rates >120-fold higher rates a PIN-fusion one. • Our artificial enzyme with the L5 linker showed the highest RNA cleavage rate. • Our artificial enzyme site-selectively cleaved influenza RNA in vitro.

  15. Regular monitoring, analysis and forecast of radioecological environment of Azgir test site

    Akhmetov, E.; Agymov, I.; Gilmanov, Zh.; Ermanov, A.; Zhetbaev, A.

    1996-01-01

    The objective of investigations: basing on the results of regular annual measurements of radiation conditions on the sites of underground nuclear cavities of the Azgir test site, specific concentrations of radionuclides and heavy metals in soil and underground aquifers on the test site and adjacent territories to obtain data on migration and transfer of radionuclides and heavy metals. This will give a real possibility to make probability predictions of ways and qualitative characteristics of spreading of radionuclides and heavy metals in the region of the northern Pricaspian lowland. The Essence of the Problem The Azgir test site is located in the arid zone of the Great Azgir salt cupola near the Azgir village of Kurmangazinskiy rayon, Atyrau region. This cupola is located in the western periphery of Pricaspian salt-bearing province situated to the north of the Caspian sea between the Volga and Emba rivers. Major Tasks: - Development of technical requirements for carrying out regular examination of radionuclide and heavy metal contamination of the Azgir test site. - Preparation of material and technical base for field works on the Azgir test site. - Radiometric measurements on the sites and around them. - Taking of soil, soil and ground waters samples both on the test site and on the adjacent territories. - Spectrometric and radiochemical investigations of soil, soil and ground water samples. - Analysis and generalization of the results creating premises for forecasting of the radioecological conditions. - Investigation of the possibility of radioactive waste disposal in underground cavities. Expected Results: - Detection and outlining of local areas of radioactive contamination on the site and adjacent territories. - Data on real structure of spreading and concentration of artificial and natural radionuclides and heavy metals in soil layer of the test site region. - Results of analytic investigations of water samples of underground sources of the site and adjacent

  16. System for the hydrogeologic analysis of uranium mill waste disposal sites

    Osiensky, J.L.

    1983-01-01

    Most of the uranium mill wastes generated before 1977 are stored in unlined tailings ponds. Seepage from some of these ponds has been of sufficient severity that the US Nuclear Regulatory Commission (NRC) has required the installation of withdrawal wells to remove the contaminated groundwater. Uranium mill waste disposal facilities typically are located in complex hydrogeologic environments. This research was initiated in 1980 to analyze hydrogeologic data collected at seven disposal sites in the US that have experienced problems with groundwater contamination. The characteristics of seepage migration are site specific and are controlled by the hydrogeologic environment in the vicinity of each tailings pond. Careful monitoring of most seepage plumes was not initiated until approximately 1977. These efforts were accelerated as a consequence of the uranium Mill Tailings Act of 1979. Some of the data collected at uranium mill waste disposal sites in the past are incomplete and some were collected by methods that are outdated. Data frequently were collected in sequences which disrupted the continuity of the hydrogeologic analysis and decreased the effectiveness of the data collection programs. Evaluation of data collection programs for seven uranium mill waste disposal sites in the US has led to the development and presentation herein of a system for the hydrogeologic analysis of disposal sites

  17. Concrete structural analysis tools and properties for Hanford site waste tank evaluation

    Moore, C.J.; Peterson, W.S.; Winkel, B.V.; Weiner, E.O.

    1995-09-01

    As Hanford Site Contractors address maintenance and future structural demands on nuclear waste tanks built as early as 1943, it is necessary to address their current safety margins and ensure safe margins are maintained. Although the current civil engineering practice has building codes for reinforced concrete design guidelines, the tanks were not constructed to today's building codes and future demands potentially result in loads and modifications to the tanks that are outside the original design basis and current practice. The Hanford Site engineering staff has embraced nonlinear finite-element modeling of concrete in an effort to obtain a more accurate understanding of the actual tank margins. This document brings together and integrates past Hanford Site nonlinear reinforced concrete analysis methods, past Hanford Site concrete testing, public domain research testing, and current concrete research directions. This document, including future revisions, provides the structural engineering overview (or survey) for a consistent, accurate approach to nonlinear finite-element modeling of reinforced concrete for Hanford Site waste storage tanks. This report addresses concrete strength and modulus degradation with temperature, creep, shrinkage, long-term sustained loads, and temperature degradation of rebar and concrete bonds. Recommendations are given for parameter studies and evaluation techniques for review of nonlinear finite-element analysis of concrete

  18. Hydrology and Oceanography Analysis Regarding The NPP Site Screening Process at Banten Province

    Yarianto-S-Budi-Susilo

    2007-01-01

    Regarding the NPP development in the future, it is needed to make inventory of potential site in the Java Island as well as in the outside Java Island. The NPP site inventory availability is to answer the energy demand challenge. Site screening process should be performed in accordance with the IAEA safety standard regarding the site selection, investigating several aspects related to the NPP safety (exclusion, safety and suitability factor) in the large area to obtain potential site candidates. For the site survey stage of hydrology and oceanography aspects, the analysis are more focused on the tidal phenomena along the north coastline, bathymetry, water resource, and hydrology system in the Banten Province. The method used are secondary data collection, field confirmation and internet searching. The result of the study showed that Tanjung Pujut and Tanjung Pasir are suitable based on the bathymetry and water intake facility consideration. Meanwhile Tanjung Kait and Tanjung Pasir more suitable considering tsunami aspects that may be generated by Krakatau Volcano. (author)

  19. On-Site Renewable Energy and Green Buildings: A System-Level Analysis.

    Al-Ghamdi, Sami G; Bilec, Melissa M

    2016-05-03

    Adopting a green building rating system (GBRSs) that strongly considers use of renewable energy can have important environmental consequences, particularly in developing countries. In this paper, we studied on-site renewable energy and GBRSs at the system level to explore potential benefits and challenges. While we have focused on GBRSs, the findings can offer additional insight for renewable incentives across sectors. An energy model was built for 25 sites to compute the potential solar and wind power production on-site and available within the building footprint and regional climate. A life-cycle approach and cost analysis were then completed to analyze the environmental and economic impacts. Environmental impacts of renewable energy varied dramatically between sites, in some cases, the environmental benefits were limited despite the significant economic burden of those renewable systems on-site and vice versa. Our recommendation for GBRSs, and broader policies and regulations, is to require buildings with higher environmental impacts to achieve higher levels of energy performance and on-site renewable energy utilization, instead of fixed percentages.

  20. Characterization of a bioactive 15 kDa fragment produced by proteolytic cleavage of chicken growth hormone.

    Arámburo, C; Carranza, M; Reyes, M; Luna, M; Martinez-Coria, H; Berúmen, L; Scanes, C G

    2001-07-01

    There is evidence for a cleaved form of GH in the chicken pituitary gland. A 25 kDa band of immunoreactive-(ir-)GH, as well as the 22 kDa monomeric form and some oligomeric forms were observed when purified GH or fresh pituitary extract were subjected to SDS-PAGE under nonreducing conditions. Under reducing conditions, the 25 kDa ir-GH was no longer observed, being replaced by a 15 kDa band, consistent with reduction of the disulfide bridges of the cleaved form. The type of protease involved was investigated using exogenous proteases and monomeric cGH. Cleaved forms of chicken GH were generated by thrombin or collagenase. The site of cleavage was found in position Arg133-Gly134 as revealed by sequencing the fragments produced. The NH2-terminal sequence of 40 amino acid residues in the 15 kDa form was identical to that of the rcGH and analysis of the remaining 7 kDa fragment showed an exact identity with positions 134-140 of cGH structure. The thrombin cleaved GH and the 15 kDa form showed reduced activity (0.8% and 0.5% of GH, respectively) in a radioreceptor assay employing a chicken liver membrane preparation. However, this fragment had a clear bioactivity in an angiogenic bioassay and was capable to inhibit the activity of deiodinase type III in the chicken liver.

  1. Philippine Wind Farm Analysis and Site Selection Analysis, 1 January 2000 - 31 December 2000

    Conover, K.

    2001-12-01

    The U.S. Department of Energy (DOE), through the National Renewable Energy Laboratory (NREL), has been working in partnership with the U.S. Agency for International Development (USAID) in an ongoing process to quantify the Philippine wind energy potential and foster wind farm development. As part of that process, NREL retained Global Energy Concepts, LLC (GEC) to review and update the policy needs as well as develop a site-screening process applicable for the Philippines. GEC worked closely with the Philippines National Power Corporation (NPC) in completing this work. This report provides the results of the policy needs and site selection analyses conducted by GEC.

  2. A web server for analysis, comparison and prediction of protein ligand binding sites.

    Singh, Harinder; Srivastava, Hemant Kumar; Raghava, Gajendra P S

    2016-03-25

    One of the major challenges in the field of system biology is to understand the interaction between a wide range of proteins and ligands. In the past, methods have been developed for predicting binding sites in a protein for a limited number of ligands. In order to address this problem, we developed a web server named 'LPIcom' to facilitate users in understanding protein-ligand interaction. Analysis, comparison and prediction modules are available in the "LPIcom' server to predict protein-ligand interacting residues for 824 ligands. Each ligand must have at least 30 protein binding sites in PDB. Analysis module of the server can identify residues preferred in interaction and binding motif for a given ligand; for example residues glycine, lysine and arginine are preferred in ATP binding sites. Comparison module of the server allows comparing protein-binding sites of multiple ligands to understand the similarity between ligands based on their binding site. This module indicates that ATP, ADP and GTP ligands are in the same cluster and thus their binding sites or interacting residues exhibit a high level of similarity. Propensity-based prediction module has been developed for predicting ligand-interacting residues in a protein for more than 800 ligands. In addition, a number of web-based tools have been integrated to facilitate users in creating web logo and two-sample between ligand interacting and non-interacting residues. In summary, this manuscript presents a web-server for analysis of ligand interacting residue. This server is available for public use from URL http://crdd.osdd.net/raghava/lpicom .

  3. Analysis of functional importance of binding sites in the Drosophila gap gene network model.

    Kozlov, Konstantin; Gursky, Vitaly V; Kulakovskiy, Ivan V; Dymova, Arina; Samsonova, Maria

    2015-01-01

    The statistical thermodynamics based approach provides a promising framework for construction of the genotype-phenotype map in many biological systems. Among important aspects of a good model connecting the DNA sequence information with that of a molecular phenotype (gene expression) is the selection of regulatory interactions and relevant transcription factor bindings sites. As the model may predict different levels of the functional importance of specific binding sites in different genomic and regulatory contexts, it is essential to formulate and study such models under different modeling assumptions. We elaborate a two-layer model for the Drosophila gap gene network and include in the model a combined set of transcription factor binding sites and concentration dependent regulatory interaction between gap genes hunchback and Kruppel. We show that the new variants of the model are more consistent in terms of gene expression predictions for various genetic constructs in comparison to previous work. We quantify the functional importance of binding sites by calculating their impact on gene expression in the model and calculate how these impacts correlate across all sites under different modeling assumptions. The assumption about the dual interaction between hb and Kr leads to the most consistent modeling results, but, on the other hand, may obscure existence of indirect interactions between binding sites in regulatory regions of distinct genes. The analysis confirms the previously formulated regulation concept of many weak binding sites working in concert. The model predicts a more or less uniform distribution of functionally important binding sites over the sets of experimentally characterized regulatory modules and other open chromatin domains.

  4. Off-site training of laparoscopic skills, a scoping review using a thematic analysis.

    Thinggaard, Ebbe; Kleif, Jakob; Bjerrum, Flemming; Strandbygaard, Jeanett; Gögenur, Ismail; Matthew Ritter, E; Konge, Lars

    2016-11-01

    The focus of research in simulation-based laparoscopic training has changed from examining whether simulation training works to examining how best to implement it. In laparoscopic skills training, portable and affordable box trainers allow for off-site training. Training outside simulation centers and hospitals can increase access to training, but also poses new challenges to implementation. This review aims to guide implementation of off-site training of laparoscopic skills by critically reviewing the existing literature. An iterative systematic search was carried out in MEDLINE, EMBASE, ERIC, Scopus, and PsychINFO, following a scoping review methodology. The included literature was analyzed iteratively using a thematic analysis approach. The study was reported in accordance with the STructured apprOach to the Reporting In healthcare education of Evidence Synthesis statement. From the search, 22 records were identified and included for analysis. A thematic analysis revealed the themes: access to training, protected training time, distribution of training, goal setting and testing, task design, and unsupervised training. The identified themes were based on learning theories including proficiency-based learning, deliberate practice, and self-regulated learning. Methods of instructional design vary widely in off-site training of laparoscopic skills. Implementation can be facilitated by organizing courses and training curricula following sound education theories such as proficiency-based learning and deliberate practice. Directed self-regulated learning has the potential to improve off-site laparoscopic skills training; however, further studies are needed to demonstrate the effect of this type of instructional design.

  5. First atmospheric diffusion experiment compaign at the Angra site. Analysis of the data measured

    Nicolli, D.

    1986-07-01

    An analysis of the data measured during the first atmospheric diffusion experiment campaign at the Angra site is presented. Some time before the diffusion experiments, with tritiated water vapor used as a tracer (HTO) could be started, many preparatory studies had to be concluded due to the local complex terrain surrounded by steep hills on three sides and a bay in the southern direction. An introductory account of these previous steps is given. Tritiated water vapor was released from a 100m-high tower and air humidity sampled at 25 locations as far as 1Km downwind of the source. An elaborated isoconcentration analysis indicates the plume spread has a Gaussian distribution in the horizontal plane up to the edge of the site's area bordered by the hill's top (about 1Km far-off). Nevertheless the Gaussian model has a restricted applicability at some parts of the site because of the terrain unevenness. In general, the results are comparable to those obtained in other countries for complex terrain. Meanwhile, the Angra's experiments seem only to validate the Gaussian model for short range dispersion from an elevated source. For releases in the lowest layer, the Gaussian model might not be valid. The isoconcentration analysis strengthens the assumption of sea breeze recirculation on the site. (Author) [pt

  6. The SocioEconomic analysis of repository siting (SEARS): Guide to data base preparation: Final draft

    Hamm, R.R.; Murdock, S.H.; Leistritz, F.L.; Kiel, B.; Parpia, B.

    1984-11-01

    This guide describes the data bases in the SocioEconomic Analysis of Repository Siting (SEARS) modeling system. This model is a user-interactive, computerized model for projecting the economic, demographic, public service, and fiscal impacts of repository siting. This guide provides a description of the data bases, sources of data, data formats, and preprocessing programs for adapting and implementing the SEARS system and is seen as an essential reference for technical users of the model. It should be used in conjunction with reports describing the model's features and characteristics. 95 refs., 3 tabs

  7. Multi-criteria analysis for the detection of the most critical European UNESCO Heritage sites

    Valagussa, Andrea; Frattini, Paolo; Berta, Nadia; Spizzichino, Daniele; Leoni, Gabriele; Margottini, Claudio; Battista Crosta, Giovanni

    2017-04-01

    A GIS-based multi-criteria analysis has been implemented to identify and to rank the most critical UNESCO Heritage sites at the European scale in the context of PROTHEGO JPI-Project. Two multi-criteria methods have been tested and applied to more than 300 European UNESCO Sites. First, the Analytic Hierarchy Procedure (AHP) was applied to the data of the UNESCO Periodic Report, in relation to 13 natural hazards that have affected or can potentially affect the Heritage sites. According to these reports, 22% of sites are without any documented hazard and 70% of the sites have at least one hazard affecting the site. The most important hazards on the European country are: fire (wildfire), storm, flooding, earthquake and erosion. For each UNESCO site, the potential risk was calculated as a weighed sum of the hazards that affect the site. The weighs of the 13 hazards were obtained by AHP procedure, which is a technique for multi-attribute decision making that enables the decomposition of a problem into hierarchy, based on the opinion of different experts about the dominance of risks. The weights are obtained by rescaling between 0 and 1 the eigenvectors relative to the maximum eigenvalue for the matrix of the coefficients. The internal coherence of the expert's attributions is defined through the calculation of the consistency ratio (Saaty, 1990). The result of the AHP method consists in a map of the UNESCO sites ranked according to the potential risk, where the site most at risk results to be the Geirangerfjord and Nærøyfjord in Norway. However, the quality of these results lies in the reliability of the Period Reports, which are produced by different experts with unknown level of scientific background. To test the reliability of these results, a comparison of the information of the periodic reports with available high-quality datasets (earthquake, volcano and landslide) at the Italian scale has been performed. Sites properly classified by the Period Reports range from

  8. Carbohydrates on Proteins: Site-Specific Glycosylation Analysis by Mass Spectrometry

    Zhu, Zhikai; Desaire, Heather

    2015-07-01

    Glycosylation on proteins adds complexity and versatility to these biologically vital macromolecules. To unveil the structure-function relationship of glycoproteins, glycopeptide-centric analysis using mass spectrometry (MS) has become a method of choice because the glycan is preserved on the glycosylation site and site-specific glycosylation profiles of proteins can be readily determined. However, glycopeptide analysis is still challenging given that glycopeptides are usually low in abundance and relatively difficult to detect and the resulting data require expertise to analyze. Viewing the urgent need to address these challenges, emerging methods and techniques are being developed with the goal of analyzing glycopeptides in a sensitive, comprehensive, and high-throughput manner. In this review, we discuss recent advances in glycoprotein and glycopeptide analysis, with topics covering sample preparation, analytical separation, MS and tandem MS techniques, as well as data interpretation and automation.

  9. Oak Ridge National Laboratory site data for safety-analysis report

    Fitzpatrick, F.C.

    1982-12-01

    The Oak Ridge National Laboratory site data contained herein were compiled in support of the United States Department of Energy (USDOE) Oak Ridge Operations Office Order OR 5481.1. That order sets forth assignment of responsibilities for safety analysis and review responsibilities and provides guidance relative to the content and format of safety analysis reports. The information presented in this document is intended for use by reference in individual safety analysis reports where applicable to support accident analyses or the establishment of design bases of significance to safety, and it is applicable only to Oak Ridge National Laboratory facilities in Bethel and Melton Valleys. This information includes broad descriptions of the site characteristics, radioactive waste handling and monitoring practices, and the organization and operating policies at Oak Ridge National Laboratory. The historical background of the Laboratory is discussed briefly and the overall physical situation of the facilities is described in the following paragraphs

  10. Oak Ridge National Laboratory site data for safety-analysis report

    Fitzpatrick, F.C.

    1982-12-01

    The Oak Ridge National Laboratory site data contained herein were compiled in support of the United States Department of Energy (USDOE) Oak Ridge Operations Office Order OR 5481.1. That order sets forth assignment of responsibilities for safety analysis and review responsibilities and provides guidance relative to the content and format of safety analysis reports. The information presented in this document is intended for use by reference in individual safety analysis reports where applicable to support accident analyses or the establishment of design bases of significance to safety, and it is applicable only to Oak Ridge National Laboratory facilities in Bethel and Melton Valleys. This information includes broad descriptions of the site characteristics, radioactive waste handling and monitoring practices, and the organization and operating policies at Oak Ridge National Laboratory. The historical background of the Laboratory is discussed briefly and the overall physical situation of the facilities is described in the following paragraphs.

  11. Transcription elongation rate has a tissue-specific impact on alternative cleavage and polyadenylation in Drosophila melanogaster.

    Liu, Xiaochuan; Freitas, Jaime; Zheng, Dinghai; Oliveira, Marta S; Hoque, Mainul; Martins, Torcato; Henriques, Telmo; Tian, Bin; Moreira, Alexandra

    2017-12-01

    Alternative polyadenylation (APA) is a mechanism that generates multiple mRNA isoforms with different 3'UTRs and/or coding sequences from a single gene. Here, using 3' region extraction and deep sequencing (3'READS), we have systematically mapped cleavage and polyadenylation sites (PASs) in Drosophila melanogaster , expanding the total repertoire of PASs previously identified for the species, especially those located in A-rich genomic sequences. Cis -element analysis revealed distinct sequence motifs around fly PASs when compared to mammalian ones, including the greater enrichment of upstream UAUA elements and the less prominent presence of downstream UGUG elements. We found that over 75% of mRNA genes in Drosophila melanogaster undergo APA. The head tissue tends to use distal PASs when compared to the body, leading to preferential expression of APA isoforms with long 3'UTRs as well as with distal terminal exons. The distance between the APA sites and intron location of PAS are important parameters for APA difference between body and head, suggesting distinct PAS selection contexts. APA analysis of the RpII215 C4 mutant strain, which harbors a mutant RNA polymerase II (RNAPII) with a slower elongation rate, revealed that a 50% decrease in transcriptional elongation rate leads to a mild trend of more usage of proximal, weaker PASs, both in 3'UTRs and in introns, consistent with the "first come, first served" model of APA regulation. However, this trend was not observed in the head, suggesting a different regulatory context in neuronal cells. Together, our data expand the PAS collection for Drosophila melanogaster and reveal a tissue-specific effect of APA regulation by RNAPII elongation rate. © 2017 Liu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  12. Prediction of functional sites in proteins using conserved functional group analysis.

    Innis, C Axel; Anand, A Prem; Sowdhamini, R

    2004-04-02

    A detailed knowledge of a protein's functional site is an absolute prerequisite for understanding its mode of action at the molecular level. However, the rapid pace at which sequence and structural information is being accumulated for proteins greatly exceeds our ability to determine their biochemical roles experimentally. As a result, computational methods are required which allow for the efficient processing of the evolutionary information contained in this wealth of data, in particular that related to the nature and location of functionally important sites and residues. The method presented here, referred to as conserved functional group (CFG) analysis, relies on a simplified representation of the chemical groups found in amino acid side-chains to identify functional sites from a single protein structure and a number of its sequence homologues. We show that CFG analysis can fully or partially predict the location of functional sites in approximately 96% of the 470 cases tested and that, unlike other methods available, it is able to tolerate wide variations in sequence identity. In addition, we discuss its potential in a structural genomics context, where automation, scalability and efficiency are critical, and an increasing number of protein structures are determined with no prior knowledge of function. This is exemplified by our analysis of the hypothetical protein Ydde_Ecoli, whose structure was recently solved by members of the North East Structural Genomics consortium. Although the proposed active site for this protein needs to be validated experimentally, this example illustrates the scope of CFG analysis as a general tool for the identification of residues likely to play an important role in a protein's biochemical function. Thus, our method offers a convenient solution to rapidly and automatically process the vast amounts of data that are beginning to emerge from structural genomics projects.

  13. Inhibition of Cellular Adhesion by Immunological Targeting of Osteopontin Neoepitopes Generated through Matrix Metalloproteinase and Thrombin Cleavage.

    Jürets, Alexander; Le Bras, Marie; Staffler, Günther; Stein, Gesine; Leitner, Lukas; Neuhofer, Angelika; Tardelli, Matteo; Turkof, Edvin; Zeyda, Maximilian; Stulnig, Thomas M

    2016-01-01

    Osteopontin (OPN), a secreted protein involved in inflammatory processes and cancer, induces cell adhesion, migration, and activation of inflammatory pathways in various cell types. Cells bind OPN via integrins at a canonical RGD region in the full length form as well as to a contiguous cryptic site that some have shown is unmasked upon thrombin or matrix metalloproteinase cleavage. Thus, the adhesive capacity of osteopontin is enhanced by proteolytic cleavage that may occur in inflammatory conditions such as obesity, atherosclerosis, rheumatoid arthritis, tumor growth and metastasis. Our aim was to inhibit cellular adhesion to recombinant truncated proteins that correspond to the N-terminal cleavage products of thrombin- or matrix metalloproteinase-cleaved OPN in vitro. We specifically targeted the cryptic integrin binding site with monoclonal antibodies and antisera induced by peptide immunization of mice. HEK 293 cells adhered markedly stronger to truncated OPN proteins than to full length OPN. Without affecting cell binding to the full length form, the raised monoclonal antibodies specifically impeded cellular adhesion to the OPN fragments. Moreover, we show that the peptides used for immunization were able to induce antisera, which impeded adhesion either to all OPN forms, including the full-length form, or selectively to the corresponding truncated recombinant proteins. In conclusion, we developed immunological tools to selectively target functional properties of protease-cleaved OPN forms, which could find applications in treatment and prevention of various inflammatory diseases and cancers.

  14. Cleavage of a recombinant human immunoglobulin A2 (IgA2)-IgA1 hybrid antibody by certain bacterial IgA1 proteases

    Senior, B; Dunlop, JI; Batten, MR

    2000-01-01

    , Streptococcus pneumoniae, S. sanguis, Neisseria meningitidis types 1 and 2, N. gonorrhoeae types 1 and 2, and Haemophilus influenzae type 2. Thus, for these enzymes the recognition site for IgA1 cleavage is contained within half of the IgA1 hinge region; additional distal elements, if required, are provided...... by either an IgA1 or an IgA2 framework. In contrast, the IgA2/A1 hybrid appeared to be resistant to cleavage with S. oralis and some H. influenzae type 1 IgA1 proteases, suggesting these enzymes require additional determinants for efficient substrate recognition....

  15. [Conformation analysis of the N-glycosylation site Asn-X-Thr/Ser in glycoproteins].

    Avanov, A Ia; Lipkind, G M

    1990-03-01

    Theoretical conformational analysis of oligopeptides CH3CO-Asn-X-Thr-NHCH3 (X = Gly, Ala, Pro), modelling N-glycosylation site, and their glycosylated derivatives CH3CO-(GlcNAc beta 1-4GlcNAc beta 1) Asn-X-Thr-NHCH3 has been carried out. Active conformations of the site are found, corresponding to structural prerequisities of N-glycosylation: Asn residue's position in beta-turn and hydrogen bond formation between side chains of Asn and Thr/Ser residues. In this case the L conformation of the central residue X is most probable. Since Pro residue does not possess this conformation, sequences with X = Pro are not glycosylated. It is shown that glycosylation of the above-mentioned sites is accompanied by reorientation of the Asn residue's side chains.

  16. Global Web Accessibility Analysis of National Government Portals and Ministry Web Sites

    Goodwin, Morten; Susar, Deniz; Nietzio, Annika

    2011-01-01

    Equal access to public information and services for all is an essential part of the United Nations (UN) Declaration of Human Rights. Today, the Web plays an important role in providing information and services to citizens. Unfortunately, many government Web sites are poorly designed and have...... accessibility barriers that prevent people with disabilities from using them. This article combines current Web accessibility benchmarking methodologies with a sound strategy for comparing Web accessibility among countries and continents. Furthermore, the article presents the first global analysis of the Web...... accessibility of 192 United Nation Member States made publically available. The article also identifies common properties of Member States that have accessible and inaccessible Web sites and shows that implementing antidisability discrimination laws is highly beneficial for the accessibility of Web sites, while...

  17. Theoretical analysis of the influence of flexoelectric effect on the defect site in nematic inversion walls

    Zheng Gui-Li; Xuan Li; Zhang Hui; Ye Wen-Jiang; Zhang Zhi-Dong; Song Hong-Wei

    2016-01-01

    Based on the experimental phenomena of flexoelectric response at defect sites in nematic inversion walls conducted by Kumar et al., we gave the theoretical analysis using the Frank elastic theory. When a direct-current electric field normal to the plane of the substrate is applied to the parallel aligned nematic liquid crystal cell with weak anchoring, the rotation of ±1 defects in the narrow inversion walls can be exhibited. The free energy of liquid crystal molecules around the +1 and –1 defect sites in the nematic inversion walls under the electric field was formulated and the electric-field-driven structural changes at the defect site characterized by polar and azimuthal angles of the local director were simulated. The results reveal that the deviation of azimuthal angle induced by flexoelectric effect are consistent with the switching of extinction brushes at the +1 and −1 defects obtained in the experiment conducted by Kumar et al. (paper)

  18. An integrated study of spatial multicriteria analysis and mathematical modelling for managed aquifer recharge site suitability mapping and site ranking at Northern Gaza coastal aquifer.

    Rahman, Mohammad Azizur; Rusteberg, Bernd; Uddin, Mohammad Salah; Lutz, Annegret; Saada, Muath Abu; Sauter, Martin

    2013-07-30

    This paper describes an integrated approach of site suitability mapping and ranking of the most suitable sites, for the implementation of Managed Aquifer Recharge (MAR) projects, using spatial multicriteria decision analysis (SMCDA) techniques and mathematical modelling. The SMCDA procedure contains constraint mapping, site suitability analysis with criteria standardization and weighting, criteria overlay by analytical hierarchy process (AHP) combined with weighted linear combination (WLC) and ordered weighted averaging (OWA), and sensitivity analysis. The hydrogeological impacts of the selected most suitable sites were quantified by using groundwater flow and transport modelling techniques. Finally, ranking of the selected sites was done with the WLC method. The integrated approach is demonstrated by a case study in the coastal aquifer of North Gaza. Constraint mapping shows that 50% of the total study area is suitable for MAR implementation. About 25% of the total area is "very good" and 25% percent is "good" for MAR, according to the site suitability analysis. Six locations were selected and ranked against six representative decision criteria. Long term (year 2003 to year 2040) groundwater flow and transport simulations were performed to quantify the selected criteria under MAR project operation conditions at the selected sites. Finally, the suitability mapping and hydrogeological investigation recommends that the location of the existing infiltration ponds, constructed near the planned North Gaza Wastewater Treatment Plant (NGWWTP) is most suitable for MAR project implementation. This paper concludes that mathematical modelling should be combined with the SMCDA technique in order to select the best location for MAR project implementation. Besides MAR project implementation, the generalised approach can be applicable for any other water resources development project that deals with site selection and implementation. Copyright © 2013 Elsevier Ltd. All rights

  19. Program Director as Webmaster? Analysis of 131 Anesthesiology Department Web Sites and Program Director Web Site Involvement and Opinion Survey.

    Daneshpayeh, Negin; Lee, Howard; Berger, Jeffrey

    2013-01-01

    The last formal review of academic anesthesiology department Web sites (ADWs) for content was conducted in 2009. ADWs have been rated as very important by medical students in researching residency training programs; however, the rapid evolution of sites require that descriptive statistics must be more current to be considered reliable. We set out to provide an updated overview of ADW content and to better understand residency program directors' (PD) role and comfort with ADWs. Two independent reviewers (ND and HL) analyzed all 131 Accreditation Council for Graduate Medical Education (ACGME) accredited ADWs. A binary system (Yes/No) was used to determine which features were present. Reviewer reliability was confirmed with inter-rater reliability and percentage agreement calculation. Additionally, a blinded electronic survey (Survey Monkey, Portland, OR) was sent to anesthesiology residency PDs via electronic mail investigating the audiences for ADWs, the frequency of updates and the degree of PD involvement. 13% of anesthesiology departments still lack a Web site with a homepage with links to the residency program and educational offerings (18% in 2009). Only half (55%) of Web sites contain information for medical students, including clerkship information. Furthermore, programs rarely contain up-to-date calendars (13%), accreditation cycle lengths (11%), accreditation dates (7%) or board pass rates (6%). The PD survey, completed by 42 of 131 PDs, noted a correlation (r = 0.36) between the number of years as PD and the frequency of Web site updates - less experienced PDs appear to update their sites more frequently (p = 0.03). Although 86% of PDs regarded a Web site as "very" important in recruitment, only 9% felt "very" comfortable with the skills required to advertise and market a Web site. Despite the overall increase in ADW content since 2009, privacy concerns, limited resources and time constraints may prevent PDs from providing the most up-to-date Web sites for

  20. Characterization of an extensin-modifying metalloprotease: N-terminal processing and substrate cleavage pattern of Pectobacterium carotovorum Prt1.

    Feng, Tao; Nyffenegger, Christian; Højrup, Peter; Vidal-Melgosa, Silvia; Yan, Kok-Phen; Fangel, Jonatan Ulrik; Meyer, Anne S; Kirpekar, Finn; Willats, William G; Mikkelsen, Jørn D

    2014-12-01

    Compared to other plant cell wall-degrading enzymes, proteases are less well understood. In this study, the extracellular metalloprotease Prt1 from Pectobacterium carotovorum (formerly Erwinia carotovora) was expressed in Escherichia coli and characterized with respect to N-terminal processing, thermal stability, substrate targets, and cleavage patterns. Prt1 is an autoprocessing protease with an N-terminal signal pre-peptide and a pro-peptide which has to be removed in order to activate the protease. The sequential cleavage of the N-terminus was confirmed by mass spectrometry (MS) fingerprinting and N-terminus analysis. The optimal reaction conditions for the activity of Prt1 on azocasein were at pH 6.0, 50 °C. At these reaction conditions, K M was 1.81 mg/mL and k cat was 1.82 × 10(7) U M(-1). The enzyme was relatively stable at 50 °C with a half-life of 20 min. Ethylenediaminetetraacetic acid (EDTA) treatment abolished activity; Zn(2+) addition caused regain of the activity, but Zn(2+)addition decreased the thermal stability of the Prt1 enzyme presumably as a result of increased proteolytic autolysis. In addition to casein, the enzyme catalyzed degradation of collagen, potato lectin, and plant extensin. Analysis of the cleavage pattern of different substrates after treatment with Prt1 indicated that the protease had a substrate cleavage preference for proline in substrate residue position P1 followed by a hydrophobic residue in residue position P1' at the cleavage point. The activity of Prt1 against plant cell wall structural proteins suggests that this enzyme might become an important new addition to the toolbox of cell-wall-degrading enzymes for biomass processing.

  1. Probabilistic and Scenario Seismic and Liquefaction Hazard Analysis of the Mississippi Embayment Incorporating Nonlinear Site Effects

    Cramer, C. H.; Dhar, M. S.

    2017-12-01

    The influence of deep sediment deposits of the Mississippi Embayment (ME) on the propagation of seismic waves is poorly understood and remains a major source of uncertainty for site response analysis. Many researchers have studied the effects of these deposits on seismic hazard of the area using available information at the time. In this study, we have used updated and newly available resources for seismic and liquefaction hazard analyses of the ME. We have developed an improved 3D geological model. Additionally, we used surface geological maps from Cupples and Van Arsdale (2013) to prepare liquefaction hazard maps. Both equivalent linear and nonlinear site response codes were used to develop site amplification distributions for use in generating hazard maps. The site amplification distributions are created using the Monte Carlo approach of Cramer et al. (2004, 2006) on a 0.1-degree grid. The 2014 National Seismic Hazard model and attenuation relations (Petersen et al., 2014) are used to prepare seismic hazard maps. Then liquefaction hazard maps are generated using liquefaction probability curves from Holzer (2011) and Cramer et al. (2015). Equivalent linear response (w/ increased precision, restricted nonlinear behavior with depth) shows similar hazard for the ME compared to nonlinear analysis (w/o pore pressure) results. At short periods nonlinear deamplification dominates the hazard, but at long periods resonance amplification dominates. The liquefaction hazard tends to be high in Holocene and late Pleistocene lowland sediments, even with lowered ground water levels, and low in Pleistocene loess of the uplands. Considering pore pressure effects in nonlinear site response analysis at a test site on the lowlands shows amplification of ground motion at short periods. PGA estimates from ME liquefaction and MMI observations are in the 0.25 to 0.4 g range. Our estimated M7.5 PGA hazard within 10 km of the fault can exceed this. Ground motion observations from

  2. Analysis of the geomorphology surrounding the Chang'e-3 landing site

    Li Chun-Lai; Mu Ling-Li; Zou Xiao-Duan; Liu Jian-Jun; Ren Xin; Zeng Xing-Guo; Yang Yi-Man; Zhang Zhou-Bin; Liu Yu-Xuan; Zuo Wei; Li Han

    2014-01-01

    Chang'e-3 (CE-3) landed on the Mare Imbrium basin in the east part of Sinus Iridum (19.51°W, 44.12°N), which was China's first soft landing on the Moon and it started collecting data on the lunar surface environment. To better understand the environment of this region, this paper utilizes the available high-resolution topography data, image data and geological data to carry out a detailed analysis and research on the area surrounding the landing site (Sinus Iridum and 45 km×70 km of the landing area) as well as on the topography, landform, geology and lunar dust of the area surrounding the landing site. A general topographic analysis of the surrounding area is based on a digital elevation model and digital elevation model data acquired by Chang'e-2 that have high resolution; the geology analysis is based on lunar geological data published by USGS; the study on topographic factors and distribution of craters and rocks in the surrounding area covering 4 km×4 km or even smaller is based on images from the CE-3 landing camera and images from the topographic camera; an analysis is done of the effect of the CE-3 engine plume on the lunar surface by comparing images before and after the landing using data from the landing camera. A comprehensive analysis of the results shows that the landing site and its surrounding area are identified as typical lunar mare with flat topography. They are suitable for maneuvers by the rover, and are rich in geological phenomena and scientific targets, making it an ideal site for exploration

  3. Analysis of the geomorphology surrounding the Chang'e-3 landing site

    Li, Chun-Lai; Mu, Ling-Li; Zou, Xiao-Duan; Liu, Jian-Jun; Ren, Xin; Zeng, Xing-Guo; Yang, Yi-Man; Zhang, Zhou-Bin; Liu, Yu-Xuan; Zuo, Wei; Li, Han

    2014-12-01

    Chang'e-3 (CE-3) landed on the Mare Imbrium basin in the east part of Sinus Iridum (19.51°W, 44.12°N), which was China's first soft landing on the Moon and it started collecting data on the lunar surface environment. To better understand the environment of this region, this paper utilizes the available high-resolution topography data, image data and geological data to carry out a detailed analysis and research on the area surrounding the landing site (Sinus Iridum and 45 km×70 km of the landing area) as well as on the topography, landform, geology and lunar dust of the area surrounding the landing site. A general topographic analysis of the surrounding area is based on a digital elevation model and digital elevation model data acquired by Chang'e-2 that have high resolution; the geology analysis is based on lunar geological data published by USGS; the study on topographic factors and distribution of craters and rocks in the surrounding area covering 4 km×4 km or even smaller is based on images from the CE-3 landing camera and images from the topographic camera; an analysis is done of the effect of the CE-3 engine plume on the lunar surface by comparing images before and after the landing using data from the landing camera. A comprehensive analysis of the results shows that the landing site and its surrounding area are identified as typical lunar mare with flat topography. They are suitable for maneuvers by the rover, and are rich in geological phenomena and scientific targets, making it an ideal site for exploration.

  4. Participation in the management of Greek Natura 2000 sites: evidence from a cross-level analysis.

    Apostolopoulou, Evangelia; Drakou, Evangelia G; Pediaditi, Kalliope

    2012-12-30

    The governance of protected areas has experienced rapid advancement over the last two decades with regard to the inclusion of stakeholders and local communities into the management process. During the same period Greek biodiversity governance has been characterized by a shift, at least on paper, towards the adoption of participatory approaches primarily through the establishment of management agencies. However, this has not been institutionalized for the majority of Natura 2000 sites, thus posing questions on the existence, nature, and effectiveness of participation in sites with no management agency. This is the first conducted large scale, cross level participation analysis for Greek Natura 2000 sites enabling the formation of a representative picture of the situation in the country. We investigated the nature and role of participation in Greek biodiversity governance by exploring both general opinions regarding the national context of participation in Greek Natura 2000 network as well as site-specific opinions regarding three case study areas where Natura 2000 sites have been established. Overall, we analyzed the results of 96 interviews, conducted with national, regional and local level stakeholders and 734 questionnaires conducted with local communities of the three case study areas. Results indicate with non-significant difference among governance levels, or between case study sites, that stakeholders' participation exists mainly on paper whereas community participation is practically absent. Stakeholder engagement seems to take place through administrational documentation across levels and to be locally confined based mainly on personal contacts and initiatives. Interviewees and survey respondents indicated a preference towards improving stakeholders' participation and the community's engagement in the management of Natura 2000 sites. Overall, the results of this study revealed the urgent need for policy initiatives towards adopting meaningful, fair and

  5. Polycystin-1 Cleavage and the Regulation of Transcriptional Pathways

    Merrick, David; Bertuccio, Claudia A.; Chapin, Hannah C.; Lal, Mark; Chauvet, Veronique; Caplan, Michael J.

    2013-01-01

    Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of end stage renal disease, affecting ~1 in 1,000 people. The disease is characterized by the development of numerous large fluid filled renal cysts over the course of decades. These cysts compress the surrounding renal parenchyma and impair its function. Mutations in two genes are responsible for ADPKD. The protein products of both of these genes, polycystin-1 and polycystin-2, localize to the primary cilium and participate in a wide variety of signaling pathways. Polycystin-1 undergoes several proteolytic cleavages that produce fragments that manifest biological activities. Recent results suggest that the production of polycystin-1 cleavage fragments is necessary and sufficient to account for at least some, although certainly not all, of the physiological functions of the parent protein. PMID:23824180

  6. Water-Level Data Analysis for the Saturated Zone Site-Scale Flow and Transport Model

    K. Rehfeldt

    2004-01-01

    This report is an updated analysis of water-level data performed to provide the ''Saturated Zone Site-Scale Flow Model'' (BSC 2004 [DIRS 170037]) (referred to as the saturated zone (SZ) site-scale flow model or site-scale SZ flow model in this report) with the configuration of the potentiometric surface, target water-level data, and hydraulic gradients for calibration of groundwater flow models. This report also contains an expanded discussion of uncertainty in the potentiometric-surface map. The analysis of the potentiometric data presented in Revision 00 of this report (USGS 2001 [DIRS 154625]) provides the configuration of the potentiometric surface, target heads, and hydraulic gradients for the calibration of the SZ site-scale flow model (BSC 2004 [DIRS 170037]). Revision 01 of this report (USGS 2004 [DIRS 168473]) used updated water-level data for selected wells through the year 2000 as the basis for estimating water-level altitudes and the potentiometric surface in the SZ site-scale flow and transport model domain based on an alternative interpretation of perched water conditions. That revision developed computer files containing: Water-level data within the model area (DTN: GS010908312332.002); A table of known vertical head differences (DTN: GS010908312332.003); and A potentiometric-surface map (DTN: GS010608312332.001) using an alternative concept from that presented by USGS (2001 [DIRS 154625]) for the area north of Yucca Mountain. The updated water-level data presented in USGS (2004 [DIRS 168473]) include data obtained from the Nye County Early Warning Drilling Program (EWDP) Phases I and II and data from Borehole USW WT-24. This document is based on Revision 01 (USGS 2004 [DIRS 168473]) and expands the discussion of uncertainty in the potentiometric-surface map. This uncertainty assessment includes an analysis of the impact of more recent water-level data and the impact of adding data from the EWDP Phases III and IV wells. In addition to being utilized

  7. Water-Level Data Analysis for the Saturated Zone Site-Scale Flow and Transport Model

    K. Rehfeldt

    2004-10-08

    This report is an updated analysis of water-level data performed to provide the ''Saturated Zone Site-Scale Flow Model'' (BSC 2004 [DIRS 170037]) (referred to as the saturated zone (SZ) site-scale flow model or site-scale SZ flow model in this report) with the configuration of the potentiometric surface, target water-level data, and hydraulic gradients for calibration of groundwater flow models. This report also contains an expanded discussion of uncertainty in the potentiometric-surface map. The analysis of the potentiometric data presented in Revision 00 of this report (USGS 2001 [DIRS 154625]) provides the configuration of the potentiometric surface, target heads, and hydraulic gradients for the calibration of the SZ site-scale flow model (BSC 2004 [DIRS 170037]). Revision 01 of this report (USGS 2004 [DIRS 168473]) used updated water-level data for selected wells through the year 2000 as the basis for estimating water-level altitudes and the potentiometric surface in the SZ site-scale flow and transport model domain based on an alternative interpretation of perched water conditions. That revision developed computer files containing: Water-level data within the model area (DTN: GS010908312332.002); A table of known vertical head differences (DTN: GS010908312332.003); and A potentiometric-surface map (DTN: GS010608312332.001) using an alternative concept from that presented by USGS (2001 [DIRS 154625]) for the area north of Yucca Mountain. The updated water-level data presented in USGS (2004 [DIRS 168473]) include data obtained from the Nye County Early Warning Drilling Program (EWDP) Phases I and II and data from Borehole USW WT-24. This document is based on Revision 01 (USGS 2004 [DIRS 168473]) and expands the discussion of uncertainty in the potentiometric-surface map. This uncertainty assessment includes an analysis of the impact of more recent water-level data and the impact of adding data from the EWDP Phases III and IV wells. In

  8. In silico analysis of Pycnoporus cinnabarinus laccase active site with toxic industrial dyes.

    Prasad, Nirmal K; Vindal, Vaibhav; Narayana, Siva Lakshmi; Ramakrishna, V; Kunal, Swaraj Priyaranjan; Srinivas, M

    2012-05-01

    Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in various industrial oxidative processes like production of fine chemicals to bioremediation of contaminated soil and water. In order to understand the mechanisms of substrate binding and interaction between substrates and Pycnoporus cinnabarinus laccase, a homology model was generated. The resulted model was further validated and used for docking studies with toxic industrial dyes- acid blue 74, reactive black 5 and reactive blue 19. Interactions of chemical mediators with the laccase was also examined. The docking analysis showed that the active site always cannot accommodate the dye molecules, due to constricted nature of the active site pocket and steric hindrance of the residues whereas mediators are relatively small and can easily be accommodated into the active site pocket, which, thereafter leads to the productive binding. The binding properties of these compounds along with identification of critical active site residues can be used for further site-directed mutagenesis experiments in order to identify their role in activity and substrate specificity, ultimately leading to improved mutants for degradation of these toxic compounds.

  9. Stormwater Pollutant Process Analysis with Long-Term Online Monitoring Data at Micro-Scale Sites

    Dominik Leutnant

    2016-07-01

    Full Text Available Stormwater runoff quality was measured with online turbidity sensors at four common types of small urban subcatchments: (i a flat roof; (ii a parking lot; (iii a residential catchment; and (iv a high-traffic street. Samples were taken to estimate site-specific correlations between total suspended solids (TSS and turbidity. Continuous TSS time series were derived from online turbidity measurements and were used to estimate event loads and event mean concentrations. Rainfall runoff event characteristics were subjected to correlation analysis to TSS loads. Significant correlations were found for rainfall intensities at sites with high imperviousness and decrease with increasing catchment size. Antecedent dry weather periods are only correlated at the parking lot site. Intra-event TSS load distributions were studied with M (V-curves. M (V-curves are grouped at runoff quantiles and statistically described with boxplots. All sites show, in general, a more pronounced first-flush effect. While wash-off of the flat roof tends to be source-limited, the parking lot and high-traffic street sites show a more transport-limited behavior. Wash-off process of the residential catchment appears to be influenced by a composition of different subcatchments.

  10. The Earliest Chinese Proto-Porcelain Excavated from Kiln Sites: An Elemental Analysis.

    Li, Yu; Zhang, Bin; Cheng, Huansheng; Zheng, Jianming

    2015-01-01

    In June 2012, the Piaoshan kiln site was excavated in Huzhou, Zhejiang Province, which hitherto proved to be the earliest known Chinese proto-porcelain kiln. Judging from the decorative patterns of unearthed impressed stoneware and proto-porcelain sherds, the site was determined to date to the late Xia (c. 2070-c. 1600 BC), the first dynasty of China. Here, we report on proton-induced X-ray emission analyses of 118 proto-porcelain and 35 impressed stoneware sherds from Piaoshan and five subsequent kiln sites in the vicinity. Using principal components analysis on the major chemical compositions, we reveal the relationships between impressed stoneware and proto-porcelain samples from the six kiln sites. The sherds from different sites have distinctive chemical profiles. The results indicate that the raw materials were procured locally. We find a developmental tendency for early glazes towards mature calcium-based glaze. It is most likely that woody plant ashes with increased calcia-potash ratios were applied to the formula.

  11. The Earliest Chinese Proto-Porcelain Excavated from Kiln Sites: An Elemental Analysis.

    Yu Li

    Full Text Available In June 2012, the Piaoshan kiln site was excavated in Huzhou, Zhejiang Province, which hitherto proved to be the earliest known Chinese proto-porcelain kiln. Judging from the decorative patterns of unearthed impressed stoneware and proto-porcelain sherds, the site was determined to date to the late Xia (c. 2070-c. 1600 BC, the first dynasty of China. Here, we report on proton-induced X-ray emission analyses of 118 proto-porcelain and 35 impressed stoneware sherds from Piaoshan and five subsequent kiln sites in the vicinity. Using principal components analysis on the major chemical compositions, we reveal the relationships between impressed stoneware and proto-porcelain samples from the six kiln sites. The sherds from different sites have distinctive chemical profiles. The results indicate that the raw materials were procured locally. We find a developmental tendency for early glazes towards mature calcium-based glaze. It is most likely that woody plant ashes with increased calcia-potash ratios were applied to the formula.

  12. Cleavage of cohesin rings coordinates the separation of centrioles and chromatids.

    Schöckel, Laura; Möckel, Martin; Mayer, Bernd; Boos, Dominik; Stemmann, Olaf

    2011-07-10

    Cohesin pairs sister chromatids by forming a tripartite Scc1-Smc1-Smc3 ring around them. In mitosis, cohesin is removed from chromosome arms by the phosphorylation-dependent prophase pathway. Centromeric cohesin is protected by shugoshin 1 and protein phosphatase 2A (Sgo1-PP2A) and opened only in anaphase by separase-dependent cleavage of Scc1 (refs 4-6). Following chromosome segregation, centrioles loosen their tight orthogonal arrangement, which licenses later centrosome duplication in S phase. Although a role of separase in centriole disengagement has been reported, the molecular details of this process remain enigmatic. Here, we identify cohesin as a centriole-engagement factor. Both premature sister-chromatid separation and centriole disengagement are induced by ectopic activation of separase or depletion of Sgo1. These unscheduled events are suppressed by expression of non-cleavable Scc1 or inhibition of the prophase pathway. When endogenous Scc1 is replaced by artificially cleavable Scc1, the corresponding site-specific protease triggers centriole disengagement. Separation of centrioles can alternatively be induced by ectopic cleavage of an engineered Smc3. Thus, the chromosome and centrosome cycles exhibit extensive parallels and are coordinated with each other by dual use of the cohesin ring complex.

  13. Relationship between synthesis and cleavage of poliovirus-specific proteins.

    Thomas, A A; Voorma, H O; Boeye, A

    1983-01-01

    Poliovirus proteinase was studied in vitro in lysates from poliovirus-infected HeLa cells. Preincubation of these lysates caused (i) a reduction in poliovirus proteinase activity and (ii) a partial dependence on exogenous mRNA for optimal translation. Proteins translated from endogenous poliovirus RNA in preincubated extracts from virus-infected HeLa cells are poorly cleaved. This cleavage deficiency is alleviated by adding fresh poliovirus RNA to the translation system, thus, allowing re-ini...

  14. Effects of Cysteamine on Sheep Embryo Cleavage Rates

    Sinem Ö. ENGİNLER

    2015-01-01

    Full Text Available Oxidative stress during in vitro culture leads to defects in development of gametes and embryos. Several antioxidants such as cysteamine, L-ascorbic acid, beta mercaptoethanol, cysteine, glutathione, proteins, vitamins have been used to supplement culture media to counter the oxidative stress. This study was conducted to detect the effect of adding cysteamine to the maturation medium to subsequent cleavage rates of sheep embryos. Totally 604 ovaries were obtained by ten replica and 2060 oocytes were collected. The cumulus oocyte complexes were recovered by the slicing method. A total of 1818 selected oocytes were divided into two groups and used for maturation (88.25%. The first group was created as supplemented with cysteamine (Group A and second group (Group B, control without cysteamine in TCM-199. The two groups were incubated for 24 h at 38.8 °C in an atmosphere of 5% CO2 in humidified air for in vitro maturation (IVM. After IVM, oocytes were fertilized with 50 x 107 / mL fresh ram semen in BSOF medium for 18 h. After fertilization, maturation groups were divided into two subgroups with different culture media: Group AI-SOF (Synthetic Oviduct Fluid medium, Group AII-CR1aa (Charles Rosencrans medium, Group BI-SOF and Group BII-CR1aa were achieved. Cleavage rates were evaluated at day 2. post insemination. The rates of cleavage were detected as 59.54% (184/309, 55.44% (173/312, 65.34% (215/329, 59.34% (200/337 respectively, with showing no statistically significant difference between the groups at the level of P>0.05. In conclusion, supplementing cysteamine to maturation media in TCM-199 did not affect the cleavage rates of sheep embryos in SOF and CR1aa culture media.

  15. Rapid Mapping and Deformation Analysis over Cultural Heritage and Rural Sites Based on Persistent Scatterer Interferometry

    D. Tapete

    2012-01-01

    Full Text Available We propose an easy-to-use procedure of “PSI-based rapid mapping and deformation analysis,” to effectively exploit Persistent Scatterer Interferometry (PSI for multispatial/temporal hazard assessment of cultural heritage and rural sites, update the condition report at the scale of entire site and single building, and address the conservation strategies. Advantages and drawbacks of the methodology are critically discussed based on feasibility tests performed over Pitigliano and Bivigliano, respectively, located in Southern and Northern Tuscany, Italy, and representative of hilltop historic towns and countryside settlements chronically affected by natural hazards. We radar-interpreted ERS-1/2 (1992–2000 and ENVISAT (2003–2010 datasets, already processed, respectively with the Permanent Scatterers (PSs and Persistent Scatterers Pairs (PSPs techniques, and assigned the levels of conservation criticality for both the sites. The PSI analysis allowed the zoning of the most unstable sectors of Pitigliano and showed a good agreement with the most updated hazard assessment of the cliff. The reconstruction of past/recent deformation patterns over Bivigliano confirmed the criticality for the Church of San Romolo, supporting the hypothesis of a correlation with local landslide phenomena, as also perceived from the annual motions observed over the entire site, where several landslide bodies are mapped.

  16. Deletion of the thrombin cleavage domain of osteopontin mediates breast cancer cell adhesion, proteolytic activity, tumorgenicity, and metastasis

    Beausoleil, Michel S; Schulze, Erika B; Goodale, David; Postenka, Carl O; Allan, Alison L

    2011-01-01

    Osteopontin (OPN) is a secreted phosphoprotein often overexpressed at high levels in the blood and primary tumors of breast cancer patients. OPN contains two integrin-binding sites and a thrombin cleavage domain located in close proximity to each other. To study the role of the thrombin cleavage site of OPN, MDA-MB-468 human breast cancer cells were stably transfected with either wildtype OPN (468-OPN), mutant OPN lacking the thrombin cleavage domain (468-ΔTC) or an empty vector (468-CON) and assessed for in vitro and in vivo functional differences in malignant/metastatic behavior. All three cell lines were found to equivalently express thrombin, tissue factor, CD44, αvβ5 integrin and β1 integrin. Relative to 468-OPN and 468-CON cells, 468-ΔTC cells expressing OPN with a deleted thrombin cleavage domain demonstrated decreased cell adhesion (p < 0.001), decreased mRNA expression of MCAM, maspin and TRAIL (p < 0.01), and increased uPA expression and activity (p < 0.01) in vitro. Furthermore, injection of 468-ΔTC cells into the mammary fat pad of nude mice resulted in decreased primary tumor latency time (p < 0.01) and increased primary tumor growth and lymph node metastatic burden (p < 0.001) compared to 468-OPN and 468-CON cells. The results presented here suggest that expression of thrombin-uncleavable OPN imparts an early tumor formation advantage as well as a metastatic advantage for breast cancer cells, possibly due to increased proteolytic activity and decreased adhesion and apoptosis. Clarification of the mechanisms responsible for these observations and the translation of this knowledge into the clinic could ultimately provide new therapeutic opportunities for combating breast cancer

  17. Inadvertent Intruder Analysis For The Portsmouth On-Site Waste Disposal Facility (OSWDF)

    Smith, Frank G. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL); Phifer, Mark A. [Savannah River Site (SRS), Aiken, SC (United States). Savannah River National Lab. (SRNL)

    2014-01-22

    The inadvertent intruder analysis considers the radiological impacts to hypothetical persons who are assumed to inadvertently intrude on the Portsmouth OSWDF site after institutional control ceases 100 years after site closure. For the purposes of this analysis, we assume that the waste disposal in the OSWDF occurs at time zero, the site is under institutional control for the next 100 years, and inadvertent intrusion can occur over the following 1,000 year time period. Disposal of low-level radioactive waste in the OSWDF must meet a requirement to assess impacts on such individuals, and demonstrate that the effective dose equivalent to an intruder would not likely exceed 100 mrem per year for scenarios involving continuous exposure (i.e. chronic) or 500 mrem for scenarios involving a single acute exposure. The focus in development of exposure scenarios for inadvertent intruders was on selecting reasonable events that may occur, giving consideration to regional customs and construction practices. An important assumption in all scenarios is that an intruder has no prior knowledge of the existence of a waste disposal facility at the site. Results of the analysis show that a hypothetical inadvertent intruder at the OSWDF who, in the worst case scenario, resides on the site and consumes vegetables from a garden established on the site using contaminated soil (chronic agriculture scenario) would receive a maximum chronic dose of approximately 7.0 mrem/yr during the 1000 year period of assessment. This dose falls well below the DOE chronic dose limit of 100 mrem/yr. Results of the analysis also showed that a hypothetical inadvertent intruder at the OSWDF who, in the worst case scenario, excavates a basement in the soil that reaches the waste (acute basement construction scenario) would receive a maximum acute dose of approximately 0.25 mrem/yr during the 1000 year period of assessment. This dose falls well below the DOE acute dose limit of 500 mrem/yr. Disposal inventory

  18. Numerical modeling of ductile tearing effects on cleavage fracture toughness

    Dodds, R.H. Jr.; Tang, M.; Anderson, T.L.

    1994-05-01

    Experimental studies demonstrate a significant effect of specimen size, a/W ratio and prior ductile tearing on cleavage fracture toughness values (J c ) measured in the ductile-to-brittle transition region of ferritic materials. In the lower-transition region, cleavage fracture often occurs under conditions of large-scale yielding but without prior ductile crack extension. The increased toughness develops when plastic zones formed at the crack tip interact with nearby specimen surfaces which relaxes crack-tip constraint (stress triaxiality). In the mid-to-upper transition region, small amounts of ductile crack extension (often c -values. Previous work by the authors described a micromechanics fracture model to correct measured J c -values for the mechanistic effects of large-scale yielding. This new work extends the model to also include the influence of ductile crack extension prior to cleavage. The paper explores development of the new model, provides necessary graphs and procedures for its application and demonstrates the effects of the model on fracture data sets for two pressure vessel steels (A533B and A515)

  19. Cleavage mechanoluminescence in elemental and III-V semiconductors

    Chandra, B.P.; Patel, R.P.; Gour, Anubha S.; Chandra, V.K.; Gupta, R.K.

    2003-01-01

    The present paper reports the theory of mechanoluminescence (ML) produced during cleavage of elemental and III-V semiconductors. It seems that the formation of crack-induced localized states is responsible for the ML excitation produced during the cleavage of elemental and III-V semiconductors. According to this mechanism, as the atoms are drawn away from each other in an advancing crack tip, the decreasing wave function overlap across the crack may result in localized states which is associated with increasing electron energy. If the energy of these localized states approach that of the conduction band, transition to the conduction band via tunnelling would be possible, creating minority carriers, and consequently the electron-hole recombination may give rise to mechanoluminescence. When an elemental or III-V semiconductor is cleaved, initially the ML intensity increases with time, attains a peak value I m at the time t m corresponding to completion of the cleavage of the semiconductor, and then it decreases following power law decay. Expressions are derived for the ML intensity I m corresponding to the peak of the ML intensity versus time curve and for the total ML intensity I T . It is shown that both I m and I T should increase directly with the area of the newly created surfaces of the crystals. From the measurements of the ML intensity, the velocity of crack propagation in material can be determined by using the relation v=H/t m

  20. Specific in vitro cleavage of Mason-Pfizer monkey virus capsid protein: evidence for a potential role of retroviral protease in early stages of infection

    Rumlova, Michaela; Ruml, Tomas; Pohl, Jan; Pichova, Iva

    2003-01-01

    Processing of Gag polyproteins by viral protease (PR) leads to reorganization of immature retroviral particles and formation of a ribonucleoprotein core. In some retroviruses, such as HIV and RSV, cleavage of a spacer peptide separating capsid and nucleocapsid proteins is essential for the core formation. We show here that no similar spacer peptide is present in the capsid-nucleocapsid (CA-NC) region of Mason-Pfizer monkey virus (M-PMV) and that the CA protein is cleaved in vitro by the PR within the major homology region (MHR) and the NC protein in several sites at the N-terminus. The CA cleavage product was also identified shortly after penetration of M-PMV into COS cells, suggesting that the protease-catalyzed cleavage is involved in core disintegration

  1. SITE-94. Discrete-feature modelling of the Aespoe site: 4. Source data and detailed analysis procedures

    Geier, J E [Golder Associates AB, Uppsala (Sweden)

    1996-12-01

    Specific procedures and source data are described for the construction and application of discrete-feature hydrological models for the vicinity of Aespoe. Documentation is given for all major phases of the work, including: Statistical analyses to develop and validate discrete-fracture network models, Preliminary evaluation, construction, and calibration of the site-scale model based on the SITE-94 structural model of Aespoe, Simulation of multiple realizations of the integrated model, and variations, to predict groundwater flow, and Evaluation of near-field and far-field parameters for performance assessment calculations. Procedures are documented in terms of the computer batch files and executable scripts that were used to perform the main steps in these analyses, to provide for traceability of results that are used in the SITE-94 performance assessment calculations. 43 refs.

  2. SITE-94. Discrete-feature modelling of the Aespoe site: 4. Source data and detailed analysis procedures

    Geier, J.E.

    1996-12-01

    Specific procedures and source data are described for the construction and application of discrete-feature hydrological models for the vicinity of Aespoe. Documentation is given for all major phases of the work, including: Statistical analyses to develop and validate discrete-fracture network models, Preliminary evaluation, construction, and calibration of the site-scale model based on the SITE-94 structural model of Aespoe, Simulation of multiple realizations of the integrated model, and variations, to predict groundwater flow, and Evaluation of near-field and far-field parameters for performance assessment calculations. Procedures are documented in terms of the computer batch files and executable scripts that were used to perform the main steps in these analyses, to provide for traceability of results that are used in the SITE-94 performance assessment calculations. 43 refs

  3. Environmental siting suitability analysis for commercial scale ocean renewable energy: A southeast Florida case study

    Mulcan, Amanda

    This thesis aims to facilitate the siting and implementation of Florida Atlantic University Southeast National Marine Renewable Energy Center (FAU SNMREC) ocean current energy (OCE) projects offshore southeastern Florida through the analysis of benthic anchoring conditions. Specifically, a suitability analysis considering all presently available biologic and geologic datasets within the legal framework of OCE policy and regulation was done. OCE related literature sources were consulted to assign suitability levels to each dataset, ArcGIS interpolations generated seafloor substrate maps, and existing submarine cable pathways were considered for OCE power cables. The finalized suitability map highlights the eastern study area as most suitable for OCE siting due to its abundance of sand/sediment substrate, existing underwater cable route access, and minimal biologic presence. Higher resolution datasets are necessary to locate specific OCE development locales, better understand their benthic conditions, and minimize potentially negative OCE environmental impacts.

  4. Field portable petroleum analysis for validation of the site characterization and analysis penetrometer system petroleum, oil and lubricant sensor

    Davis, W.M.; Jones, P.; Porter, B.

    1995-01-01

    A petroleum, oil and lubricant (POL) sensor for the Site Characterization and Analysis Penetrometer System (SCAPS) has been developed by the Tri-Services (e.g. Army, Navy and Air Force) to characterize the distribution of POL contaminants on military sites. The sensor is based on the detection of POL contaminants using a laser induced fluorescence (LIF) spectrometer. The SCAPS POL sensor has been shown to be a valuable tool for the rapid screening of POL contamination in the subsurface. However, many factors can affect the LIF response of a particular fuel at a particular site. These include fuel type, age of spill (e.g. weathering) and soil type. The LIF sensor also detects fluorescence from any naturally occurring fluorophores, including humic substances and fluorescent minerals. These factors lead to the development of an independent procedure for the verification of the POL sensor response. This paper describes a field portable total recoverable petroleum hydrocarbon (TRPH) method based on EPA Method 418.1 and its application to on site validation of the SCAPS POL sensor response at a number of contaminated sites

  5. Analysis of external flooding events occurred in foreign nuclear power plant sites

    Li Dan; Cai Hankun; Xiao Zhi; An Hongzhen; Mao Huan

    2013-01-01

    This paper screens and studies 17 external flooding events occurred in foreign NPP sites, analysis the characteristic of external flooding events based on the source of the flooding, the impact on the building, systems and equipment, as well as the threat to nuclear safety. Furthermore, based on the experiences and lessons learned from Fukushima nuclear accident relating to external flooding and countermeasures carried out in the world, some suggestions are proposed in order to improve external flooding response capacity for Chinese NPPs. (authors)

  6. Tentative job analysis for a high-level, fixed-site, nuclear security officer

    Adams, K.G.; Trujillo, A.A.

    1977-10-01

    A tentative job analysis for a high-level, fixed-site, nuclear security officer is presented. The primary objective of the report is to provide a framework for evaluating the functions of a security officer in physical protection systems. Several job requirements related to duties, basic skills, personal contacts, supervision, working conditions, and decision making are presented. Individual character traits desirable in security officers are described

  7. Biosphere analysis - a complementary assessment of dose conversion factors for the Olkiluoto site

    Kylloenen, J.; Keto, V.

    2010-04-01

    The Olkiluoto site is currently the primary candidate for the final disposal site for spent nuclear fuel from the Olkiluoto and Loviisa NPPs. Safety analysis calculations must be performed to verify the compliance with the long-term safety requirements. The behaviour and distribution of radionuclides in the biosphere is of high importance in these calculations. The aim of this study was to perform a complementary assessment of dose conversion factors for the Olkiluoto site. Posiva has performed extensive analysis on the different ecosystems. In this work the biosphere analysis model of Fortum Nuclear Services (FNS) is used to give an independent estimate of biosphere dose conversion factors for the Olkiluoto site. The following nuclides are analysed: Cl-36, Ni-59, Se-79, Mo-93, Nb-94, Sn-126, I-129 and Cs-135. The FNS model is an equilibrium compartment model in which a steady annual release of 1 Bq of each radionuclide is distributed in different scenarios. The scenarios are the well scenario, which models a small agricultural ecosystem, the lake scenario which models a larger ecosystem with both agriculture and lake use, and sea and transition scenario, which models the behaviour of the radionuclides in marine environments. The scenarios are described and the transfer equations written for the lake scenario. The parameter values are taken from the FNS biosphere database, which has been used in the Finnish L/ILW waste repository safety analyses since mid 1990's. The results of the FNS analysis are compared to those presented in Posiva working report 2000-20 (POSIVA-WR-00-20). The results are of the same order of magnitude for all nuclides except I-129. Since the Posiva and FNS models were independently constructed, the results can be considered as convincing, and the compliance of the results give confidence to the modelling results. (orig.)

  8. Fuel Assemblies Thermal Analysis in the New Spent Fuel Storage Facility at Inshass Site

    Khattab, M.; Mariy, Ahmed

    1999-01-01

    New Wet Storage Facility (NSF) is constructed at Inshass site to solve the problem of spent fuel storage capacity of ETRR-1 reactor . The Engineering Safety Heat Transfer Features t hat characterize the new facility are presented. Thermal analysis including different scenarios of pool heat load and safety limits are discussed . Cladding temperature limit during handling and storage process are specified for safe transfer of fuel

  9. Active site specificity profiling datasets of matrix metalloproteinases (MMPs 1, 2, 3, 7, 8, 9, 12, 13 and 14

    Ulrich Eckhard

    2016-06-01

    Full Text Available The data described provide a comprehensive resource for the family-wide active site specificity portrayal of the human matrix metalloproteinase family. We used the high-throughput proteomic technique PICS (Proteomic Identification of protease Cleavage Sites to comprehensively assay 9 different MMPs. We identified more than 4300 peptide cleavage sites, spanning both the prime and non-prime sides of the scissile peptide bond allowing detailed subsite cooperativity analysis. The proteomic cleavage data were expanded by kinetic analysis using a set of 6 quenched-fluorescent peptide substrates designed using these results. These datasets represent one of the largest specificity profiling efforts with subsequent structural follow up for any protease family and put the spotlight on the specificity similarities and differences of the MMP family. A detailed analysis of this data may be found in Eckhard et al. (2015 [1]. The raw mass spectrometry data and the corresponding metadata have been deposited in PRIDE/ProteomeXchange with the accession number http://www.ebi.ac.uk/pride/archive/projects/PXD002265.

  10. Sequence adaptations affecting cleavage of the VP1/2A junction by the 3C protease in foot-and-mouth disease virus-infected cells

    Gullberg, Maria; Polacek, Charlotta; Belsham, Graham

    2014-01-01

    The foot-and-mouth disease virus (FMDV) capsid protein precursor P1-2A is cleaved by the virus-encoded 3C protease to VP0, VP3, VP1 and 2A. It was shown previously that modification of a single amino acid residue (K210E) within the VP1 protein and close to the VP1/2A cleavage site, inhibited...... cleavage of this junction and produced 'self-tagged' virus particles. A second site substitution (E83K) within VP1 was also observed within the rescued virus [Gullberg et al. (2013). J Virol 87: , 11591-11603]. It was shown here that introduction of this E83K change alone into a serotype O virus resulted...... in the rapid accumulation of a second site substitution within the 2A sequence (L2P), which also blocked VP1/2A cleavage. This suggests a linkage between the E83K change in VP1 and cleavage of the VP1/2A junction. Cells infected with viruses containing the VP1 K210E or the 2A L2P substitutions contained...

  11. A Ten Step Protocol and Plan for CCS Site Characterization, Based on an Analysis of the Rocky Mountain Region, USA

    McPherson, Brian; Matthews, Vince

    2013-09-15

    This report expresses a Ten-Step Protocol for CO2 Storage Site Characterization, the final outcome of an extensive Site Characterization analysis of the Rocky Mountain region, USA. These ten steps include: (1) regional assessment and data gathering; (2) identification and analysis of appropriate local sites for characterization; (3) public engagement; (4) geologic and geophysical analysis of local site(s); (5) stratigraphic well drilling and coring; (6) core analysis and interpretation with other data; (7) database assembly and static model development; (8) storage capacity assessment; (9) simulation and uncertainty assessment; (10) risk assessment. While the results detailed here are primarily germane to the Rocky Mountain region, the intent of this protocol is to be portable or generally applicable for CO2 storage site characterization.

  12. Overheads, Safety Analysis and Engineering FY 1995 Site Support Program Plan WBS 6.3.5

    DiVincenzo, E.P.

    1994-09-27

    The Safety Analysis & Engineering (SA&E) department provides core competency for safety analysis and risk documentation that supports achievement of the goals and mission as described in the Hanford Mission Plan, Volume I, Site Guidance (DOE-RL 1993). SA&E operations are integrated into the programs that plan and conduct safe waste management, environmental restoration, and operational activities. SA&E personnel are key members of task teams assigned to eliminate urgent risks and inherent threats that exist at the Hanford Site. Key to ensuring protection of public health and safety, and that of onsite workers, are the products and services provided by the department. SA&E will continue to provide a leadership role throughout the DOE complex with innovative, cost-effective approaches to ensuring safety during environmental cleanup operations. The SA&E mission is to provide support to direct program operations through safety analysis and risk documentation and to maintain an infrastructure responsive to the evolutionary climate at the Hanford Site. SA&E will maintain the appropriate skills mix necessary to fulfill the customers need to conduct all operations in a safe and cost-effective manner while ensuring the safety of the public and the onsite worker.

  13. Overheads, Safety Analysis and Engineering FY 1995 Site Support Program Plan WBS 6.3.5

    DiVincenzo, E.P.

    1994-01-01

    The Safety Analysis ampersand Engineering (SA ampersand E) department provides core competency for safety analysis and risk documentation that supports achievement of the goals and mission as described in the Hanford Mission Plan, Volume I, Site Guidance (DOE-RL 1993). SA ampersand E operations are integrated into the programs that plan and conduct safe waste management, environmental restoration, and operational activities. SA ampersand E personnel are key members of task teams assigned to eliminate urgent risks and inherent threats that exist at the Hanford Site. Key to ensuring protection of public health and safety, and that of onsite workers, are the products and services provided by the department. SA ampersand E will continue to provide a leadership role throughout the DOE complex with innovative, cost-effective approaches to ensuring safety during environmental cleanup operations. The SA ampersand E mission is to provide support to direct program operations through safety analysis and risk documentation and to maintain an infrastructure responsive to the evolutionary climate at the Hanford Site. SA ampersand E will maintain the appropriate skills mix necessary to fulfill the customers need to conduct all operations in a safe and cost-effective manner while ensuring the safety of the public and the onsite worker

  14. Draft site characterization analysis of the site characterization report for the Basalt Waste Isolation Project, Hanford, Washington site. Appendices E through W

    1983-03-01

    Volume 2 contains Appendices E through W: potential for large-scale pump tests in the Grande Ronde; review of hydrochemical characterization related to flow system interpretation in Hanford basalts; limitations of packer-testing for head evaluation in Hanford basalts; hydrogeologic data integration for conceptual groundwater flow models; drilling mud effects on hydrogeologic testing; site issue analyses related to the nature at the present groundwater system at the Hanford site, Washington; structural and stratigraphic characteristics related to groundwater flow at the Hanford site, Washington; seismic hazard and some examples of hazard studies at Hanford; earthquake swarms in the Columbia Plateau; seismic ground motion at depth; failure modes for the metallic waste package component; degradation mechanisms of borosilicate glass; transport and retardation of radionuclides in the waste package; determination and interpretation of redox conditions and changes in underground high-level repositories; determination and interpretation of sorption data applied to radionuclide migration in underground repositories; solubility of radionuclide compounds presented in the BWIP site characterization report; and release rate from engineered system

  15. [Recent knowledge about intestinal absorption and cleavage of carotenoids].

    Borel, P; Drai, J; Faure, H; Fayol, V; Galabert, C; Laromiguière, M; Le Moël, G

    2005-01-01

    Our knowledge about intestinal absorption and cleavage of carotenoids has rapidly grown during the last years. New facts about carotenoid absorption have emerged while some controversies about cleavage are close to end. The knowledge of the absorption and conversion processes is indispensable to understand and interpret the perturbations that can occur in the metabolism of carotenoids and vitamin A. Recently, it has been shown that the absorption of certain carotenoids is not passive - as believed for a long time - but is a facilitated process that requires, at least for lutein, the class B-type 1 scavenger receptor (SR-B1). Various epidemiological and clinical studies have shown wide variations in carotenoid absorption from one subject to another, such differences are now explained by the structure of the concerned carotenoid, by the nature of the food that is absorbed with the carotenoid, by diverse exogenous factors like the intake of medicines or interfering components, by diet factors, by genetic factors, and by the nutritional status of the subject. Recently, the precise mechanism of beta-carotene cleavage by betabeta-carotene 15,15' monooxygenase (EC 1.14.99.36) - formerly called beta-carotene 15,15' dioxygenase (ex EC 1.13.11.21) - has been discovered, and a second enzyme which cleaves asymmetrically the beta-carotene molecule has been found. beta-carotene 15,15' monooxygenase only acts on the 15,15' bond, thus forming two molecules of retinal from one molecule of beta-carotene by central cleavage. Even though the betabeta-carotene 15,15' monooxygenase is much more active on the beta-carotene molecule, a study has shown that it can act on all carotenoids. Searchers now agree that other enzymes that can catalyse an eccentric cleavage of carotenoids probably exist, but under physiological conditions the betabeta-carotene 15,15' monooxygenase is by far the most active, and it is mainly effective in the small bowel mucosa and in the liver. However the

  16. Conserved nucleation sites reinforce the significance of Phi value analysis in protein-folding studies.

    Gianni, Stefano; Jemth, Per

    2014-07-01

    The only experimental strategy to address the structure of folding transition states, the so-called Φ value analysis, relies on the synergy between site directed mutagenesis and the measurement of reaction kinetics. Despite its importance, the Φ value analysis has been often criticized and its power to pinpoint structural information has been questioned. In this hypothesis, we demonstrate that comparing the Φ values between proteins not only allows highlighting the robustness of folding pathways but also provides per se a strong validation of the method. © 2014 International Union of Biochemistry and Molecular Biology.

  17. [Analysis of genetico-demographic structure of rural populations living near the Semipalatinsk nuclear test site].

    Sviatova, G S; Berezina, G M; Abil'dinova, G Zh

    2001-12-01

    Rural populations neighboring the Semipalatinsk nuclear test site were used as a model to develop and test an integrated population-genetic approach to analysis of the medical genetic situation and environmental conditions in the areas studied. The contributions of individual factors of population dynamics into the formation of the genetic load were also assessed. The informative values of some genetic markers were estimated. Based on these estimates, a mathematical model was constructed that makes it possible to calculate numerical scores for analysis of the genetic loads in populations differing in environmental exposure.

  18. Analysis and prediction of Multiple-Site Damage (MSD) fatigue crack growth

    Dawicke, D. S.; Newman, J. C., Jr.

    1992-08-01

    A technique was developed to calculate the stress intensity factor for multiple interacting cracks. The analysis was verified through comparison with accepted methods of calculating stress intensity factors. The technique was incorporated into a fatigue crack growth prediction model and used to predict the fatigue crack growth life for multiple-site damage (MSD). The analysis was verified through comparison with experiments conducted on uniaxially loaded flat panels with multiple cracks. Configuration with nearly equal and unequal crack distribution were examined. The fatigue crack growth predictions agreed within 20 percent of the experimental lives for all crack configurations considered.

  19. Analysis and prediction of Multiple-Site Damage (MSD) fatigue crack growth

    Dawicke, D. S.; Newman, J. C., Jr.

    1992-01-01

    A technique was developed to calculate the stress intensity factor for multiple interacting cracks. The analysis was verified through comparison with accepted methods of calculating stress intensity factors. The technique was incorporated into a fatigue crack growth prediction model and used to predict the fatigue crack growth life for multiple-site damage (MSD). The analysis was verified through comparison with experiments conducted on uniaxially loaded flat panels with multiple cracks. Configuration with nearly equal and unequal crack distribution were examined. The fatigue crack growth predictions agreed within 20 percent of the experimental lives for all crack configurations considered.

  20. Hanford Site Composite Analysis Technical Approach Description: Groundwater Pathway Dose Calculation.

    Morgans, D. L. [CH2M Hill Plateau Remediation Company, Richland, WA (United States); Lindberg, S. L. [Intera Inc., Austin, TX (United States)

    2017-09-20

    The purpose of this technical approach document (TAD) is to document the assumptions, equations, and methods used to perform the groundwater pathway radiological dose calculations for the revised Hanford Site Composite Analysis (CA). DOE M 435.1-1, states, “The composite analysis results shall be used for planning, radiation protection activities, and future use commitments to minimize the likelihood that current low-level waste disposal activities will result in the need for future corrective or remedial actions to adequately protect the public and the environment.”

  1. Data base for site specific migration analysis of radioactive elements in repositories

    Hadermann, J

    1982-01-01

    Migration analysis is of considerable importance in long-term safety aspects of radioactive waste repositories. The present work gives the data base for a selected part of a comprehensive geosphere transport calculation. We restrict ourselves to a critical evaluation of parameters pertinent to the migration analysis of the /sup 245/Cm chain. This includes the important nuclide /sup 237/Np. With these we are able to perform a site specific calculation for repositories planned in deep geologic formations in Switzerland. It is shown that the granitic basement induces strong time delays in nuclide migration. In contrast to that, the overlaying sedimentary layers cause primarily a dilution of the radionuclide concentrations.

  2. Array analysis of regional Pn and Pg wavefields from the Nevada Test Site

    Leonard, M.A.

    1991-06-01

    Small-aperture high-frequency seismic arrays with dimensions of a few kilometers or less, can improve our ability to seismically monitor compliance with a low-yield Threshold Test Ban Treaty. This work studies the characteristics and effectiveness of array processing of the regional Pn and Pg wavefields generated by underground nuclear explosions at the Nevada Test Site. Waveform data from the explosion HARDIN (m b = 5.5) is recorded at a temporary 12-element, 3-component, 1.5 km-aperture array sited in an area of northern Nevada. The explosions VILLE (m b = 4.4) and SALUT (m b = 5.5) are recorded at two arrays sited in the Mojave desert, one a 96-element vertical-component 7 km-aperture array and the other a 155-element vertical-component 4 km-aperture array. Among the mean spectra for the m b = 5.5 events there are significant differences in low-frequency spectral amplitudes between array sites. The spectra become nearly identical beyond about 6 Hz. Spectral ratios are used to examine seismic source properties and the partitioning of energy between Pn and Pg. Frequency-wavenumber analysis at the 12-element array is used to obtain estimates of signal gain, phase velocity, and source azimuth. This analysis reveals frequency-dependent biases in velocity and azimuth of the coherent Pn and Pg arrivals. Signal correlation, the principal factor governing array performance, is examined in terms of spatial coherence estimates. The coherence is found to vary between the three sites. In all cases the coherence of Pn is greater than that for Pg. 81 refs., 92 figs., 5 tabs

  3. Array analysis of regional Pn and Pg wavefields from the Nevada Test Site

    Leonard, M.A. (California Univ., Berkeley, CA (United States). Dept. of Geology and Geophysics Lawrence Berkeley Lab., CA (United States))

    1991-06-01

    Small-aperture high-frequency seismic arrays with dimensions of a few kilometers or less, can improve our ability to seismically monitor compliance with a low-yield Threshold Test Ban Treaty. This work studies the characteristics and effectiveness of array processing of the regional Pn and Pg wavefields generated by underground nuclear explosions at the Nevada Test Site. Waveform data from the explosion HARDIN (m{sub b} = 5.5) is recorded at a temporary 12-element, 3-component, 1.5 km-aperture array sited in an area of northern Nevada. The explosions VILLE (m{sub b} = 4.4) and SALUT (m{sub b} = 5.5) are recorded at two arrays sited in the Mojave desert, one a 96-element vertical-component 7 km-aperture array and the other a 155-element vertical-component 4 km-aperture array. Among the mean spectra for the m{sub b} = 5.5 events there are significant differences in low-frequency spectral amplitudes between array sites. The spectra become nearly identical beyond about 6 Hz. Spectral ratios are used to examine seismic source properties and the partitioning of energy between Pn and Pg. Frequency-wavenumber analysis at the 12-element array is used to obtain estimates of signal gain, phase velocity, and source azimuth. This analysis reveals frequency-dependent biases in velocity and azimuth of the coherent Pn and Pg arrivals. Signal correlation, the principal factor governing array performance, is examined in terms of spatial coherence estimates. The coherence is found to vary between the three sites. In all cases the coherence of Pn is greater than that for Pg. 81 refs., 92 figs., 5 tabs.

  4. Visibility Analysis of Domestic Satellites on Proposed Ground Sites for Optical Surveillance

    Jung Hyun Jo1

    2011-12-01

    Full Text Available The objectives of this study are to analyze the satellite visibility at the randomly established ground sites, to determine the five optimal ground sites to perform the optical surveillance and tracking of domestic satellites, and to verify the acquisition of the optical observation time sufficient to maintain the precise ephemeris at optimal ground sites that have been already determined. In order to accomplish these objectives, we analyzed the visibility for sun-synchronous orbit satellites, low earth orbit satellites, middle earth orbit satellites and domestic satellites as well as the continuous visibility along with the fictitious satellite ground track, and calculate the effective visibility. For the analysis, we carried out a series of repetitive process using the satellite tool kit simulation software developed by Analytical Graphics Incorporated. The lighting states of the penumbra and direct sun were set as the key constraints of the optical observation. The minimum of the observation satellite elevation angle was set to be 20 degree, whereas the maximum of the sun elevation angle was set to be -10 degree which is within the range of the nautical twilight. To select the candidates for the optimal optical observation, the entire globe was divided into 84 sectors in a constant interval, the visibility characteristics of the individual sectors were analyzed, and 17 ground sites were arbitrarily selected and analyzed further. Finally, five optimal ground sites (Khurel Togoot Observatory, Assy-Turgen Observatory, Tubitak National Observatory, Bisdee Tier Optical Astronomy Observatory, and South Africa Astronomical Observatory were determined. The total observation period was decided as one year. To examine the seasonal variation, the simulation was performed for the period of three days or less with respect to spring, summer, fall and winter. In conclusion, we decided the optimal ground sites to perform the optical surveillance and tracking

  5. Multielemental analysis to the profile sediments from several sites on the Havana Bay, by the Neutron activation analysis

    Diaz Riso, O.; Gelen, A.; Lopez, N.; Manso, M.V.; Graciano, A.M.; Nogueira, C.A.; Beltran, J.

    2003-01-01

    Instrumental neutron activation analysis (INAA) technique was employed to analyze the profile sediments from several sites on the Havana Bay, Cuba. Measurements of 24 heavy and trace elements in the sediments are reported. The results show the increment in the sediment pollution occurred in the 70-80 years of the last century. The data confirm that an anthropogenic input into the bay from domestic sewage and industries occurred

  6. Efficient analysis using custom interactive visualization tools at a Superfund site

    Williams, G.; Durham, L.

    1992-01-01

    Custom visualization analysis programs were developed and used to analyze contaminant transport calculations from a three-dimensional numerical groundwater flow model developed for a Department of Energy Superfund site. The site hydrogeology, which is highly heterogenous, includes both fractured limestone and dolomite and alluvium deposits. Three-dimensional interactive visualization techniques were used to understand and analyze the three-dimensional, double-porosity modeling results. A graphical object oriented programming environment was applied to efficiently develop custom visualization programs in a coarse-grained data structure language. Comparisons were made, using the results from the three-dimensional, finite-difference model, between traditional two-dimensional analyses (contour and vector plots) and interactive three-dimensional techniques. Subjective comparison areas include the accuracy of analysis, the ability to understand the results of three-dimensional contaminant transport simulation, and the capability to transmit the results of the analysis to the project management. In addition, a quantitative comparison was made on the time required to develop a thorough analysis of the modeling results. The conclusions from the comparative study showed that the visualization analysis provided an increased awareness of the contaminant transport mechanisms, provided new insights into contaminant migration, and resulted in a significant time savings

  7. Efficient analysis using custom interactive visualization tools at a Superfund site

    Williams, G. [Northwestern Univ., Evanston, IL (United States); Durham, L. [Argonne National Lab., IL (United States)

    1992-12-01

    Custom visualization analysis programs were developed and used to analyze contaminant transport calculations from a three-dimensional numerical groundwater flow model developed for a Department of Energy Superfund site. The site hydrogeology, which is highly heterogenous, includes both fractured limestone and dolomite and alluvium deposits. Three-dimensional interactive visualization techniques were used to understand and analyze the three-dimensional, double-porosity modeling results. A graphical object oriented programming environment was applied to efficiently develop custom visualization programs in a coarse-grained data structure language. Comparisons were made, using the results from the three-dimensional, finite-difference model, between traditional two-dimensional analyses (contour and vector plots) and interactive three-dimensional techniques. Subjective comparison areas include the accuracy of analysis, the ability to understand the results of three-dimensional contaminant transport simulation, and the capability to transmit the results of the analysis to the project management. In addition, a quantitative comparison was made on the time required to develop a thorough analysis of the modeling results. The conclusions from the comparative study showed that the visualization analysis provided an increased awareness of the contaminant transport mechanisms, provided new insights into contaminant migration, and resulted in a significant time savings.

  8. Life cycle cost analysis changes mixed waste treatment program at the Savannah River Site

    Pickett, J.B.; England, J.L.; Martin, H.L.

    1992-01-01

    A direct result of the reduced need for weapons production has been a re-evaluation of the treatment projects for mixed (hazardous/radioactive) wastes generated from metal finishing and plating operations and from a mixed waste incinerator at the Savannah River Site (SRS). A Life Cycle Cost (LCC) analysis was conducted for two waste treatment projects to determine the most cost effective approach in response to SRS mission changes. A key parameter included in the LCC analysis was the cost of the disposal vaults required for the final stabilized wasteform(s) . The analysis indicated that volume reduction of the final stabilized wasteform(s) can provide significant cost savings. The LCC analysis demonstrated that one SRS project could be eliminated, and a second project could be totally ''rescoped and downsized.'' The changes resulted in an estimated Life Cycle Cost saving (over a 20 year period) of $270,000,000

  9. Exploring the feasibility of multi-site flow cytometric processing of gut associated lymphoid tissue with centralized data analysis for multi-site clinical trials.

    Ian McGowan

    Full Text Available The purpose of this study was to determine whether the development of a standardized approach to the collection of intestinal tissue from healthy volunteers, isolation of gut associated lymphoid tissue mucosal mononuclear cells (MMC, and characterization of mucosal T cell phenotypes by flow cytometry was sufficient to minimize differences in the normative ranges of flow parameters generated at two trial sites. Forty healthy male study participants were enrolled in Pittsburgh and Los Angeles. MMC were isolated from rectal biopsies using the same biopsy acquisition and enzymatic digestion protocols. As an additional comparator, peripheral blood mononuclear cells (PBMC were collected from the study participants. For quality control, cryopreserved PBMC from a single donor were supplied to both sites from a central repository (qPBMC. Using a jointly optimized standard operating procedure, cells were isolated from tissue and blood and stained with monoclonal antibodies targeted to T cell phenotypic markers. Site-specific flow data were analyzed by an independent center which analyzed all data from both sites. Ranges for frequencies for overall CD4+ and CD8+ T cells, derived from the qPBMC samples, were equivalent at both UCLA and MWRI. However, there were significant differences across sites for the majority of T cell activation and memory subsets in qPBMC as well as PBMC and MMC. Standardized protocols to collect, stain, and analyze MMC and PBMC, including centralized analysis, can reduce but not exclude variability in reporting flow data within multi-site studies. Based on these data, centralized processing, flow cytometry, and analysis of samples may provide more robust data across multi-site studies. Centralized processing requires either shipping of fresh samples or cryopreservation and the decision to perform centralized versus site processing needs to take into account the drawbacks and restrictions associated with each method.

  10. Exploring the feasibility of multi-site flow cytometric processing of gut associated lymphoid tissue with centralized data analysis for multi-site clinical trials.

    McGowan, Ian; Anton, Peter A; Elliott, Julie; Cranston, Ross D; Duffill, Kathryn; Althouse, Andrew D; Hawkins, Kevin L; De Rosa, Stephen C

    2015-01-01

    The purpose of this study was to determine whether the development of a standardized approach to the collection of intestinal tissue from healthy volunteers, isolation of gut associated lymphoid tissue mucosal mononuclear cells (MMC), and characterization of mucosal T cell phenotypes by flow cytometry was sufficient to minimize differences in the normative ranges of flow parameters generated at two trial sites. Forty healthy male study participants were enrolled in Pittsburgh and Los Angeles. MMC were isolated from rectal biopsies using the same biopsy acquisition and enzymatic digestion protocols. As an additional comparator, peripheral blood mononuclear cells (PBMC) were collected from the study participants. For quality control, cryopreserved PBMC from a single donor were supplied to both sites from a central repository (qPBMC). Using a jointly optimized standard operating procedure, cells were isolated from tissue and blood and stained with monoclonal antibodies targeted to T cell phenotypic markers. Site-specific flow data were analyzed by an independent center which analyzed all data from both sites. Ranges for frequencies for overall CD4+ and CD8+ T cells, derived from the qPBMC samples, were equivalent at both UCLA and MWRI. However, there were significant differences across sites for the majority of T cell activation and memory subsets in qPBMC as well as PBMC and MMC. Standardized protocols to collect, stain, and analyze MMC and PBMC, including centralized analysis, can reduce but not exclude variability in reporting flow data within multi-site studies. Based on these data, centralized processing, flow cytometry, and analysis of samples may provide more robust data across multi-site studies. Centralized processing requires either shipping of fresh samples or cryopreservation and the decision to perform centralized versus site processing needs to take into account the drawbacks and restrictions associated with each method.

  11. A novel carotenoid cleavage activity involved in the biosynthesis of Citrus fruit-specific apocarotenoid pigments

    Rodrigo, María J.

    2013-09-04

    Citrus is the first tree crop in terms of fruit production. The colour of Citrus fruit is one of the main quality attributes, caused by the accumulation of carotenoids and their derivative C30 apocarotenoids, mainly ?-citraurin (3-hydroxy-?-apo-8?-carotenal), which provide an attractive orange-reddish tint to the peel of oranges and mandarins. Though carotenoid biosynthesis and its regulation have been extensively studied in Citrus fruits, little is known about the formation of C30 apocarotenoids. The aim of this study was to the identify carotenoid cleavage enzyme(s) [CCD(s)] involved in the peel-specific C30 apocarotenoids. In silico data mining revealed a new family of five CCD4-type genes in Citrus. One gene of this family, CCD4b1, was expressed in reproductive and vegetative tissues of different Citrus species in a pattern correlating with the accumulation of C30 apocarotenoids. Moreover, developmental processes and treatments which alter Citrus fruit peel pigmentation led to changes of ?-citraurin content and CCD4b1 transcript levels. These results point to the involvement of CCD4b1 in ?-citraurin formation and indicate that the accumulation of this compound is determined by the availability of the presumed precursors zeaxanthin and ?-cryptoxanthin. Functional analysis of CCD4b1 by in vitro assays unequivocally demonstrated the asymmetric cleavage activity at the 7?,8? double bond in zeaxanthin and ?-cryptoxanthin, confrming its role in C30 apocarotenoid biosynthesis. Thus, a novel plant carotenoid cleavage activity targeting the 7?,8? double bond of cyclic C40 carotenoids has been identified. These results suggest that the presented enzyme is responsible for the biosynthesis of C30 apocarotenoids in Citrus which are key pigments in fruit coloration. The Author 2013.

  12. A novel carotenoid cleavage activity involved in the biosynthesis of Citrus fruit-specific apocarotenoid pigments

    Rodrigo, Marí a J.; Alqué zar, Berta; Aló s, Enriqueta; Medina, Ví ctor; Carmona, Lourdes; Bruno, Mark; Al-Babili, Salim; Zacarí as, Lorenzo

    2013-01-01

    Citrus is the first tree crop in terms of fruit production. The colour of Citrus fruit is one of the main quality attributes, caused by the accumulation of carotenoids and their derivative C30 apocarotenoids, mainly ?-citraurin (3-hydroxy-?-apo-8?-carotenal), which provide an attractive orange-reddish tint to the peel of oranges and mandarins. Though carotenoid biosynthesis and its regulation have been extensively studied in Citrus fruits, little is known about the formation of C30 apocarotenoids. The aim of this study was to the identify carotenoid cleavage enzyme(s) [CCD(s)] involved in the peel-specific C30 apocarotenoids. In silico data mining revealed a new family of five CCD4-type genes in Citrus. One gene of this family, CCD4b1, was expressed in reproductive and vegetative tissues of different Citrus species in a pattern correlating with the accumulation of C30 apocarotenoids. Moreover, developmental processes and treatments which alter Citrus fruit peel pigmentation led to changes of ?-citraurin content and CCD4b1 transcript levels. These results point to the involvement of CCD4b1 in ?-citraurin formation and indicate that the accumulation of this compound is determined by the availability of the presumed precursors zeaxanthin and ?-cryptoxanthin. Functional analysis of CCD4b1 by in vitro assays unequivocally demonstrated the asymmetric cleavage activity at the 7?,8? double bond in zeaxanthin and ?-cryptoxanthin, confrming its role in C30 apocarotenoid biosynthesis. Thus, a novel plant carotenoid cleavage activity targeting the 7?,8? double bond of cyclic C40 carotenoids has been identified. These results suggest that the presented enzyme is responsible for the biosynthesis of C30 apocarotenoids in Citrus which are key pigments in fruit coloration. The Author 2013.

  13. Analysis of elemental composition of porcelains unearthed from Waguantan kiln site by PIXE–RBS

    Zhou, Z.; Zhang, K.; Xia, C.D.; Liu, M.T.; Zhu, J.J.; An, Z.; Bai, B.

    2015-01-01

    Highlights: •We analyzed 61 unearthed porcelain shards in Yuan Dynasty by PIXE–RBS. •An electron gun was installed to solve the electric charge accumulations. •The factor analysis was performed for the element compositions. •The “exotic group” porcelain samples unearthed were produced locally. -- Abstract: A method combining proton-induced X-ray emission spectrometry (PIXE) and Rutherford backscattering spectrometry (RBS) was used to determine the composition of 61 porcelain shards from the Yuan Dynasty (1271–1368 A.D.) unearthed from the Waguantan kiln site at Tianzhu County in Guizhou Province, China. Based on our previous experimental setup, an electron gun device with a LaB 6 crystal cathode was installed to solve the problem created when the incident proton beams generated electric charge accumulations on the surfaces of the insulating porcelain samples, which induced a large bremsstrahlung background. The use of the electron gun has largely eliminated the large bremsstrahlung background and has therefore improved the detection limits for elements, especially for trace elements, and made it possible to determine the origin of the porcelains based on the trace elements. Major and trace elemental compositions of the porcelain bodies and glazes measured by PIXE and RBS were analyzed by the factor analysis method. The factor analysis showed that a few pieces of porcelain with a style similar to the porcelain of the Longquan kiln among the unearthed porcelains from the Waguantan kiln site did not have obvious differences in elemental compositions from other remaining porcelains unearthed from the Waguantan kiln site, indicating that the pieces of unearthed porcelain with the Longquan kiln style did in fact belong to the product fired locally by imitating the model of the Longquan celadon with local raw materials. This result therefore indicated that the Longquan kiln technology that originated from the Five Dynasties (907–960 A.D.) had been

  14. A possibilistic analysis approach for environmental risk assessment of petroleum-contaminated sites

    Liu, L.; Huang, G.H.; Fuller, G.A.

    1999-01-01

    A possibilistic approach for assessing risks associated with petroleum-contaminated sites is provided. The approach is applied to an illustrative case study in order to show its applicability and implementation for decision making in effective site remediation and management. The approach consists of three main parts: (1) the determination of fuzzy steady state contaminant concentrations in a aquifer based on an analytical solute transport model; (2) possibilistic analysis of fuzzy criteria for different risk levels; and (3) environmental risk assessment based on the Euclidian method. Based on results from an illustrative case study, environmental risks at a petroleum-contaminated site can be effectively evaluated using the developed methodology. The risk assessment framework can effectively handle uncertainities presented as fuzzy numbers. The fuzzy nature of water quality and risk level criteria were seen in the related simulation and evaluation models, and the framework is especially useful for situations in which probabilistic information is not available. This use of this approach is new in the area of petroleum waste management under uncertainty, and its results are useful for the related site remediation and management decisions. 36 refs., 1 tab., 4 figs

  15. Anaerobic digester systems (ADS) for multiple dairy farms: A GIS analysis for optimal site selection

    Thompson, Ethan; Wang, Qingbin; Li, Minghao

    2013-01-01

    While anaerobic digester systems (ADS) have been increasingly adopted by large dairy farms to generate marketable energy products, like electricity, from animal manure, there is a growing need for assessing the feasibility of regional ADS for multiple farms that are not large enough to capitalize their own ADS. Using geographical information system (GIS) software, this study first identifies potential sites in a dairy region in Vermont, based on geographical conditions, current land use types, and energy distribution infrastructure criteria, and then selects the optimal sites for a given number of ADS, based on the number of dairy farms to be served, the primary energy input to output (PEIO) ratio of ADS, and the existing transportation network. This study suggests that GIS software is a valid technical tool for identifying the potential and optimal sites for ADS. The empirical findings provide useful information for assessing the returns of alternative numbers of ADS in this region, and the research procedures can be modified easily to incorporate any changes in the criteria for this region and can be applied in other regions with different conditions and criteria. - Highlights: • This study examines the feasibility of regional ADS for multiple dairy farms. • GIS is used to identify candidate sites and optimal locations for ADS in a dairy region. • Model includes environmental, social, infrastructure, and energy return criteria. • Empirical analysis provides scenario results on 1–15 ADS in the study region. • Method could be applied to other regions with different conditions and criteria

  16. Soil pollution with trace elements at selected sites in Romania studied by instrumental neutron activation analysis

    Pantelica, A.; Carmo Freitas, M. do; Ene, A.; Steinnes, E.

    2013-01-01

    Instrumental neutron activation analysis (INAA) was used to determine concentrations of 42 elements in samples of surface soil collected at seven sites polluted from various anthropogenic activities and a control site in a relatively clean area. Elements studied were Ag, Al, As, Au, Ba, Br, Ca, Cd, Ce, Co, Cr, Cs, Eu, Fe, Gd, Hf, Hg, K, La, Lu, Mg, Mn, Mo, Na, Nd, Ni, Rb, Sb, Sc, Se, Sm, Sr, Ta, Tb, Th, Ti, U, V, W, Yb, Zn, and Zr. The results are compared with data for trace elements atmospheric deposition in lichen transplants from the same sites. The most severe soil contamination was observed at Copsa Mica from non-ferrous metallurgy. Appreciable soil contamination was also indicated at Baia Mare (non-ferrous mining and metallurgy), Deva (coal-fired power plant, cement and building materials industry), Galati (ferrous metallurgy), Magurele and Afumati (general urban pollution), and Oradea (chemical and light industries). In most cases excessive levels of toxic metals in soils matched correspondingly high values in lichen transplants. Compared to Romanian norms, legal upper limits were exceeded for Zn and Cd at Copsa Mica. Also, As and Sb occurred in excessive levels at given sites. (orig.)

  17. Soil pollution with trace elements at selected sites in Romania studied by instrumental neutron activation analysis

    Pantelica, A. [Horia Hulubei National Institute for Physics and Nuclear Engineering (IFIN-HH), Magurele, Ilfov County (Romania); Carmo Freitas, M. do [Technological and Nuclear Institute (ITN), Sacavem (Portugal); Ene, A. [Dunarea de Jos Univ. of Galati (Romania). Dept. of Chemistry, Physics and Environment; Steinnes, E. [Norwegian Univ. of Science and Technology, Trondheim (Norway). Dept. of Chemistry

    2013-03-01

    Instrumental neutron activation analysis (INAA) was used to determine concentrations of 42 elements in samples of surface soil collected at seven sites polluted from various anthropogenic activities and a control site in a relatively clean area. Elements studied were Ag, Al, As, Au, Ba, Br, Ca, Cd, Ce, Co, Cr, Cs, Eu, Fe, Gd, Hf, Hg, K, La, Lu, Mg, Mn, Mo, Na, Nd, Ni, Rb, Sb, Sc, Se, Sm, Sr, Ta, Tb, Th, Ti, U, V, W, Yb, Zn, and Zr. The results are compared with data for trace elements atmospheric deposition in lichen transplants from the same sites. The most severe soil contamination was observed at Copsa Mica from non-ferrous metallurgy. Appreciable soil contamination was also indicated at Baia Mare (non-ferrous mining and metallurgy), Deva (coal-fired power plant, cement and building materials industry), Galati (ferrous metallurgy), Magurele and Afumati (general urban pollution), and Oradea (chemical and light industries). In most cases excessive levels of toxic metals in soils matched correspondingly high values in lichen transplants. Compared to Romanian norms, legal upper limits were exceeded for Zn and Cd at Copsa Mica. Also, As and Sb occurred in excessive levels at given sites. (orig.)

  18. Developing a GIS to facilitate data analysis for environmental restoration of a large waste site

    Tzemos, S.; Evans, B.J.; White, M.E.

    1991-05-01

    The Hanford Site area located southeastern Washington State and is a US Department of Energy Superfund cleanup site. The site contains a complex mixture of chemical and radioactive pollutants, which makes environmental monitoring, analysis, and restoration a complicated task. The Hanford Environmental Information System (HEIS) has been developed to manage the data generated from the characterization and environmental monitoring of the Hanford Site. The Geographic Information Systems (GIS) component of HEIS, known as HEISGIS, is being developed to help scientists spatially analyze the environmental data stored in the HEIS database and provide an integrated system for environmental restoration. HEISGIS is in its second stage of development. The first stage produced a working prototype. The second stage focuses on improvements in data accuracy, data standardization, analytical modeling capabilities, interfaces between the various software packages, and the graphical user interface. This paper describes the development of the HEISGIS and summarizes the considerations and difficulties encountered in the integration of the GIS with a large relational database. 1 ref., 1 fig., 3 tabs

  19. Pathway analysis and exposure assessment: MEPAS modeling for nonradiological chemical contaminants at the Hanford Site

    Blanton, M.L.; Dirkes, R.; Buck, J.; Cooper, A.; Castieton, K.; Glantz, C.

    1995-01-01

    A Chemical Pathway Analysis and Exposure Assessment was performed by the Surface Environmental Surveillance Project (SESP). The SESP monitors air, surface water, sediment, agricultural products, vegetation, soil, and wildlife in order to assess onsite of offsite environmental impacts and offsite human health risk at the Hanford Site. The objectives of this study are (1) determine if a nonradiological chemical monitoring program is warranted for the Hanford Site, (2) ensure that the selection of surveillance parameters such as media, sampling location, and analytes are chosen in a manner that is scientifically sound and cost-efficient, and (3) identify specific nonradiological chemicals of concern (COC) for the Hanford Site. The basis for identification of COC for the Hanford Site was an extensive literature review. The model was also used to predict COC concentrations required onsite to achieve an offsite cancer incidence of 1 E-6 and a hazard quotient of 1.0. This study indicated that nonradiological chemical contamination occurring onsite does not pose a significant offsite human health risk. The highest cancer incidence to the offsite maximally exposed individual from COC was from arsenic (1.76E-1 0); the highest hazard quotient was chromium VI (1.48E-04)

  20. A sensitivity analysis of hazardous waste disposal site climatic and soil design parameters using HELP3

    Adelman, D.D.; Stansbury, J.

    1997-01-01

    The Resource Conservation and Recovery Act (RCRA) Subtitle C, Comprehensive Environmental Response, Compensation, And Liability Act (CERCLA), and subsequent amendments have formed a comprehensive framework to deal with hazardous wastes on the national level. Key to this waste management is guidance on design (e.g., cover and bottom leachate control systems) of hazardous waste landfills. The objective of this research was to investigate the sensitivity of leachate volume at hazardous waste disposal sites to climatic, soil cover, and vegetative cover (Leaf Area Index) conditions. The computer model HELP3 which has the capability to simulate double bottom liner systems as called for in hazardous waste disposal sites was used in the analysis. HELP3 was used to model 54 combinations of climatic conditions, disposal site soil surface curve numbers, and leaf area index values to investigate how sensitive disposal site leachate volume was to these three variables. Results showed that leachate volume from the bottom double liner system was not sensitive to these parameters. However, the cover liner system leachate volume was quite sensitive to climatic conditions and less sensitive to Leaf Area Index and curve number values. Since humid locations had considerably more cover liner system leachate volume than and locations, different design standards may be appropriate for humid conditions than for and conditions

  1. A GIS- Based suitability analysis for siting a solid waste in an urban area

    M. Salemi

    2017-01-01

    Full Text Available An appropriate solid waste disposal has been a major problem in municipal environment. The use of landfills is the most economical and environmentally acceptable method for the disposal of solid waste all over the world. The analysis of spatial data and consideration of regulation and accepted criteria are part of the important elements in the site selection. The aim of this paper is to show how application of geographic information system could be used for siting solid waste disposal in Abadan city. In this paper, we consider types of soil suitable for solid waste disposals, land use/ land cover, transportation routes and proximity to surface water. Relative importance weight of each criteria in the geographic information system was determined and finally suitability map was prepared. Based on the final suitability map, appropriate solid waste landfill site was located in north east part of the study area. Select the best landfill site among the candidate ones, and   the output results can enable decision makers to make appropriate decisions to reduce the costs both in   economic and environmental criteria.

  2. Analysis of Air Particles Around Site Plan of Gold Mining, North Sumatera

    Gatot-Suhariyono; Erizal-Tanjung

    2004-01-01

    Analysis of air particles around site plan of gold mining, North Sumatra has been conducted. Air particles of TSP (Total Suspended Particulate), which has maximum diameter around 45 μm (PM 2.5 ) was sampled in four places using impactor cascade. The measurement results indicate that concentration of TSP and PM 10 /PM 2.5 were in site plan center of mining smaller than quality standard of ambient air (PP RI no. 41/1999), while the concentration in areas of around it was on the contrary. The concentration in areas of around the mining was not because of air particle from in site plan center of mining. Based on regulatory of BAPEDAL head no. Kep-107/BAPEDAL/11/1997, concentration of PM 10 /PM 2.5 and TSP in site plan center of mining is in moderate category, while in areas of around the mining are in unhealthy category. Unhealthy category affects decrease at view distance and happened dust defilement everywhere, while moderate category is only happened degradation of view distance. (author)

  3. Sampling and Analysis Plan for U.S. Department of Energy Office of Legacy Management Sites

    2012-01-01

    This plan incorporates U.S. Department of Energy (DOE) Office of Legacy Management (LM) standard operating procedures (SOPs) into environmental monitoring activities and will be implemented at all sites managed by LM. This document provides detailed procedures for the field sampling teams so that samples are collected in a consistent and technically defensible manner. Site-specific plans (e.g., long-term surveillance and maintenance plans, environmental monitoring plans) document background information and establish the basis for sampling and monitoring activities. Information will be included in site-specific tabbed sections to this plan, which identify sample locations, sample frequencies, types of samples, field measurements, and associated analytes for each site. Additionally, within each tabbed section, program directives will be included, when developed, to establish additional site-specific requirements to modify or clarify requirements in this plan as they apply to the corresponding site. A flowchart detailing project tasks required to accomplish routine sampling is displayed in Figure 1. LM environmental procedures are contained in the Environmental Procedures Catalog (LMS/PRO/S04325), which incorporates American Society for Testing and Materials (ASTM), DOE, and U.S. Environmental Protection Agency (EPA) guidance. Specific procedures used for groundwater and surface water monitoring are included in Appendix A. If other environmental media are monitored, SOPs used for air, soil/sediment, and biota monitoring can be found in the site-specific tabbed sections in Appendix D or in site-specific documents. The procedures in the Environmental Procedures Catalog are intended as general guidance and require additional detail from planning documents in order to be complete; the following sections fulfill that function and specify additional procedural requirements to form SOPs. Routine revision of this Sampling and Analysis Plan will be conducted annually at the

  4. Sampling and Analysis Plan for U.S. Department of Energy Office of Legacy Management Sites

    None

    2012-10-24

    This plan incorporates U.S. Department of Energy (DOE) Office of Legacy Management (LM) standard operating procedures (SOPs) into environmental monitoring activities and will be implemented at all sites managed by LM. This document provides detailed procedures for the field sampling teams so that samples are collected in a consistent and technically defensible manner. Site-specific plans (e.g., long-term surveillance and maintenance plans, environmental monitoring plans) document background information and establish the basis for sampling and monitoring activities. Information will be included in site-specific tabbed sections to this plan, which identify sample locations, sample frequencies, types of samples, field measurements, and associated analytes for each site. Additionally, within each tabbed section, program directives will be included, when developed, to establish additional site-specific requirements to modify or clarify requirements in this plan as they apply to the corresponding site. A flowchart detailing project tasks required to accomplish routine sampling is displayed in Figure 1. LM environmental procedures are contained in the Environmental Procedures Catalog (LMS/PRO/S04325), which incorporates American Society for Testing and Materials (ASTM), DOE, and U.S. Environmental Protection Agency (EPA) guidance. Specific procedures used for groundwater and surface water monitoring are included in Appendix A. If other environmental media are monitored, SOPs used for air, soil/sediment, and biota monitoring can be found in the site-specific tabbed sections in Appendix D or in site-specific documents. The procedures in the Environmental Procedures Catalog are intended as general guidance and require additional detail from planning documents in order to be complete; the following sections fulfill that function and specify additional procedural requirements to form SOPs. Routine revision of this Sampling and Analysis Plan will be conducted annually at the

  5. Explorative analysis of microbes, colloids and gases together with microbial modelling. Site description model SDM-Site Laxemar

    Hallbeck, Lotta; Pedersen, Karsten (Microbial Analytics Sweden AB, Goeteborg (Sweden))

    2008-08-15

    The work has involved the development of descriptive and mathematical models for groundwaters in relation to rock domains, fracture domains and deformation zones. Past climate changes are the major driving force for hydrogeochemical changes and therefore of fundamental importance for understanding the palaeohydrogeological, palaeohydrogeochemical and present evolution of groundwater in the crystalline bedrock of the Fennoscandian Shield. Understanding current undisturbed hydrochemical conditions at the proposed repository site is important when predicting future changes in groundwater chemistry. The causes of copper corrosion and/or bentonite degradation are of particular interest as they may jeopardise the long-term integrity of the planned SKB repository system. Thus, the following variables are considered for the hydrogeochemical site descriptive modelling: pH, Eh, sulphur species, iron, manganese, carbonate, phosphate, nitrogen species, total dissolved solids (TDS), isotopes, colloids, fulvic and humic acids and microorganisms. In addition, dissolved gases (e.g. carbon dioxide, methane and hydrogen) are of interest because of their likely participation in microbial reactions. In this series of reports, the final hydrogeochemical evaluation work of the site investigation at the Laxemar site, is presented. The work was conducted by SKB's hydrogeochemical project group, ChemNet, which consists of independent consultants and Univ. researchers with expertise in geochemistry, hydrochemistry, hydrogeochemistry, microbiology, geomicrobiology, analytical chemistry etc. The resulting site descriptive model version, mainly based on available primary data from the extended data freeze L2.3 (Nov 2007). This report focuses on microbiology, colloids and gases. Several methods must be used to characterise active microbial communities in groundwater. Microbial parameters of interest are the total number of cells (TNC) and the presence of various metabolic groups of

  6. Explorative analysis of microbes, colloids and gases together with microbial modelling. Site description model SDM-Site Laxemar

    Hallbeck, Lotta; Pedersen, Karsten

    2008-08-01

    The work has involved the development of descriptive and mathematical models for groundwaters in relation to rock domains, fracture domains and deformation zones. Past climate changes are the major driving force for hydrogeochemical changes and therefore of fundamental importance for understanding the palaeohydrogeological, palaeohydrogeochemical and present evolution of groundwater in the crystalline bedrock of the Fennoscandian Shield. Understanding current undisturbed hydrochemical conditions at the proposed repository site is important when predicting future changes in groundwater chemistry. The causes of copper corrosion and/or bentonite degradation are of particular interest as they may jeopardise the long-term integrity of the planned SKB repository system. Thus, the following variables are considered for the hydrogeochemical site descriptive modelling: pH, Eh, sulphur species, iron, manganese, carbonate, phosphate, nitrogen species, total dissolved solids (TDS), isotopes, colloids, fulvic and humic acids and microorganisms. In addition, dissolved gases (e.g. carbon dioxide, methane and hydrogen) are of interest because of their likely participation in microbial reactions. In this series of reports, the final hydrogeochemical evaluation work of the site investigation at the Laxemar site, is presented. The work was conducted by SKB's hydrogeochemical project group, ChemNet, which consists of independent consultants and Univ. researchers with expertise in geochemistry, hydrochemistry, hydrogeochemistry, microbiology, geomicrobiology, analytical chemistry etc. The resulting site descriptive model version, mainly based on available primary data from the extended data freeze L2.3 (Nov 2007). This report focuses on microbiology, colloids and gases. Several methods must be used to characterise active microbial communities in groundwater. Microbial parameters of interest are the total number of cells (TNC) and the presence of various metabolic groups of

  7. Live births after polar body biopsy and frozen-thawed cleavage stage embryo transfer: case report.

    Guimarães, Fernando; Roque, Matheus; Valle, Marcello; Kostolias, Alessandra; Azevedo, Rodrigo A de; Martinhago, Ciro D; Sampaio, Marcos; Geber, Selmo

    2016-12-01

    Pre-implantation genetic diagnosis (PGD) or screening (PGS) technology, has emerged and developed in the past few years, benefiting couples as it allows the selection and transfer of healthy embryos during IVF treatments. These techniques can be performed in oocytes (polar-body biopsy) or embryos (blastomere or trophectoderm biopsy). In this case report, we describe the first two live births to be published in Brazil after a polar-body (PB) biopsy. In case 1, a 42-year-old was submitted to PB biopsy with PGS due to advanced maternal age and poor ovarian reserve. Five MII oocytes underwent first and second polar body biopsy and four cleavage embryos were cryopreserved. The PGS analysis resulted in two euploid embryos (next generation sequence). A frozen-thawed embryo transfer (FET) was performed after endometrial priming and a healthy baby was delivered after a cesarean section (37 weeks, female, 3390g, 47.5 cm). In case 2, a 40-year old patient with balanced translocation and poor ovarian response was submitted to PB biopsy. Two MII oocytes underwent first and second polar body biopsy and two embryos were cryopreserved in cleavage stage. The analysis resulted in one euploid embryo that was transferred after endometrial priming. A preterm healthy baby (34 weeks, female, 2100g, 40 cm) was delivered via cesarean section. In conclusion, although the blastocyst biopsy is the norm when performing PGS/PGD during IVF treatments, other alternatives (as PB biopsy) should be considered in some specific situations.

  8. Egypt's Red Sea coast: phylogenetic analysis of cultured microbial consortia in industrialized sites.

    Mustafa, Ghada A; Abd-Elgawad, Amr; Abdel-Haleem, Alyaa M; Siam, Rania

    2014-01-01

    The Red Sea possesses a unique geography, and its shores are rich in mangrove, macro-algal and coral reef ecosystems. Various sources of pollution affect Red Sea biota, including microbial life. We assessed the effects of industrialization on microbes along the Egyptian Red Sea coast at eight coastal sites and two lakes. The bacterial communities of sediment samples were analyzed using bacterial 16S rDNA pyrosequencing of V6-V4 hypervariable regions. The taxonomic assignment of 131,402 significant reads to major bacterial taxa revealed five main bacterial phyla dominating the sampled sites: Proteobacteria (68%), Firmicutes (13%), Fusobacteria (12%), Bacteriodetes (6%), and Spirochetes (0.03%). Further analysis revealed distinct bacterial consortia that primarily included (1) marine Vibrio spp.-suggesting a "marine Vibrio phenomenon"; (2) potential human pathogens; and (3) oil-degrading bacteria. We discuss two divergent microbial consortia that were sampled from Solar Lake West near Taba/Eilat and Saline Lake in Ras Muhammad; these consortia contained the highest abundance of human pathogens and no pathogens, respectively. Our results draw attention to the effects of industrialization on the Red Sea and suggest the need for further analysis to overcome the hazardous effects observed at the impacted sites.

  9. Egypt’s Red Sea Coast: Phylogenetic analysis of cultured microbial consortia in industrialized sites

    Ghada A. Mustafa

    2014-08-01

    Full Text Available The Red Sea has a unique geography and ecosystem and its shores are very rich in mangrove, macro-algae and coral reefs. Different sources of pollution are affecting the Red Sea shores and waters which impacts biological life including microbial life. We assessed the effects of industrialization, along the Egyptian Red Sea coast in eight coastal sites and two lakes, on microbial life. The bacterial community in sediment samples was analyzed using bacterial 16S rDNApyrosequencing of V6-V4 hypervariable regions. Taxonomical assignment of 131,402 significant reads to major bacterial taxa revealed five main bacterial phyla dominating the sampled Red Sea sites. This includes Proteobacteria (68%, Firmicutes (13%, Fusobacteria (12%, Bacteriodetes (6% and Spirochetes (0.03%. Further analysis revealed distinct bacterial consortium formed mainly of: 1 marine Vibrio’s- suggesting a Marine Vibrio phenomenon 2 potential human pathogens and 3 oil-degrading bacteria. We discuss a distinct microbial consortium in Solar Lake West near Taba/Eilat and Saline Lake in Ras Muhammad; revealing the highest abundance of human pathogens versus no pathogens, respectively. Our results draw attention to the affects of industrialization on the Red Sea, and suggest further analysis to overcome hazardous affects on the impacted sites.

  10. Sampling and analysis plan for Wayne Interim Storage Site (WISS), Wayne, New Jersey

    Brown, K.S.; Murray, M.E.; Rodriguez, R.E.

    1998-10-01

    This field sampling plan describes the methodology to perform an independent radiological verification survey and chemical characterization of a remediated area of the subpile at the Wayne Interim Storage Site, Wayne, New Jersey.Data obtained from collection and analysis of systematic and biased soil samples will be used to assess the status of remediation at the site and verify the final radiological status. The objective of this plan is to describe the methods for obtaining sufficient and valid measurements and analytical data to supplement and verify a radiological profile already established by the Project Remediation Management Contractor (PMC). The plan describes the procedure for obtaining sufficient and valid analytical data on soil samples following remediation of the first layer of the subpile. Samples will be taken from an area of the subpile measuring approximately 30 m by 80 m from which soil has been excavated to a depth of approximately 20 feet to confirm that the soil beneath the excavated area does not exceed radiological guidelines established for the site or chemical regulatory limits for inorganic metals. After the WISS has been fully remediated, the Department of Energy will release it for industrial/commercial land use in accordance with the Record of Decision. This plan provides supplemental instructions to guidelines and procedures established for sampling and analysis activities. Procedures will be referenced throughout this plan as applicable, and are available for review if necessary

  11. Modelling and analysis of radionuclide dispersion from PWR on abnormal condition in Bojanegara Serang site

    Sri Kuntjoro

    2010-01-01

    Additional of electrical power especially Nuclear Power Plant will give radiological consequence sto population and environment due to radioactive release in normal and abnormal condition. In consequence the management of nuclear power plant must supply data and strong argumentation to clarify the safety of nuclear power plant to environment. For that purpose it needs to be carried out an analysis of abnormal condition in nuclear power plant and its radiological consequences to the environment. That analysis is done using abnormal condition simulation model postulated on 1000 MWe nuclear power plant.That simulation model is used also to evaluate environmental potential as site capability in supporting the radiological consequences. Radionuclide transport modeling from reactor core to containment uses EMERALD computer code. Other computer codes are Wind rose, PC-COSYMA and Arc View are used to simulate meteorology condition, radionuclide release to population distribution of food production and consumption and distribution of radiation dose received to population around nuclear power plant. Application of that simulation is carried out to NPP candidate site in Bojanegara-Kramatwatu, Serang Banten peninsula. Using source term data, meteorology data, dispersion data and pathways modeling are resulting radionuclide dispersion model and radiation pathway acceptance at the surrounding nuclear power plant site (Bojanegara-Serang peninsula). The result shows that maximum radiation dose received is lower than dose permitted in accordance with regulatory body (BAPETEN). (author)

  12. Coastal flooding as a parameter in multi-criteria analysis for industrial site selection

    Christina, C.; Memos, C.; Diakoulaki, D.

    2014-12-01

    Natural hazards can trigger major industrial accidents, which apart from affecting industrial installations may cause a series of accidents with serious impacts on human health and the environment far beyond the site boundary. Such accidents, also called Na-Tech (natural - technical) accidents, deserve particular attention since they can cause release of hazardous substances possibly resulting in severe environmental pollution, explosions and/or fires. There are different kinds of natural events or, in general terms, of natural causes of industrial accidents, such as landslides, hurricanes, high winds, tsunamis, lightning, cold/hot temperature, floods, heavy rains etc that have caused accidents. The scope of this paper is to examine the coastal flooding as a parameter in causing an industrial accident, such as the nuclear disaster in Fukushima, Japan, and the critical role of this parameter in industrial site selection. Land use planning is a complex procedure that requires multi-criteria decision analysis involving economic, environmental and social parameters. In this context the parameter of a natural hazard occurrence, such as coastal flooding, for industrial site selection should be set by the decision makers. In this paper it is evaluated the influence that has in the outcome of a multi-criteria decision analysis for industrial spatial planning the parameter of an accident risk triggered by coastal flooding. The latter is analyzed in the context of both sea-and-inland induced flooding.

  13. Radioactive dispersion analysis for hypothetical nuclear power plant (NPP) candidate site in Perak state, Malaysia

    Shamsuddin, Shazmeen Daniar; Basri, Nor Afifah; Omar, Nurlyana; Koh, Meng-Hock; Ramli, Ahmad Termizi; Saridan Wan Hassan, Wan Muhamad

    2017-10-01

    Malaysia is planning to build a nuclear power plant (NPP) by 2030 to diversify the national electricity supply and resources. Selection of an NPP site must consider various factors, especially nuclear safety consideration to fulfil the nuclear safety objectives. Environmental Risk Assessment Analysis is a part of safety requirements by the International Atomic Energy Agency (IAEA) prior to the NPP commissioning process. Risk Assessments Analysis (RIA) is compulsory for the NPP site evaluation. One of RIA methods are Radioactive Dispersion Analysis using probabilistic risk analysis software. It is also important to perform studies to estimate the impact to the neighbouring population in the case of a nuclear accident at the power plant. In the present work, aimed to study the impact of a hypothetical nuclear accident by simulating the dispersion pattern of radionuclides originated from a candidate site at Manjung, Perak. The work has been performed using the HotSpot Health Physics codes. Two types of radionuclides have been considered namely 137Cs and 131I. In calculations, the initial concentration of radioactive materials of Fukushima Daiichi accident data are used which are 2.06 x 1016 Bq and 1.68 x 1017 Bq respectively for the two radionuclides. The result shows that the dispersion distance obtained from both software are not the same. It shows that 137Cs and 131I can be dispersed as far as 16 km and 80 km away from the site during radiological accident respectively, reaching major towns in Perak. Using HOTSPOT, the estimated total effective dose equivalent (TEDE) for 137Cs and 131I at major towns in Perak such as Lumut and Sitiawan are 1.2 mSv and 9.9 mSv. As for Taiping, Ipoh, Kampar, and Teluk Intan the estimated TEDE is around 0.2 mSv and 1.6 mSv respectively. In conclusion, the dispersion can reach as far as 80 km from the site. However, estimated annual effective dose is not more than 1 mSv limit, which is considered acceptable in the point of view of

  14. Radioactive dispersion analysis for hypothetical nuclear power plant (NPP candidate site in Perak state, Malaysia

    Shamsuddin Shazmeen Daniar

    2017-01-01

    Full Text Available Malaysia is planning to build a nuclear power plant (NPP by 2030 to diversify the national electricity supply and resources. Selection of an NPP site must consider various factors, especially nuclear safety consideration to fulfil the nuclear safety objectives. Environmental Risk Assessment Analysis is a part of safety requirements by the International Atomic Energy Agency (IAEA prior to the NPP commissioning process. Risk Assessments Analysis (RIA is compulsory for the NPP site evaluation. One of RIA methods are Radioactive Dispersion Analysis using probabilistic risk analysis software. It is also important to perform studies to estimate the impact to the neighbouring population in the case of a nuclear accident at the power plant. In the present work, aimed to study the impact of a hypothetical nuclear accident by simulating the dispersion pattern of radionuclides originated from a candidate site at Manjung, Perak. The work has been performed using the HotSpot Health Physics codes. Two types of radionuclides have been considered namely 137Cs and 131I. In calculations, the initial concentration of radioactive materials of Fukushima Daiichi accident data are used which are 2.06 x 1016 Bq and 1.68 x 1017 Bq respectively for the two radionuclides. The result shows that the dispersion distance obtained from both software are not the same. It shows that 137Cs and 131I can be dispersed as far as 16 km and 80 km away from the site during radiological accident respectively, reaching major towns in Perak. Using HOTSPOT, the estimated total effective dose equivalent (TEDE for 137Cs and 131I at major towns in Perak such as Lumut and Sitiawan are 1.2 mSv and 9.9 mSv. As for Taiping, Ipoh, Kampar, and Teluk Intan the estimated TEDE is around 0.2 mSv and 1.6 mSv respectively. In conclusion, the dispersion can reach as far as 80 km from the site. However, estimated annual effective dose is not more than 1 mSv limit, which is considered acceptable in the point

  15. Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow.

    Suryawanshi, Gajendra W; Xu, Song; Xie, Yiming; Chou, Tom; Kim, Namshin; Chen, Irvin S Y; Kim, Sanggu

    2017-06-14

    Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

  16. Numerical and on-site experimental dynamic analysis of the Italian PEC fast reactor building

    Castoldi, A.; Muzzi, F.; Orsi, R.; Panzeri, P.; Pezzoli, P.; Ruggeri, G.; Martelli, A.; Masoni, P.; Brancati, V.

    1988-01-01

    On-site dynamic tests and three-dimensional numerical analysis have been performed by ISMES on behalf of ENEA on the building of the Italian PEC fast reactor test facility. These studies aimed at evaluating the safety margins in the PEC reactor seismic analysis and at providing data for the optimization of the PEC seismic monitoring system. The paper describes the on-site dynamic tests carried out using various excitation methods (two eccentric back-rotating-mass mechanical vibrator, blasting in bore-hole and hydraulic actuators at the building foundations). It highlights the purposes of the four tests campaigns performed at various construction stages and reports the main experimental results. In connection with the experimental tests, a detailed 3D finite element model was set up for fixed base analysis; from the results of the 3D model a simplified equivalent model of the structure was then derived for soil-structure interaction analysis. The mathematical model was validated and calibrated by using the results of the experimental dynamic tests. The main numerical results and the comparisons with the experimental data are presented. (author)

  17. Cleavage preference distinguishes the two-component NS2B-NS3 serine proteinases of Dengue and West Nile viruses.

    Shiryaev, Sergey A; Kozlov, Igor A; Ratnikov, Boris I; Smith, Jeffrey W; Lebl, Michal; Strongin, Alex Y

    2007-02-01

    Regulated proteolysis of the polyprotein precursor by the NS2B-NS3 protease is required for the propagation of infectious virions. Unless the structural and functional parameters of NS2B-NS3 are precisely determined, an understanding of its functional role and the design of flaviviral inhibitors will be exceedingly difficult. Our objectives were to define the substrate recognition pattern of the NS2B-NS3 protease of West Nile and Dengue virises (WNV and DV respectively). To accomplish our goals, we used an efficient, 96-well plate format, method for the synthesis of 9-mer peptide substrates with the general P4-P3-P2-P1-P1'-P2'-P3'-P4'-Gly structure. The N-terminus and the constant C-terminal Gly of the peptides were tagged with a fluorescent tag and with a biotin tag respectively. The synthesis was followed by the proteolytic cleavage of the synthesized, tagged peptides. Because of the strict requirement for the presence of basic amino acid residues at the P1 and the P2 substrate positions, the analysis of approx. 300 peptide sequences was sufficient for an adequate representation of the cleavage preferences of the WNV and DV proteinases. Our results disclosed the strict substrate specificity of the WNV protease for which the (K/R)(K/R)R/GG amino acid motifs was optimal. The DV protease was less selective and it tolerated well the presence of a number of amino acid residue types at either the P1' or the P2' site, as long as the other position was occupied by a glycine residue. We believe that our data represent a valuable biochemical resource and a solid foundation to support the design of selective substrates and synthetic inhibitors of flaviviral proteinases.

  18. Addendum to Composite Analysis for Low-Level Waste Disposal in the 200 Area Plateau of the Hanford Site

    Bergeron, Marcel P.; Freeman, Eugene J.; Wurstner, Signe K.; Kincaid, Charles T.; Coony, Mike M.; Strenge, Dennis L.; Aaberg, Rosanne L.; Eslinger, Paul W.

    2001-09-28

    This report summarizes efforts to complete an addendum analysis to the first iteration of the Composite Analysis for Low-Level Waste Disposal in the 200 Area Plateau of the Hanford Site (Composite Analysis). This document describes the background and performance objectives of the Composite Analysis and this addendum analysis. The methods used, results, and conclusions for this Addendum analysis are summarized, and recommendations are made for work to be undertaken in anticipation of a second analysis.

  19. Essential Structural and Functional Roles of the Cmr4 Subunit in RNA Cleavage by the Cmr CRISPR-Cas Complex

    Nancy F. Ramia

    2014-12-01

    Full Text Available Summary: The Cmr complex is the multisubunit effector complex of the type III-B clustered regularly interspaced short palindromic repeats (CRISPR-Cas immune system. The Cmr complex recognizes a target RNA through base pairing with the integral CRISPR RNA (crRNA and cleaves the target at multiple regularly spaced locations within the complementary region. To understand the molecular basis of the function of this complex, we have assembled information from electron microscopic and X-ray crystallographic structural studies and mutagenesis of a complete Pyrococcus furiosus Cmr complex. Our findings reveal that four helically packed Cmr4 subunits, which make up the backbone of the Cmr complex, act as a platform to support crRNA binding and target RNA cleavage. Interestingly, we found a hook-like structural feature associated with Cmr4 that is likely the site of target RNA binding and cleavage. Our results also elucidate analogies in the mechanisms of crRNA and target molecule binding by the distinct Cmr type III-A and Cascade type I-E complexes. : Ramia et al. show that the helical core of the type III-B Cmr CRISPR-Cas effector complex, made up of multiple Cmr4 subunits, forms the platform for a corresponding number of cleavages of the target RNA. Comparison with the type I-E Cascade structure reveals strikingly similar mechanisms of crRNA and target binding.

  20. Targeted cleavage of hepatitis E virus 3' end RNA mediated by hammerhead ribozymes inhibits viral RNA replication

    Sriram, Bandi; Thakral, Deepshi; Panda, Subrat Kumar

    2003-01-01

    The 3' end of hepatitis E virus (HEV) contains cis-acting regulatory element, which plays an important role in viral replication. To develop specific replication inhibitor at the molecular level, mono- and di-hammerhead ribozymes (Rz) were designed and synthesized against the conserved 3' end sequences of HEV, which cleave at nucleotide positions 7125 and 7112/7125, respectively. Di-hammerhead ribozyme with two catalytic motifs in tandem was designed to cleave simultaneously at two sites spaced 13 nucleotides apart, which increases the overall cleavage efficiency and prevents the development of escape mutants. Specific cleavage products were obtained with both the ribozymes in vitro at physiological conditions. The inactive control ribozymes showed no cleavage. The ribozymes showed specific inhibition of HEV 3' end fused-luciferase reporter gene expression by ∼37 and ∼60%, respectively in HepG2 cells. These results demonstrate a feasible approach to inhibit the HEV replication to a limited extent by targeting the cis-acting 3' end of HEV with hammerhead ribozymes